Electrophoresis '79: Advanced methods, biochemical and clinical applications. Proceedings of the Second International Conference on Electrophoresis, Munich, Germany, October 15–17, 1979 9783111713625, 9783110081541

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Electrophoresis '79 Advanced Methods Biochemical and Clinical Applications

Electrophoresis '79 Advanced Methods Biochemical and Clinical Applications Proceedings of the Second International Conference on Electrophoresis Munich, Germany, October 15-17,1979 Editor Bertold J. Radola

W DE

G Walter de Gruyter • Berlin • New York 1980

Editor Prof. Dr. Bertold J. Radola Institut für Lebensmitteltechnologie und Analytische Chemie Technische Universität München D-8050 Freising-Weihenstephan Federal Republic of Germany

CIP-Kurztitelaufnahme

der Deutschen

Bibliothek

[Electrophoresis seventy-nine] Electrophoresis '79 : advanced methods, biochem. and clin. applications; proceedings of the 2. Internat. Conference on Electrophoresis, Munich, Germany, October 15-17,1979/ed. Bertold J. Radola. - Berlin, New York: de Gruyter, 1980. ISBN 3-11-008154-7 NE: Radola, Bertold J. [Hrsg.]; International Conference on Electrophoresis

Library of Congress Cataloging in Publication

Data

International Conference on Elektrophoresis, 2d, Munich, 1979. Electrophoresis '79. Includes index. 1. Electrophoresis-Congresses. 2. Isoelectric Focusing-Congresses. 3. Biological Chemistry-Technique-Congresses. 4. Chemistry ClinicalTechnique-Congresses. 1. Radola, B.J., 1931QP 519.9. E. 434 157 1979 574.19'28 80-13830 ISBN 3-11-008154-7

© Copyright 1980 by Walter de Gruyter & Co., Berlin 30. All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form - by photoprint, microfilm, or any other means - nor transmitted nor translated into a machine language without written permission from the publisher. Printing: Color-Druck, Berlin. - Binding: Ltideritz & Bauer, Buchgewerbe GmbH. Printed in Germany.

PREFACE

The International Conference "Electrophoresis '79" was held at the Technical University in Munich on October 15 - 17, 1979. It was the second of a projected series of international meetings organized with the objective to stimulate communication, information exchange and advancement of knowledge in all areas of electrophoresis. The need for this type of conferences is just an inference of a development in the field of electrophoretic methodology. In recent years not only have different techniques emerged but some have fused to powerful combinations, and it therefore seems justified to deal with different aspects of electrophoresis under a broader heading. The conference in Munich was the largest of all electrophoretic meetings so far held. Much of the success of "Electrophoresis '79" has been achieved because it attracted most of the recognized leaders in the field. The conference was attended by participants from all parts of the world and from a broad range of academic disciplines. The format of the conference as well as the balanced representation from laboratories involved in the development and the application of electrophoresis- has led to excellent personal interaction and much intellectual stimulation and benefit to the participants. The conference was certainly successful. This volume contains a large portion of the papers presented either as oral reports or as posters at "Electrophoresis '79". The manuscripts have been compiled in four sections entitled: I. Theory and Methods, II. High Resolution Two-Dimensional Electrophoresis, III. Preparative Separations and IV. Biomedical and Biological Applications. These sections correspond to the major topics on which emphasis was placed at the conference. Not every manuscript fits precisely into one or another of the four sections but the most appropriate assignment has been made

VI wherever possible. This volume could be produced because most authors were cooperative and admirably disciplined with regard to the deadline set for the submission of manuscripts. I take pleasure in expressing my appreciations and thanks to all contributors. The electrophoretic community owes much to them. I also greatly appreciate the efforts of the staff of Walter de Gruyter, Berlin, which led to the rapid publication of this volume.

Freising-Weihenstephan Bertold J. Radola

ACKNOWLEDGEMENTS

I would like to acknowledge the splendid help of my laboratory staff in organizing the conference. I am indebted to Helga Piontek for excellent typing. I also thank my wife for volunteering her time and talent in helping to organize the conference and to prepare this volume for publication. Financial help for the conference was provided by: Bio-Rad Laboratories, München; Desaga, Heidelberg; Deutsche Pharmacia, Freiburg; Immuno Diagnostika, Heidelberg; LKB Instrument, Gräfelfing; Serva Feinbiochemica, Heidelberg; Shandon Labortechnik, Frankfurt; Shimadzu (Europa), Düsseldorf.

VII CONTENTS

SECTION I, THEORY AND METHODS

GEL ELECTROFOCUSING WITH INCREASED DEGREES OF FREEDOM A. Chrambach, L. Hjelmeland and N. Y. Nguyen pH-MOBILITY CURVES OF PROTEINS BY ISOELECTRIC FOCUSING COMBINED WITH ELECTROPHORESIS AT RIGHT ANGLES P. G. Righetti and E. Gianazza

23

PARTITIONING AND ELECTROPHORESIS IN FLEXIBLE POLYMER NETWORKS H. J. Bode

39

DOUBLE ONE-DIMENSIONAL SLAB-GEL ELECTROPHORESIS K. Altland and R. Hackler

53

ULTRATHIN-LAYER HORIZONTAL ELECTROPHORESIS, ISOELECTRIC FOCUSING, AND PROTEIN MAPPING IN POLYACRYLAMIDE GELS ON CELLOPHANE A. Görg, W. Postel and R. Westermeier

67

ULTRATHIN-LAYER ISOELECTRIC FOCUSING IN 50 - 100 urn POLYACRYLAMIDE GELS ON SILANIZED GLASS PLATES OR POLYESTER FILMS B. J. Radola

79

ISOELECTRIC FOCUSING OF PROTEINS IN AGAROSE GELS AND DETECTION OF THE PROTEINS BY DIFFERENT STAINING PROCEDURES 0. Vesterberg

95

AGAROSE ISOELECTRIC FOCUSING OF MONOCLONAL IgM AND IgG ANTIBODIES A. Rosen

105

IMMUNOPEROXIDASE STAINING AND RADIOIMMUNOBINDING OF HUMAN TUMOR MARKERS SEPARATED BY DIRECT TISSUE AGAROSE ISOELECTRIC FOCUSING C. A. Saravis, C. G. Cunningham, P. V. Marasco, R. B. Cook and N. Zamcheck

117

Vili AGAROSE AS SUPPORTING MEDIUM FOR ANALYTICAL ISOELECTRIC FOCUSING: DETERMINATION OF THE RECENTLY DISCOVEREC SUBTYPES IN THE HUMAN SERUM PROTEIN Gc (GROUP SPECIFIC COMPONENT) M. Thymann

123

FACTS AND ARTEFACTS IN ISOELECTRIC FOCUSING E. Gianazza and P. G. Righetti

129

ISOELECTRIC FOCUSING PATTERNS OF SIMIAN VIRUS 40 (SV 40) TUMOR ANTIGEN: VARIOUS ALKYLATION PROCEDURES CAUSE REVERSIBLE ARTEFACTS K. Palme and R. Hanning

141

THE ROLE OF SIALIC ACID IN THE MICROHETEROGENEITY OF ALPHA. ACID GLYCOPROTEIN: STUDY BY ISOELECTRIC FOCUSING AND TITRATION CURVES P. Arnaud, E. Gianazza, P. G. Righetti and H. H. Fudenberg

151

THE EFFECT OF ELECTRODE SOLUTIONS ON THE pH DRIFT IN THIN-LAYER ISOELECTRIC FOCUSING H. Delincêe

165

QUANTITATIVE IMMUNOELECTROPHORESIS: A SURVEY AND SOME APPLICATIONS T. C. B?Sg-Hansen, I. Lorenc-Kubis and 0. J. Bjerrum

173

AFFINITY ELECTROPHORESIS: QUANTIFICATION AND CHARACTERIZATION OF GLYCOPROTEINS WITH LECTINS T. C. Btfg-Hansen

193

RECENT ADVANCES IN MICROELECTROPHORESIS V. Neuhoff

203

A SIMPLE, EFFECTIVE AND INEXPENSIVE EQUIPMENT FOR POLYACRYLAMIDE GEL ELECTROPHORESIS IN COOLED MICROCAPILLARIES R. Peter

219

LINEAR POLYACRYLAMIDE GRADIENT GELS FOR MOLECULAR WEIGHT ESTIMATION OF PROTEINS P. Lambin

227

IX SOLUBLE POLYACRYLAMIDE GRADIENT GELS IN THE SEPARATION AND ESTIMATION OF PROTEINS M. P. Brown and A. V. Ernes

235

QUANTIFICATION OF PROTEINS SEPARATED BY TWO-DIMENSIONAL PAGE: A NOVEL DYE ELUTION TECHNIQUE COMPARED WITH A DENSITOMETRY PROCEDURE O. H. W. Martini, J. Kruppa and R. Temkin

241

PREPARATION AND ANALYSIS OF PROTEINS AND PROTEIN FRAGMENTS BY TWO-DIMENSIONAL THIN-LAYER GEL FILTRATION AND ISOELECTRIC FOCUSING K. G. Welider

249

ENHANCEMENT OF LOAD CAPACITY IN ISOTACHOPHORESIS F. M. Everaerts, F. E. P. Mikkers and Th. P. E. M. Verhegen

255

A MODEL FOR THE USE OF MORE THAN ONE SINGLE BUFFERING COMMON COUNTERION IN ISOTACHOPHORESIS P. J. Svendsen and C. Schafer-Nielsen

265

SEPARATION OF MOLECULES IN STEADY STATE ELECTROPHORESIS SYSTEMS WITH ZONES CONTAINING MORE THAN ONE TERMINATING ION C. Schafer-Nielsen and P. J. Svendsen

275

FLOW-THROUGH CONTINUOUS THIN LAYER CARRIER-FREE ISOTACHOPHORESIS Z. Prusik, J. Stëpânek and V. KaSiäka

287

SECTION II, HIGH RESOLUTION TWO-DIMENSIONAL ELECTROPHORESIS

TWO-DIMENSIONAL ELECTROPHORESIS: HIGH RESOLUTION OF PROTEINS AND AUTOMATIC EVALUATION OF THE PATTERNS UNDER DIFFERENT METHODICAL CONDITIONS J. Klose, J. Nowak and W. Kade

297

HIGH-RESOLUTION TWO-DIMENSIONAL ELECTROPHORETIC MAPPING OF HUMAN PROTEINS N. Anderson, J. J. Edwards, C. S. Giometti, K. E. Willard, S. L. Tollaksen, S. L. Nance, B. J. Hickman, J. Taylor, B. Coulter, A. Scandora and N. G. Anderson 313

X ESTIMATION OF TWO-DIMENSIONAL ELECTROPHORETIC SPOT INTENSITIES AND POSITIONS BY MODELING J. Taylor, N. L. Anderson, B. P. Coulter, A. E. Scandora, Jr. and N. G. Anderson

329

INTERNAL CHARGE STANDARDIZATION FOR TWO-DIMENSIONAL ELECTROPHORESIS B. J. Hickman, N. L. Anderson, K. E. Willard and N. G. Anderson

341

A METHOD FOR STUDYING PROTEINS IN 2-D GELS USING THERMAL DENATURATION ANALYSIS S. L. Nance, B. J. Hickman, N. L. Anderson

351

THE QUANTITATION OF RADIOACTIVELY LABELED PROTEINS ON TWO-DIMENSIONAL GELS: TESTS OF A METHOD FOR ANALYZING CHANGES IN PROTEIN SYNTHESIS AND GENE EXPRESSION G. C. Stone, D. L. Wilson and G. W. Perry

361

TWO-DIMENSIONAL ELECTROPHORETIC MAPPING OF HUMAN ERYTHROCYTE PROTEINS J. J. Edwards, H. J. Hahn and N. G. Anderson

383

TWO-DIMENSIONAL ELECTROPHORESIS OF HUMAN SALIVA C. S. Giometti and N. G. Anderson

395

TWO-DIMENSIONAL ELECTROPHORESIS OF HUMAN URINARY PROTEINS IN HEALTH AND DISEASE S. L. Tollaksen and N. G. Anderson

405

ALTERATIONS OF TWO-DIMENSIONAL ELECTROPHORETIC MAPS IN HUMAN PERIPHERAL BLOOD LYMPHOCYTES INDUCED BY CONCANAVALIN A K. E. Willard and N. L. Anderson

415

HIGH RESOLUTION TWO-DIMENSIONAL ELECTROPHORESIS (ISO-DALT) IN CLINCAL CHEMISTRY: COMPARISON WITH ROUTINE ELECTROPHORETIC AND IMMUNOLOGICAL METHODS A. K. Thorsrud, H. F. Haugen and E. Jellum

425

TWO-DIMENSIONAL MAPS OF HUMAN APOLIPOPROTEINS IN NORMAL AND DISEASED STATES V. I. Zannis and J. L. Breslow

437

XI ISOELECTRIC FOCUSING AND TWO-DIMENSIONAL ELECTROPHORESIS OF VIRAL PROTEINS AND VIRUS-INFECTED CELLS R. Drzeniek, C. Reichel, K. J. Wiegers, A. Hamann and M. Hilbrig

475

SECTION III, PREPARATIVE SEPARATIONS

ISOELECTRIC FOCUSING, ISOELECTRIC POINTS, AND THE PREPARATIVE SEPARATION OF PROTEINS D. H. Leaback

493

PREPARATIVE MULTIPHASIC ZONE ELECTROPHORESIS AND ISOELECTRIC FOCUSING IN DENSITY GRADIENTS USING THE POLY-PREP N. Catsimpoolas, A. Stamatopoulou and A. L. Griffith

503

MULTIPHASIC COLUMN ELECTROFOCUSING: AN APPROACH TO COMPOSITE FRACTIONATION PROBLEMS D. Stathakos, A. Vellios and S. Koussoulakos

517

PREPARATIVE ELECTROFOCUSING OF ENZYMES INVOLVED IN AROMATIC DEGRADATION IN T. CUTANEUM A. B. Gaal and H. Y. Neujahr

529

PREPARATIVE FLAT-BED ISOELECTRIC FOCUSING OF LDH, MDH, ALD, SDH AND G-6.PDH FROM RABBIT LENS J. Bours, M. Garbers and 0. Hockwin

539

CONTINUOUS-FLOW ISOELECTRIC FOCUSING OF PROTEINS P. Basset, J. Zwiller, C. Froissart, M. Recasens, R. Massarelli and G. Vincendon

545

RECYCLING OF CARRIER AMPHOLYTES BY ULTRAFILTRATION K. Goerth and B. J. Radola

555

REMOVAL OF SALTS AND CARRIER AMPHOLYTES FROM BILIPEPTIDES OF LOW MOLECULAR WEIGHT H.-P. Kost and E. Kost-Reyes

565

PREPARATIVE ELECTROPHORESIS IN GEL-BLOCKS WITH DISCONTINUOUS ELUTION H. Stegemann

571

XII FURTHER DEVELOPMENTS IN PREPARATIVE DESPLACEMENT ELECTROPHORESIS IN GEL F. Hampson

583

VELOCITY GRADIENT STABILISED CONTINUOUS ELECTROPHORESIS A. R. Thomson, P. Mattock and G. F. Aitchison

591

PREPARATIVE DENSITY GRADIENT ELECTROPHORESIS OF LYMPHOID CELLS N. Catsimpoolas, A. L. Griffith, S. Gupta, R. A. Good and C. D. Platsoucas

607

PREPARATIVE CELL ELECTROPHORESIS IN A STATIC VERTICAL COLUMN STABILIZED WITH A HEAVY WATER GRADIENT C. J. van Oss and P. M. Bronson

623

SECTION IV, BIOMEDICAL AND BIOLOGICAL APPLICATIONS

PAST, PRESENT AND FUTURE USES OF ELECTROPHORESIS IN CLINICAL MEDICINE R. C. Allen

631

TWO-DIMENSIONAL PROTEIN PATTERNS OF HUMAN BODY FLUIDS OBTAINED UNDER DISSOCIATING AND NON-DISSOCIATING CONDITIONS K. Felgenhauer

647

SIMPLIFIED DETERMINATION OF HEMOGLOBIN A, IN 1c DIABETIC PATIENTS BY USE OF ELECTROFOCUSING J. 0. Jeppson, B. Franzen and A. B. Gaal

655

A COMPARISON OF ISOELECTRIC FOCUSING AND ELECTROCHROMATOGRAPHY FOR THE SEPARATION AND QUANTIFICATION OF HEMOGLOBIN A. 1c R. C. Allen, M. Stastny, D. Hallett and M. A. Simmons

663

INVESTIGATIONS OF HAEMOGLOBIN A„ ANALOGUES BY 1C ELECTROFOCUSING ON POLYACRYLAMIDE GELS C. J. Holloway, U. Schianstedt-Jahn, I. Haeger, H.-J. Mitzkat and I. Trautschold

669

XIII ISOELECTRIC ENZYME PATTERN: A NEW CONCEPT FOR TUMOR CLASSIFICATION H. J. Radzun, M. R. Parwaresch and D. Schmidt

679

MONOCYTE-SPECIFIC ISOELECTRIC FOCUSING PATTERN OF LYSOSOMAL ACID ESTERASE: A NEW POSSIBILITY FOR CELL-SPECIFIC IMMUNOHISTOCHEMICAL IDENTIFICATION J. W. Wittke, H. J. Radzun and M. R. Parwaresch

683

ISOELECTRIC FOCUSING IN CELLULOSE ACETATE MEMBRANE AND FLAT-BED GRANULATED GEL: APPLICATION TO THE STUDY OF HUMAN ALDEHYDE DEHYDROGENASE ISOZYMES S. Harada, D. P. Agarwal and H. W. Goedde

687

CEREBROSPINAL FLUID (CSF) PROTEIN PATTERNS DETERMINED BY ISOELECTRIC FOCUSING IN MULTIPLE SCLEROSIS B. Perthen and W. Müller-Beißenhirtz

695

BINDING AFFINITIES BETWEEN VDBP AND VITAMIN D3, 25-(OH)-D3, 24-25-(OH)2-D3 and 1-25-(OH)2-D3 STUDIED BY ELECTROPHORETIC METHODS (PAGE-IE-F, COMBINED IEFELECTROPHORESIS) AND PRINT-IMMUNOFIXATION J. Constans, M. Viau, C. Gouaillard, C. Bouissou and A. Clerc

701

IMMUNOFIXATION AFTER ELECTROFOCUSING: APPLICATION TO THE STUDY OF THE MICROHETEROGENEITY AND PLYMORPHISM OF SERUM PROTEINS C. Chapuis-Cellier and A. Francina

711

MICROHETEROGENEITY AND POLYMORPHISM OF THYROXINEBINDING GLOBULIN: STUDY IN HUMAN SERUM BY RADIOPRINT IMMUNOFIXATION Y. Lasne, F. Lasne, 0. Benzerara and P. Arnaud

727

TWO-DIMENSIONAL IMMUNELECTROPHORESIS OF PLASMAANTITHROMBIN III IN THE PRESENCE OF HEPARIN-AGAROSE H. Bleyl and H. Peichl

733

DETERMINATION OF STREPTOKINASE ANTIBODY TITRE IN PLASMA OR SERUM BY AN IMMUNOELECTROPHORETIC METHOD B. Skoog, A. Soderlindh and B. Granstrand

743

XIV TWO-DIMENSIONAL IMMUNOELECTROPHORESIS APPLIED TO THE SEMI-QUANTITATIVE DETERMINATION OF BENCE JONES PROTEIN IN PATHOLOGICAL URINES M. Campens, A. Francina, H. Cloppet, C. Chapuis Cellier and P. Arnaud

753

APPLICATION OF PHA IN ELECTROPHORETIC DIFFERENTIATION OF HUMORAL IMMUNOGLOBULINS G. A. Spengler and R.-M. Weber

761

FEASIBILITY OF STUDYING THE FUNCTIONAL NATURE OF HUMAN IgM SUBCLASS RESPONSES BY MEANS OF ANALYTICAL ISOTACHOPHORESIS K. W. Hedlund, R. Wistar, Jr. and D. Nichelson

765

IN VITRO INVESTIGATIONS OF THE GLUCURONIC ACID PATHWAY WITH THE AID OF ANALYTICAL CAPILLARY ISOTACHOPHORESIS C. J. Holloway

775

QUANTITATIVE RESOLUTION OF 1- AND 2-NAPHTHYLGLUCURONIDES FROM SINGLE MIXED ZONES IN ISOTACHOPHEROGRAMS C. J. Holloway, S. Husmann-Holloway, G. Brunner, I. Trautschold and A. Baldesten

781

A RAPID METHOD FOR QUANTITATIVE DETERMINATION OF URINARY OXALATE BY ANALYTICAL ISOTACHOPHORESIS K. Schmidt and G. Bruchelt

791

STUDIES ON SERUM CHOLINESTERASE ISOZYMES AND GENETIC VARIANTS IN HUMAN TISSUES USING POLYACRYLAMIDE GEL ELECTROPHORESIS D. P. Agarwal, T. Münch, V. Stein and H. W. Goedde

797

MATERNAL PLASMA PROTEIN VARIATIONS DURING NORMAL AND PATHOLOGIC PREGNANCY: A COMPARATIVE STUDY BY MEANS OF SDS-PAA ELECTROPHORESIS AND OSMOMETRIC EVALUATION OF NUMBER AVERAGE MOLECULAR WEIGHT R. Schroeck and E. Feicht

805

SERUM PROTEIN ANALYSIS BY MOLECULAR INTERPRETATION OF CELLULOSE ACETATE ELECTROPHORESIS: EVALUATION OF ITS CLINICAL USEFULNESS F. Aguzzi, C. Petrini, G. Merlini, S. Perolini, F. Pozzi and S. D. Jayakar

811

XV GENETICAL AND BIOCHEMICAL" CHARACTERIZATION OF A DOMINANT MUTATION OF MOUSE LACTATE DEHYDROGENASE W. Pretsch and D. Charles

817

VERTEBRATE MITOCHONDRIAL 55S RIBOSOMES: COMPARISON OF PROTEIN PATTERNS WITH BACTERIAL ANC CYTOPLASMIC R-PROTEINS BY 2-D PAGE W. Czempiel and B. Ulbrich

825

ISOELECTRIC PEROXIDASE PATTERNS AS INDICATORS FOR GROWTH AND FOR THE EFFECT OF GROWTH REGULATORS IN TOBACO TISSUE CULTURES W. Rvicker and J. Markotai

833

STUDY OF HEMOGLOBIN AND A SOLUBLE HEART PEROXIDASE IN LACERTA GALLOTI AND LACERTA STHELINI BY ISOELECTRIC FOCUSING Z. Gonzalez-Lama, R. H. Lopez Orge and A. M. Lamas

841

AUTHOR INDEX

84 7

SUBJECT INDEX

84 9

Section I Theory and Methods

GEL ELECTROFOCUSING WITH INCREASED DEGREES OF FREEDOM

A. Chrambach, L. Hjelmeland and N. Y. Nguyen National Institute of Child Health and Human Development, Bethesda, MD 20205 USA B. An der Lan Bureau of Biologies, Food and Drug Administration, Bethesda, MD 20205 USA

Introduction Electrofocusing started out as, and is widely applied as, a cookbook method of separation, which is controlled by the pi-ranges of the commercially available carrier ampholytes:

Take one spoonful of such a prepara-

tion in the pi-range closest to your requirements, plus one spoonful of a "standard" polymerization mixture containing acrylamide, a pinch of Bis, a sprinkling of persulfate and a dash of TEMED, and bake for 30 min.

Place

it between strong acid and base, apply the protein, then an electric potential of 479.2 V, wait again for 2.3 h, stain-destain, or slice and measure pHs; count bands; publish the band pattern to demonstrate again history's most highly resolving method; publish the pi.

End of recipe.

In this review of electrofocusing, we will take the opposite viewpoint. To us it appears axiomatic that, if one is aiming at optimal resolution, each separation problem requires its particular adaptation of a more general method.

This review will deal, therefore, with ways to make elec-

trofocusing more flexible, and to adapt it to particular separation problems.

We will discuss flexible choice of apparatus, the various modes of

"pH gradient engineering" and of stabilizing pH gradients, the choice of gel media, the detergents applicable to electrofocusing, the ways of circumventing conductance gaps, and of preparing proteins from each of the zones in analytical gel electrofocusing.

With the ability to vary all

these elements, electrofocusing becomes extremely adaptable to the specific

© 1980 Walter de Gruyter & Co., Berlin • New York Electrophoresis '79

k s e p a r a t i o n , a n d its resolving capacity is a u g m e n t e d .

1) A p p a r a t u s

If e l e c t r o f o c u s i n g is to be used in a flexible m a n n e r , this m u s t be r e flected i n the c h o i c e of a p p a r a t u s .

In o t h e r w o r d s , d i f f e r e n t types of

a p p a r a t u s are suited to d i f f e r e n t s e p a r a t i o n p r o b l e m s .

If the object of

e x p e r i m e n t a t i o n is, for e x a m p l e , the m e a s u r e m e n t of p i , then the gel tube a p p a r a t u s c o n t i n u e s to e x c e l , b e c a u s e of the ease w i t h w h i c h p r o t e i n p o s i tions a n d r e s o l u t i o n , as w e l l as p H g r a d i e n t s , c a n be m o n i t o r e d a t v a r i able e l e c t r o f o c u s i n g

times, a n d because of the load v o l u m e c a p a c i t y , a n d

of the e c o n o m y of c a r r i e r c o n s t i t u e n t s .

W h e n using c y l i n d r i c a l g e l s , p H

g r a d i e n t s c a n be m o n i t o r e d s e m i - a u t o m a t i c a l l y w i t h the c o m m e r c i a l l y able p H g r a d i e n t m e a s u r i n g d e v i c e .

A n o t h e r advantage of tube

avail-

apparatus,

w h i c h c o u l d also be imparted to horizontal slab a p p a r a t i , a r e

electrolyte

c h a m b e r s of s u f f i c i e n t c a p a c i t y to prevent a p p r e c i a b l e c h a n g e s of a n o l y t e and c a t h o l y t e c o m p o s i t i o n during the course of e l e c t r o f o c u s i n g for further d i s c u s s i o n see b e l o w ] .

[(1) and

A n important r e c e n t a d d i t i o n to the

a r m o r y o f g e l tube e l e c t r o f o c u s i n g e q u i p m e n t is a p l a t i n u m w i r e a t t a c h m e n t a l l o w i n g v o l t a g e s a c r o s s the gel (excluding the r e s e r v o i r s ) to be m o n i t o r e d c o n t i n u o u s l y during e l e c t r o f o c u s i n g

( F i g . 1).

This is i m p o r -

tant w h e n r e s i s t i v e u n c h a r g e d a n o l y t e s and c a t h o l y t e s are u s e d below).

(see

A n o t h e r a d v a n c e is a m o d i f i c a t i o n of a commercial e l e c t r i c

slicing d e v i c e (Fig. 2) c a p a b l e of p r o v i d i n g u n i f o r m slices of r e s t r i c t i v e " 6 %T, 15 ^Cjj^TD gels (see b e l o w ) .

gel

"non-

If, o n the o t h e r h a n d ,

e l e c t r o f o c u s i n g k i n e t i c s and the rate of a t t a i n m e n t of the

steady-state

are a l r e a d y k n o w n , a n d c o n s e q u e n t l y e l e c t r o f o c u s i n g time can be k e p t constant, h o r i z o n t a l slab a p p a r a t u s offers the a d v a n t a g e that, a t any o n e time of e l e c t r o f o c u s i n g , d i r e c t c o m p a r i s o n s of m u l t i p l e b a n d p a t t e r n s w i t h i n the same gel a r e possible

(2).

F u r t h e r m o r e , t h e r e is g r e a t e r

f r e e d o m i n the choice of m a t r i x m a t e r i a l , b e c a u s e the h o r i z o n t a l e l i m i n a t e s p r o b l e m s of m e c h a n i c a l s u p p o r t and w a l l a d h e r e n c e

support

(see b e l o w ) .

A m o r e d e t a i l e d d i s c u s s i o n of the r e l a t i v e m e r i t s and d i s a d v a n t a g e s tube and s l a b a p p a r a t u s c a n be found e l s e w h e r e

(1).

of

5

Fig. 1. Device for voltage measurement across the gel in gel electrofocusing w i t h weakly acidic/basic or amphoteric anolyte and catholyte at a pH at w h i c h their net charge approximates zero.

GEL SUPPORT PLATES (FRONT AND BACK OF GEL)

Fig. 2. Modification of an electric gel slicer (Hoefer Instruments, San Francisco) to facilitate cutting of "non-restrictive" 6 % T , 15 % C D A D T gels in electrofocusing. The cutting wires pass through the gel into grooves.

6 2) pH-Gradient Engineering The proposition that the flatter the pH gradient around the steady-state pi-positions of the proteins, the better the resolution, appears selfevident.

This concept appears to hold for separation, but is not sup-

ported by the available evidence for resolution.

However, present data

are still limited to 2 protein systems (3).

Here it is important to dis-

tinguish between resolution and separation.

Separations of either iso-

hormones of human growth hormone or BSA and ovalbumin have been compared on either flat or steep pH gradients (Fig. 3).

Although these data are

too sparse to make generalizations, they allow us to define some of the conditions under which "the flatter, the better" holds.

Fig. 3 shows

that, although resolution (defined as R = peak separation/average standard deviation of each peak) is not enhanced on a flat pH gradient, separation is greatly improved, and preparative separation by a practical slicing method becomes possible.

The price of improved separation, how-

ever, is that the average mobility of proteins is decreased.

Hence, to

reach their steady-state, longer electrofocusing times are required than are necessary using steeper gradients.

This requirement is illustrated

by Fig.6(3) where the pH values associated with the peaks of BSA and ovalbumin after 24 h of electrofocusing do not correspond to their pi' values, whereas after 48 h they do.

The separation is better at 48 h,

presumably because the steady-state has been reached.

In view of the

increased electrofocusing time, stabilization is a sine qua non for flat pH gradients.

Fortunately, flatter pH gradients tend to be more stable

than steeper ones (see below).

A second and related problem concerns

the rate of migration of the protein into the gel.

Human growth hormone,

for example, does not enter into flat pH-gradients unless it is first charged by dissolving in 0.1 M arginine (pH 10.5).

Thus, it appears

that the protein is titrated to a lower pH by the pH gradient sufficiently slowly to be able to maintain adequate mobility in penetrating the gel at a practical rate. How does one go about making a flat pH gradient?

There are three ways

of tailoring pH gradients to the pH-range which circumscribes, as narrowly as possible, the pi's of the proteins that one wants to separate:

7

BSA — Ovalbumin

hGH

R-5.1

STEEP

1.0

o

0.5

1.0

RELATIVE GEL LENGTH

Fig. 3. Separation and resolution between isohormones of human growth hormone (hGH) and between BSA and ovalbumin: A comparison between flat and steep pH gradients. Gel concentration: 5 %T, 15 ^ c d a t D ' F l a t P H gradients: Ampholine (pi-range 4.5-5.0), threonine (pH 5.6) catholyte, diiodotyrosine (pH 4.4) anolyte. Electrofocusing time 48, 50 h respectively. Steep pH gradients: Ampholine (pi-range 3.5-10), 0.2 N KOH catholyte, 0.2 N H2SO4 anolyte. Electrofocusing time: 10 h. Resolution, R, is defined as the distance between peaks/average standard deviation of the two peaks. Data of ref. (3).

8 A) Choice of carrier constituents 2 .

B) Constituent

displacement.

C) Selection of anolyte pH. A ) Mixtures of simple buffer have been used as carrier constituents to make natural pH gradients in a particular pH-range; this is known as "buffer electrofocusing" of proteins (4).

As one would expect, the addi-

tion of acidic carrier constituents to the mixture causes a shift of the pH gradient in the acidic direction, and of basic constituents a shift in the basic direction (Fig. 4 ) (3).

MES 3.6

ACES 3.6

TES 4.3

TAUR 5.12

TRIC ~5.2

Also, a single constituent, added

BIC ~5.4

GLY 6.20

GABA ASPNH 2 7.29 5.4 CAC BISTRIS HFAH pk6.21 pk6.50 pk6.80 HOAC pk 4.72

PROP pk 4.83

PH

RELATIVE GEL LENGTH Fig. 4. Selection of specific carrier constituents in buffer electrofocusing. A common carrier constituent mixture of 8 amphoteric buffers was modified by adding basic ( • • ), neutral (Jk A ) and acidic ( • • ) constituents. Gel concentration and electrolytes as in Fig. 3. Data of ref. (3).

Since natural pH gradients can be formed by mixtures of non-amphoteric buffers alone (4), we prefer the term "carrier constituent" to "carrier ampholyte."

9

6 66

50 122

0.01 M G A B A (pH 2 5 . c *6.40)

0 2N

KOH

0.01M M E S

0.2N

H2S04

(pH 2 5 . c =4,50)

RELATIVE GEL LENGTH Fig. 5. pH G r a d i e n t d e s i g n by c o n s t i t u e n t d i s p l a c e m e n t . Buffer e l e c t r o focusing (pi-range 3 . 6 - 7 . 3 ) . G e l c o n c e n t r a t i o n 5 % T , 15 Left panel: S t r o n g l y v s . w e a k l y a c i d i c a n d b a s i c a n o l y t e a n d c a t h o l y t e . Right panel: Fine a d j u s t m e n t of pH g r a d i e n t s , u s i n g terminal carrier c o n s t i t u e n t s as the a n o l y t e a n d c a t h o l y t e . D a t a of r e f . (23).

i n large a m o u n t s c o m p a r e d to the c o n c e n t r a t i o n s of the other

constituents,

m u s t f l a t t e n the pH g r a d i e n t in the v i c i n i t y of its s t e a d y - s t a t e (5).

position

T h u s , the a d d i t i o n of " s e p a r a t o r s " to m u l t i c o m p o n e n t s y n t h e t i c m i x -

tures of carrier a m p h o l y t e s a l s o falls into this c a t e g o r y of pH g r a d i e n t design

(5).

B) T h e p H - r a n g e of a g r a d i e n t f o r m e d w i t h either s y n t h e t i c

multicomponent

m i x t u r e s of carrier c o n s t i t u e n t s of the A m p h o l i n e type, or w i t h b u f f e r s (4), can be n a r r o w e d by suitable c h o i c e of a n o l y t e and c a t h o l y t e

(Fig. 5).

For this p u r p o s e , the pH of a n o l y t e a n d c a t h o l y t e are c h o s e n so that they a r e w i t h i n the p H - r a n g e of the g r a d i e n t , and so that they c o r r e s p o n d the d e s i r e d t e r m i n a l pH v a l u e s of the g r a d i e n t (6-8).

Since anolytes

to and

c a t h o l y t e s at s u i t a b l e pH v a l u e s c a u s e d i s p l a c e m e n t from the gel of all carrier c o n s t i t u e n t s w h o s e s t e a d y - s t a t e pH is less than the pH of the a n o l y t e , or h i g h e r t h a n the pH of the c a t h o l y t e , this m e t h o d of the pH g r a d i e n t h a s b e e n termed " c o n s t i t u e n t d i s p l a c e m e n t . "

The

delimiting success

of " c o n s t i t u e n t d i s p l a c e m e n t " d e p e n d s o n c o n s t a n c y of e l e c t r o l y t e pH w i t h

10 time and thus requires relatively large (500-2,000 ml) electrolyte reservoirs [Fig. 13 of (9)].

In this regard soaked filter paper strips do not

suffice as electrolyte reservoirs (10).

For the same reason, i.e., main-

taining a constant pH in the electrolyte reservoirs, it is advantageous to use amphoteric anolytes and catholytes (e.g., aminoacids) at their pi; w h e n using fully deprotonated bases or fully protonated acids, catholyte and anolyte may have to be renewed once electrolysis causes

significant

pH changes.

C) It is possible to shift the pH gradient along the pH axis by varying the pH of the anolyte in a range outside the steady-state pH-range of the gradient (Fig. 6).

Thus, the more acid the anolyte, the more acidic the

pH gradient; the more closely the anolyte pH approximates the steady-state anodic terminal pH of the gradient, the more the pH gradient shifts in the cathodic direction (11).

The stability of the gradient is also enhanced

as the pH of the anolyte approaches that of the anodic terminus of the gradient [(11) and see below].

Anolyte control over the pH gradient

primarily depends on the pH, rather than on the nature of the anion; progressive acidification of pH gradients with increasing concentration of phosphoric acid is evidence for this (12).

However, the ability of the

anolyte to control the pH gradient appears also to depend on its buffering capacity.

Thus, weak acid anolytes (e.g., carboxylic acids) and strongly

acidic anolytes w i t h buffering capacity (e.g., phosphoric acid) exhibit this effect, whereas it is impossible to demonstrate a systematic shift of pH gradients as a function of pH with various concentrations of sulfuric acid, probably because the gradients are relatively unstable.

In

all experiments to date the catholyte failed to influence the pH gradient. It is important to note, however, that the catholyte was a relatively weak base, i.e., either histidine, lysine or arginine, or amines w i t h pKs ranging from 6.8 to 8.1, w h e n carboxylic acids [pKs (25°C) 3.7 to 6.2] or phosphoric acid were used as anolyte.

It is still possible that the oppo-

site effect may occur, w h e n a strongly basic catholyte is combined with a weakly acidic anolyte, i.e., the catholyte may be used to induce an anodic pH gradient shift, whose extent depends on the alkalinity of the catholyte.

11 This expectation is supported by the fact that, under just these conditions (11), the pH gradient drifted toward the anode, rather than the cathode.

3) pH Gradient

Stability

Although m o s t users of gel electrofocusing will concede that pH gradient stability is a function of electrofocusing time and voltage, they would deny that this is of any consequence within the relatively short times allegedly required for protein separations.

We take the opposite position

Fig. 6 . Systematic shift of pH gradients as a function of the pH of the anolyte. Gel concentration: 6 %T, 15 Ampholine pi-range 4-8. Catholyte: 0.1 M lysine. Left panel: Weakly acidic anolytes of varying pK. Right panel: H3PO4 at varying concentrations (11, 12).

12 on the following grounds:

a) The plot of pH

electrofocusing time

shows [e.g., (14-16)] that proteins in so-called "non-restrictive" gels attain a constant pH, i.e., an apparent pi (pi'), after a time that is roughly one order of magnitude greater than that required for the formation of the pH gradient (Fig. 7).

In the representative cases shown the

pi* was attained after 10 - 20 h of focusing at about 20 V/cm of gel and at 0-4°C, while the gradients formed after 2 - 3 h (17).

b) When a pro-

tein occurs in multiple aggregation states (as most proteins probably do), it may take 80 h of electrofocusing

[under the conditions described in

Fig. 1 of ref. (18)] for protein patterns to condense and become uniform, c) The rate of migration, at a given voltage, is specific for each protein system and depends on the charge on the protein and on the degree to which it is retarded by the support medium.

Those proteins with flat titration

curves must, in addition, approach their isoelectric positions more slowly than those with relatively steep titration curves, as "poor and good carrier ampholytes" do respectively (19).

All proteins approach true pl-

positions on the pH gradient asymptotically,

d) A further impediment to

migration of proteins is the progressive appearance of conductance gaps (see below) which arise in the neutral pH-range at approximately onethird of the rate, at which the pH gradient forms (20).

Thus, it appears

necessary in each particular application of gel electrofocusing to stabilize the pH gradient for a time period sufficient for the attainment of the isoelectric endpoint (i.e., the position on the pH gradient where pH = pi') under the chosen conditions of gel concentration, voltage and temperature.

Such stabilization can be brought about, or at least en-

hanced, by the following measures:

a ) Flattening pH-range gradients

(3, 21, 22) as demonstrated by comparing the degree of stability with that of wide pH gradients (3).

b) Replacing the conventional strongly acidic

anolyte and strongly basic catholyte by those constituting the steadystate anodic and cathodic terminals of the gradient (3, 21, 22). c) Approximating the anolyte pH to the steady-state anodic terminal pH of the pH gradient (11). catholyte (23).

d) Increasing the concentration of anolyte and

e) Increasing the carrier constituent concentration,

although the time required to form the gradient is increased proportionately [Fig. 1 of (24)].

f) Increasing the viscosity (sucrose, urea) of

the gel (2, 25), although apparently this is not effective in all cases

13

6.8

\

6.7

-

6.6

o o

6.5

-

126

I-hPRL (pituitary)

^^^

Sucrose Density Gradient

-

6.4

R O G O L et al. ~

20

t

i

I

40

60

80

8 7 6 5 4 3 pH

2

Tetanus toxins

6

6%T

-

15%Cdatd

5 A N DER L A N et al.

10

20

30

40

50

ELECTROFOCUSING TIME (hours)

Fig. 7. Asymptotic approach of proteins towards their pi-positions in electrofocusing. Top panel: Radioiodinated human prolactin in sucrose density gradient focusing. Ampholine pi-range 5-8 (14). Center panel: The 6 major isohormones of human chorionic gonadotropin in electrofocusing on polyacrylamide gel. Ampholine pi-range 2-6 (16). Bottom panel: Radioiodinated tetanus toxin, Ampholine pi-range 3.5-10 (15).

14 (17, 23).

g) Improving the wall adherence of the gel (13, 26).

4) Choice of Gel Media

Since the protein approaches its pi-position slowly and since optimal resolution depends on attainment of the steady-state [see above and (3, 4)], it is imperative to provide an effectively "non-restrictive" pore size in electrofocusing.

This is not easily defined.

adopted working definition was to designate a pore size

A previously "non-restrictive"

w h e n it enabled maximally charged proteins (at either low or high pH) to migrate within the same Stacking Limits as a tracking dye (27).

In elec-

trofocusing, where proteins are relatively less charged, this criterion proves to be inadequate, i.e., mobilities on so-called

"non-restrictive"

gels appear low compared w i t h mobilities on such media as granulated gels. A) Polyacrylamide as gel matrix has the unique property of enabling one to vary pore size continuously over a very wide range.

However, a wide

variety of pore sizes is irrelevant for electrofocusing which demands "non-restrictiveness," rather than a tight fitting molecular sieve (in contradistinction to polyacrylamide gel electrophoresis).

The popular

low (2 to 5) "i Bis-crosslinked polyacrylamide gels, even at low concentration (3.5 to 5 %T), provide more restrictive gels than the highly (15-50) % crosslinked gels (1, 27, 28).

Of the latter types, the ones

crosslinked w i t h DATD provide the best mechanical stability and wall adherence, especially w h e n gel tubes are pre-coated w i t h linear polyacrylamide (13, 27).

[The good adherence properties ("stickiness") of

allyl-crosslinked gels may be due to a low average chain length compared to methylene-crosslinked gels.

Allyl groups react at a lower rate than

vinyl groups, so that allyl-pools accumulate during polymerization, which gives rise to an uneven distribution of crosslinks along the length of the polymer and to chain termination (29, 30).]

As the degree of cross-

linking w i t h Bis increases above 10%, gels become progressively less "restrictive" and could be used for electrofocusing, if they could be firmly attached to the surrounding glass walls.

This has been achieved

by covalently bonding gel to glass w i t h the vinyl siliconating agent,

15 A-174 (Pharmacia).

However, in the single case (5 %T, 30

sub-

jected to electrofocusing, the wall adherence was lost after 6 h under conditions, where a 5 %T, 15 ^Cp^TD gel proved perfectly stable (26). B) Agarose has the advantage over polyacrylamide in providing enormous pore sizes (compounds of molecular weight 150 x 10^ and above are excluded from a 1% gel), in not swelling differentially as the pH varies along the gradient, in ease of gelation and possibly of protein recovery. Agarose gels excel in the ease with which they can be sliced.

Since they

can be liquefied by heating, measurement of radioactivity does not present the problems of gel solubilization or elution from gel slices encountered with polyacrylamide gels.

Agarose has the disadvantage, compared with

polyacrylamide, of a residual bound sulfate concentration sufficient to give rise to electroendosmosis (31), and of not adhering to glass. ever, both of these problems are manageable.

How-

The sulfate concentration

in recent commercial preparations has been reduced to a level which decreases electroendosmosis to such a low value (R^ = -0.005 in the buffer system used for comparison), that it may be negligible in the practice of gel electrofocusing (32).

Presumably, the manufacture of special

carrier ampholyte preparations for electrofocusing on agarose relates to this electroendosmosis problem.

The wall adhesion problem can be ne-

glected if agarose is either used on horizontal slab apparatus, particularly with commercially available coated plates, to which agarose adheres, or in vertical tubes or slabs supported by a strong polyacrylamide plug (L. Hjelmeland, unpublished data). C) Granulated gels:

Granulated dextran (Sephadex, Ultrodex, etc.), gran-

ulated polyacrylamide-agarose copolymer (Ultrogel, Indubiose, etc.), granulated agarose gels (Sepharose, etc.), and polyvinylchloride and acetate (Pevikon) mixed with dextran (33) can be used for electrofocusing on horizontal gel beds.

Compared to non-granulated polyacrylamide gels,

these materials excel in the ease of protein recovery and non-restrictiveness, providing suitable pore sizes are selected to exclude the protein. However, as anticonvective media, their efficiency is not as high as continuous gel media, thus giving rise to broader zones.

Conventionally,

granulated gels are used in a horizontal slab apparatus.

Here the packing

16 of horizontal beds evenly and reproduclbly, and maintaining a uniform field across all channels of the conventional, relatively large beds may present problems.

Cylindrical columns of Sephadex, supported by a poly-

acrylamide plug, can be used for electrofocusing of one or a few samples in a conventional gel tube apparatus (34).

5) Electrofocusing in Detergents

Recently, electrofocusing has been applied to hydrophobic proteins.

Pre-

viously, it had been limited to those rare cases where non-ionic detergents could disaggregate and solubilize the protein.

Even in those rare cases,

the very large micellar sizes of non-ionic detergents gave rise to complexes containing several different protein molecules, and thus to uninterpretable pi values (35).

Non-ionic detergents had the further disadvantage

that they appeared to associate with the relatively non-polar basic carrier ampholytes (36), thus interfering with the formation of natural pH gradients in the alkaline range.

Ionic detergents, on the other hand, suffer

from the disadvantage that they increase the current and Joule heat. Furthermore, anionic detergents prevent formation of basic pH gradients presumably by binding to the carrier ampholyte species of the opposite net charge, and shifting their positions or displacing them (37).

Amphoteric

detergents of the alkyl-sulfobetaine type (Calbiochem. Cat. No. 693015693023) are suitable for electrofocusing, since their large pI-pKp values should allow them to condense across the entire pH gradient (37).

How-

ever, in the absence of assays for these detergents, there is at present no direct evidence for their immobilization across the pH gradient.

The

spreading of the methyl sulfobetaine, TMAPS (see below), across the gradient can be viewed as indirect evidence that long chain alkyl sulfobetaine detergents will also behave in such a manner.

Similarly, the assumption

that the presence of amphoteric detergents does not affect the pH gradient has only been verified for TMAPS.

Although it is unlikely that the polar

headgroups of such detergents, which resemble each other chemically, will affect the gradient, non-polar tails may do so, e.g., by binding to basic carrier ampholytes (36).

The claim that amphoteric detergents would not

denature proved to be unjustified, since some protein activities were

17

Solubility

Zwitterionic Detergent 0 / W W W W V \ N Ì V \ S - O

Sqluble ^\N®AA|-Oe

HO

Insoluble

\

0

-N®/\A S

/

e

- o

II ° Soluble

10.000

Native

yV\N®/\A|-Oe

HO

HO"'" ^ ^ ^ —

—

'OH

-N^V\|-O

e

Fig. 8. Structural characteristics of a non-denaturing amphoteric detergent suitable for electrofocusing. The flexible alkyl tail group of sulfobetaine-14 (SB14) renders this detergent denaturing to the hydrophobic membrane protein, cytochrome P-450 (37). In contrast, a rigid cholicacid-like tail group makes the detergent non-denaturing. However, the relative insolubility of the molecule brought about by the rigid tail group needs to be compensated for by a n increase in the polarity of the amphoteric polar head group of the detergent. (L. Hjelmeland, in preparation) .

found to be destroyed (37).

This defect has now been eliminated by re-

placing the flexible hydrocarbon chain of the amphoteric detergents w i t h rigid cholic acid-type tails and by enhancing the polarity with multiple sulfobetaine polar head groups (Fig. 8).

Such detergents are non-

denaturing and suitable for gel electrofocusing (L. Hjelmeland, in preparation).

Amphoteric detergents have the further advantage that micellar

sizes are small compared with those of ionic detergents (40), thus ensuring that only a single copy of a protein, and particularly not several different proteins, can associate w i t h (dissolve in) a single detergent

18 micelle

(Fig. 8 ) .

E l e c t r o f o c u s i n g in d e t e r g e n t s , h o w e v e r , s t i l l

presents

p r o b l e m s of p r o t e i n f i x a t i o n a n d s t a i n i n g , b e c a u s e d e t e r g e n t s b i n d to d y e s , and because they counteract

the p r o t e i n f i x a t i o n r e q u i r e d for s t a i n i n g .

is p o s s i b l e t h a t t h i s d i l e m m a c a n be r e s o l v e d by c r o s s l i n k i n g prior

to s t a i n i n g , t h e r e b y p r o t e c t i n g

gent, and by reducing

protein

it f r o m s o l u b i l i z a t i o n b y the

the d e t e r g e n t c o n c e n t r a t i o n to b e l o w the

micelle concentration, allowing to

the

It

deter-

critical

for its r a p i d d i f f u s i o n f r o m the g e l

prior

staining.

6) C o n d u c t a n c e

Gaps

A l l n a t u r a l pH g r a d i e n t s a n a l y z e d to d a t e , w h e t h e r g e n e r a t e d w i t h

simple

b u f f e r m i x t u r e s or w i t h s y n t h e t i c c a r r i e r a m p h o l y t e s , h a v e d e v e l o p e d d u c t a n c e m i n i m a a f t e r some time of e l e c t r o f o c u s i n g

(20, 3 8 ) .

con-

C l a i m s of

"even conductance" across pH gradients all rely o n analysis at a

single

electrofocusing

voltage

time p r i o r , it is s u s p e c t e d ,

m a x i m a i n the n e u t r a l pH r e g i o n

[e.g. ( 3 9 ) ] .

t i o n of c o n d u c t a n c e g a p s a p p e a r s e x t r a n e o u s

to the f o r m a t i o n of Mechanistically,

the

forma-

to the f o r m a t i o n of n a t u r a l

g r a d i e n t s , s i n c e it h a s b e e n o b s e r v e d i n g e l s c o n t a i n i n g one a m i n o

acid

o n l y , a n d e v e n w h e n no c a r r i e r c o n s t i t u e n t s w e r e p r e s e n t at a l l (20). conductance gaps

(voltage maxima) develop after

pH

the pH g r a d i e n t h a s

The

formed

a n d , i n m a n y c a s e s , b e f o r e the p r o t e i n c a n r e a c h its i s o e l e c t r i c pH o n the pH g r a d i e n t .

D e p e n d i n g o n the p o s i t i o n of the p r o t e i n r e l a t i v e

to

these

v o l t a g e m a x i m a (20) r e s o l u t i o n m a y e i t h e r be d e c r e a s e d b y i m p e d i n g m i g r a t i o n of the p r o t e i n to i t s p i - p o s i t i o n o n the g r a d i e n t or be (40).

T h i s p r o b l e m c a n be o v e r c o m e e x p e r i m e n t a l l y

by c o n d u c t i n g

the enhanced

electro-

f o c u s i n g e i t h e r i n h i g h (0.1 M ) s a l t (19), or i n the p r e s e n c e of a n t e r i c " u n c h a r g e d s a l t " of the t r i m e t h y l a m i n o - p r o p i o n y l - s u l f o n a t e t y p e (40).

S i n c e 1 M T M A P S o n l y c o n d u c t s a s w e l l a s 0 . 0 1 M K C 1 , it is

not surprising

that its a b i l i t y to b r i d g e c o n d u c t a n c e g a p s is n o t

c i e n t to e n s u r e t h a t the g r a d i e n t is p e r m a n e n t l y f r e e of gaps (40).

B u t m o r e h i g h l y c o n d u c t i v e a n a l o g s of T M A P S w i t h

electrofocusing.

suffi-

conductance

p o l a r i t y h a v e b e e n s y n t h e s i z e d a n d are e x p e c t e d to p e r f o r m a s KC1" in

ampho-

(TMAPS)

increased "amphoteric

19 7) Preparative Gel Electrofocusing

The recovery of protein from non-granulated electrofocusing gel slices by diffusion is slow, and yields appear inversely related to the degree to which the isoelectric endpoint (and thus full resolution) has been attained (41).

Presumably, this is due to the isoelectric precipitation of the pro-

tein in an efficiently anticonvective medium.

(It is still open to ques-

tion whether the granulated gel technique is similarly affected.) tion to this problem has recently been developed.

The procedure

A solufirst

deals w i t h the problem of the isoelectric precipitation of the protein by applying either an alkaline Steady-State Stacking gel, or a quick exposure to alkali at 0°C, in order to charge the isoelectric protein. tions may, of course, denature active proteins.

These condi-

Further work is needed to

determine whether detergent solubilization of the isoelectric protein w i t h maintenance of activities is possible or preferable to exposure to an extreme of pH.

After solubilization, the protein on electrofocusing gel

slices can be extracted electrophoretically and concentrated by SteadyState Stacking (isotachophoresis) at any pH (18).

A n apparatus has been

constructed which allows one to extract 10 slices, or pools of slices in any amount, simultaneously and in h i g h yield (42).

Since Steady-State

Stacking is carried out on polyacrylamide, the concentrated protein fractions are contaminated by non-proteinaceous impurities derived from that medium.

These are removed chromatographically prior to lyophilization.

The overall yield of human growth hormone, the protein applied in this study, was 70%.

The concentration step alone, tested on 10 and 100 mg

loads of BSA per gel of 2.54 cm

surface area, yielded 80% (42).

For

electrofocusing separations on polyacrylamide gel, a protein load of 8 mg/zone/cm

appears tolerable; thus for m o s t preparative purposes in

biochemistry, the m e t h o d applied to a single cylindrical

electrofocusing

gel of wide diameter (18 m m or less) yields the required milligram amounts of protein.

Further application of this preparative gel electrofocusing

method to multiple zones simultaneously will require development of a wide-diameter slab w i t h e v e n field strength across its length, and a guidestrip and horizontal gel slicing device designed to translate m i g r a tion distances on a fixed and stained guidestrip to the original zone m i gration distances.

Alternatively, protein detection on the entire prepara-

20 tive slab may be possible without need for a guidestrip, using as a detection tool the natural fluorescence of proteins at very low temperatures (43). Among the preparative gel electrofocusing techniques applicable to granular gels, the continuous method of Fawcett (44) remains promising as a tool for preparing gram amounts.

Progress has recently been made on the

auxiliary techniques of carrier ampholyte recycling, continuous optical and conductance scanning by Bier, et al. (45).

However, to make this

approach widely applicable, ways must be found to maintain simplicity of operation and cost-effectiveness.

Conclusions

The possibility of engineering pH gradients to fit one's needs, the dynamics of pH gradients, the multiplicity of gel media and apparatus types, the application of amphoteric detergents, even conductance gaps can be turned to use in electrofocusing to improve protein separations. However, all of these devices remain empirical.

A real control over

electrofocusing must await a full physical-chemical understanding of pH gradient formation and decay, a prediction and computer simulation of its dynamics and its changes under the influence of the parameters w i t h which this review has dealt.

The recent analysis by Mürel, et^ _al. (46) may well

signal the beginning of the necessary theoretical advance.

References

J. Mol. Cell. Biol., in press

(1979). 1

1.

Chrambach, A.:

2.

Righetti, P.G., Gianazza, E . , Bosisio, A.B.: New Developments on Chromatography and Electrophoresis (Frigerio, A., ed.), Vol. 2, Elsevier North Holland, New York-Amsterdam, in press (1979).

3.

Nguyen, N.Y., Chrambach, A.: mitted (1979). 1

4.

Chrambach, A . , Nguyen, N.Y.: Electrophoresis '78 (Catsimpoolas, N, , ed.), Elsevier North Holland, New York-Amsterdam, pp. 3-18 (1978).

"''Available upon request.

J. Biochem. Biophys. Methods, sub-

21 5.

Caspers, M.L., Posey, Y., Brown, R.K.: (1977).

Anal. Biochem. 79_, 166-180

6.

McCormick, A., Miles, L.E.M., Chrambach, A.: 314-324 (1976).

7.

McCormick, A.G., Wachslicht, H., Chrambach, A.: 209-218 (1978).

8.

Chrambach, A., Nguyen, N.Y.: Proc. International Workshop on Technology for Protein Separation and Improvement of Blood Plasma Fractionation (Sandberg, H., ed.), Reston, VA, pp. 484-507 (1977). 1

9.

Chrambach, A., Jovin, T.M., Svendsen, P.J., Rodbard, D.: Methods of Protein Separation (Catsimpoolas, N., ed.), Vol. 2, Plenum Press, New York, NY, pp. 27-144 (1976). 1

Anal. Biochem. 75, Anal. Biochem. 85,

10.

Arosio, P., Gianazza, E., Righetti, P.G.: 55-64 (1978).

J. Chromatography 166,

11.

An der Lan, B.. Chrambach, A.: mitted (1979). 1

J. Biochem. Biophys. Methods, sub-

12.

An der Lan, B., Chrambach, A.:

This Symposium, Abstract No. 3 (1979).

13.

Nguyen, N.Y., McCormick, A.G., Chrambach, A.: 186-195 (1978).

14.

Rogol, A.D., Ben-David, M., Sheats, R., Rodbard, D., Chrambach, A.: Endocrine Res. Comm. 2_, 379-402 (1975).

15.

An der Lan, B., Habig, W.H., Hardegree, M.C., Chrambach, A.: Biochem. Biophys., submitted (1979).

16.

Nwokoro. N., Chen, C.-H., Chrambach, A.: (1979). 1

17.

Baumann, G., Chrambach, A.: Progress in Isoelectric Focusing and Isotachophoresis (Righetti, P.G., ed.), Elsevier North Holland, New York-Amsterdam, pp. 13-23 (1975).

18.

Nguyen, N.Y., Chrambach, A.: 187 (1979).

Anal. Biochem. 88,

Arch.

Endocrinology, submitted

J. Biochem. Biophys. M e t h o d s ^ , 171~~

19.

Righetti, P.G., Chrambach, A.:

20.

Jackiw, B.A., Brown, R.K.: (1979).

Anal. Biochem. 90, 633-643 (1978).

21.

Nguyen, N.Y., Chrambach, A.

Anal. Biochem. 82, 226-235 (1977).

J. Biochem. Biophys. Methods, submitted

22.

Nguyen, N.Y., Chrambach, A.

Anal. Biochem. 82, 54-62 (1977).

23.

Nguyen, N.Y., Chrambach, A.

Anal. Biochem. 79, 462-469 (1977).

24.

Nguyen, N.Y., Chrambach, A.

Anal. Biochem., 74, 145-153 (1976).

25.

Doerr, P., Chrambach, A.:

26

An der Lan, B.. Nguyen, N.Y., Chrambach, A.: mitted (1979).

27.

Baumann, G., Chrambach, A.:

Anal. Biochem. 42, 96-107 (1971). Anal. Biochem., sub-

Anal. Biochem. 70, 32-38 (1976).

22 28.

Rodbard, D., Levitov, C., Chrambach, A.: 723 (1972).

Separation Sei. ]_, 705-

29.

Tuszynski, G.P., Buck, O.A., Warren, L.: 408 (1979).

Anal. Biochem. 9^, 329-

30.

Schildknecht, C.E.: Allyl Compounds and Their Polymers, WileyInterscience, New York, NY, p. 14 (1973).

31.

Ghosh, S., Moss, D.B.:

32.

Rosen, A., Ek, K., Aman, P.:

33.

Otavsky, W.I., Bell, T., Saravis, C., Drysdale, J.W.: 78, 302-307 (1977).

34.

Jackiw, A.: (1979).

35.

Hjelmeland, L.M., Nebert, D.W., Chrambach, A.: Electrophoresis '78 (Catsimpoolas, N., ed.), Elsevier North Holland, New York-Amsterdam, pp. 29-56 (1978).

36.

Gianazza, E., Astorri, C., Righetti, P.G.: 161-169 (1979).

37.

Hjelmeland, L.M., Nebert, D.W., Chrambach, A.: 201-208 (1979).

38.

Jackiw, B.A., Chrambach, A.: (1979).1

39.

Gelsema, W.J., De Ligny, C.L., Van der Veen, N.G.: 173, 33-41 (1979).

40.

Nguyen, N.Y., Hjelmeland, L.M., Chrambach, A.: submitted (1979).1

41.

Salokangas, A., Eppenberger, U., Chrambach, A.: Acta, submitted (1979).

42.

Nguyen, N.Y., DiFonzo, J., Chrambach, A.: (1979).1

43.

Mardian, J.K.W., Isenberg, I.:

44.

Fawcett, J.S.:

45.

Egen, N.B., Twitty, G.E., Bier, M.: 17th Ann. Aerospace Sei. Meet., New Orleans, LA, American Inst. Aeronautics and Astronautics, Publ., New York, NY, Doc. No. 79-0405 (1979).

46.

Mürel, A., Kirjanen, I., Kirret, 0.: (1979).

_

Anal. Biochem. 62^, 365-370 (1974). J. Immunol. Methods 28, 1-10 (1979). Anal. Biochem.

Abstr. Xlth Interntl. Congr. Biochem., Toronto, p. 716

J. Chromatography 171, Anal. Biochem. 95,

J. Biochem. Biophys. Methods, submitted J. Chromatography

J. Chromatography, Biochim. Biophys.

Anal. Biochem., submitted

Anal. Biochem. 91, 1-12 (1978).

Ann. N.Y. Acad. Sei. 209, 112-126 (1973).

J. Chromatography 174, 1-11

pH-MOBILITY CURVES OF PROTEINS BY ISOELECTRIC FOCUSING COMBINED WITH ELECTROPHORESIS AT RIGHT ANGLES

Pier Giorgio Righetti and Elisabetta Gianazza Department of Biochemistry, University of Milano, Via Celoria 2, Milano 20133, Italy

Introduction A direct knowledge of a given protein, and of its contaminants, titration curves would be a great strategic help for a biochemist attempting to purify it, since it would allow selection of the proper pH conditions for any subsequent purification work based on charge-dependent methods, such as electrophoresis, isotachophoresis, equilibrium isoelectric focusing (IEF) and ion-exchange chromatography. Unfortunately, only a handful of proteins have been titrated so far and only once they had been purified to homogeneity. In fact, in conventional potentiometric acid-base titrations, mixtures of different proteins would exhibit a single titration curve averaged over the amino acid compositions and the relative ratios of the various components in the mixture. There are hints that things are rapidly changing. A major breakthrough came in 1976, at a meeting in Hamburg, when Rosengren et dl. (1) presented "a simple method of choosing optimum pH conditions for electrophoresis", which was in fact a direct display in a polyacrylamide gel slab of the titration curves of all the proteins present in a mixture. This was achieved by a two dimensional technique by which the sample would move electrophoretically perpendicular to a pH gradient, generated by a stack of stationary carrier ampholytes. The sigmoidal, pH-mobility curves thus generated were indeed proportional to titration curves. By further exploiting this idea, we have demonstrated the possibility of revealing the mutant charged amino acid in mutant phenotypes (2), of detecting liganded states of proteins (3) and of studying macromolecule-macromolecule

© 1980 Walter de Gruyter & Co., Berlin Electrophoresis '79

New York

2k

2

4

6

8

10

12

PH

Fig. 1. Comparison of electrophoretic mobilities and dissociation curves of trypsin. Solid line: dissociation curve; points: electrophoretic mobilities in various buffers, ionic strength 0.13. X: NaCl-HCl; A, Na acetateH C 1 ; 0 , CaCl 2 "barbiturate; O , MgCl 2 ~barbiturate; V, CaCl 2 -glycine : , CaCl 2 NH^Cl (from Duke, J.A., Bier, M. and Nord, F.F.: Arch. Biochem. Biophys. 40, 424-430 (1952)).

interactions (4). Moreover, in the case of simple uni- uni- valent amphoteric molecules, we have derived an equation linking the electrophoretic mobility of the titrated ions with the pK's of their anionic and cationic groups (5, 6). Thus, mobility data could be used to study protolytic equilibria of weak ionizable groups. In the present article we will review the results obtained so far and the possible future applications of this technique .

Historical

In the early fifties, attempts were made to correlate the electrophoretic mobility to protein charge, by using theories and equations derived by Smoluchowski (7), Debye and Hückel (8), Hückel (9) and Henry (10). The pH-mobility curves generated by electrophoresis at a series of different pH's in the pH 2-11 range were correlated to Potentiometrie titration data. In some cases, as shown in Fig. 1 for trypsin titration, a quite remarkable correlation among the two sets of data was achieved. However, generally,

25

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59

Fig.5: Short distance PAG-electrophoresis as first separating step to obtain routine double-one-dimensional patterns of transferrin. Albumin has migrated 3 cm. The gel strip w i t h the Tf-region to be transferred to the EF-gel is indicated. Note that the extreme right and left samples contain hemoglobin at a level visible in unstained gels. The Hb-marker is used as a guide for reproducable positioning of the gel strips. The upper gel contains 12, the lower gel 24 samples w h i c h had been applied using a 12-unit multiple syringe. 4 of these gels are run simultaneously in the PAG-electrophoresis system HAVANA ( DESAGA,Heidelberg ).Two properly adjusted variable multiple syringes for multiple handling of samples are accessory of this high speed vertical PAGE-system.

it follows that the latter gels are less sensitive than 4.5 M urea gels for detecting the variants used in this comparative

study.

From figure 3b, c and d it can be followed that the detection of electrophoretic variants by one-dimensional PAG electrofocusing is very difficult and impossible in some cases w h e n a variant Tf-compound occupies a position i dentical w i t h that of a non-Tf-compound. This problem is overcome by double one-dimensional electrophoresis.

Figure

5 demonstrates a short distance PAGE and figure 6 a long distance PAGE of sera from different individuals. The strip indicated in figure 5 w h i c h contains the Tf region and a reduced number of non-Tf-compounds is eliminated

6o

0 X-MQ

-oC

J f a C ( C 3 )

1 M •

*

m

m

-

T

f

c

F i g . 6 : P A G - e l e c t r o p h o r e s i s ( T=5, C=3 ) of 1 ul s e r u m samples w i t h a m i g r a t i o n d i s t a n c e of 10 c m for a l b u m i n . Samples No. 1-3 are identical w i t h No. 4-6 e x c e p t that h e m o g l o b i n has b e e n a d d e d to 1-3 as w e l l as to samples N o . 7-12 to d e m o n s t r a t e g e n e t i c a l l y d e t e r m i n e d h a p t o g l o b i n e - p h e n o t y p e s as H b H p - c o m p l e x e s ( No.lrHp 1-1 ;No. 2 :Hp2-l ;No. 3 :Hp2-2 ). N o t e that this s e p a r a t i o n d i s t a n c e is s u f f i c i e n t l y large to cut out p r o p e r strips also for other n o n - T f - p r o t e i n s and thus to o b t a i n h i g h r e s o l u t i o n E F - p a t t e r n s by the d o u b l e - o n e - d i m e n s i o n a l slab gel t e c h n i q u e .

f r o m the u n s t a i n e d gel and u s e d as sample for slab-gel P A G The r e s u l t is seen in figure 7 - 9 .

electrofocusing.

In figure 7 the m e t h o d is u s e d as a

s c r e e n i n g technique. Short d i s t a n c e P A G E is p e r f o r m e d d u r i n g the

prefo-

c u s i n g of the e l e c t r o f o c u s i n g gel. U s i n g this s c r e e n i n g m e t h o d m o r e

than

2 . 5 0 0 sera from d i f f e r e n t p o p u l a t i o n s w e r e s c r e e n e d for T f - v a r i a n t s b y three w o r k e r s of our l a b o r a t o r y w i t h i n one w e e k i n c l u d i n g all w o r k i n g steps

from

thawing of the sera till d o c u m e n t a t i o n on film, d a t a s h e e t s . c o n s e r v a t i o n

of

the gels b y d r y i n g a n d r e p e t i t i o n of u n c e r t a i n s . F i g u r e 8 shows samples No. 2 - 1 4

f r o m figure 3 b y r o u t i n e d o u b l e o n e - d i m e n s i o n a l e l e c t r o p h o r e s i s

t r a n s f e r r i n . The v a r i a n t s can be c l e a r l y i d e n t i f i e d w i t h o u t

for

difficulties.

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Fig. 5: Ultrathin-layer protein mapping of two different varieties of Phaseolus coccineus. For separation conditions, see figs. 2 and 3.

•IEF PAGE

A

B

Fig. _6: Ultrathin-layer protein mapping of wheat flour. (A) Stained for proteins. (B) Esterase isoenzymes. For separation conditions, see figs. 2 and 3.

77 In conclusion, for ultrathin-layer techniques in Polyacrylamide gels on foils we can summarize:

1) Preparation of ultrathin gels on cellophane is performed easily with standard equipment. Because of their mechanical stability, the gels can be handled very conveniently. 2) The method allows great flexibility in gel size, pore size, pH-gradients, and usage of additives. 3) Compared with conventional gel slabs, separation and resolution are improved. 4) Separation as well as staining, destaining, and drying are completed in substantially shorter time. 5) The considerably reduced demand for reagents, especially carrier ampholytes for isoelectric focusing, drastically cuts costs for electrophoretic methods.

References 1. Awdeh, Z.L., Williamson, A.R., and Askonas, B.A.; "Isoelectric focusing in Polyacrylamide gel and its application to Immunglobulins." Nature (London) 219, 66-67 (1968). 2. Leaback, D.H. and Rutter, A.C.: "Polyacrylamide- isoelectric-focusing. A new technique for the electrophoresis of proteins." Biochiir. Biophys. Res. Commun. 32, 447-453 (1 968) 3. Frater, R.: "Artifacts in isoelectric focusing." J. Chromatogr. 50, 469-474 (1970). 4. Bours, J.: "Isoelectric focusing of lens crystallins in thin-layer Polyacrylamide gels. A method for detection of soluble proteins in eye lens extracts." J. Chromatogr. 60, 225-233 (1971). 5. Vesterberg, 0.: "Isoelectric focusing of proteins in Polyacrylamide gels." Biochim. Biophys. Acta 257, 11-19 (1972)

78 6. Righetti, P.G. and Drysdale, J.: "Isoelectric focusing" North-Holland, Amsterdam, Oxford; American Elsevier, New York (1976). 7. Görg, A., Postel, W., and Westermeier, R.: "Einfache Herstellung von Polyacrylamidgel-Folien für die isoelektrische Fokussierung." Z. Lebensm. Unters. Forsch. 164, 160-162 (1977). 8. Görg, A., Postel, W., and Westermeier, R.: "Ultrathinlayer isoelectric focusing in Polyacrylamide gels on cellophane." Anal. Biochem. 89, 60-70 (1978). 9. Görg, A., Postel, W., and Westermeier, R.: "Herstellung von ultradünnen PAA-Gelen auf Cellophanfolie." LKB Workshop, Munich, May 1978. 10. Söderholm, J., Allestam, P., and Wadström, T.: "A rapid method for isoelectric focusing in Polyacrylamide gel." FEBS Lett. 24, 89-92 (1972). 11. Righetti, P.G. and Righetti, A.B.B.: "High voltage analytical and preparative isoelectric focusing." in "Isoelectric focusing" (Arbuthnott, J.P. and Beeley, J.A., eds.) Butterworths, London, pp. 114-131 (1975). 12. Görg, A., Postel, W., and Westermeier, R.: "Ultradünnschicht isoelektrische Fokussierung." Communication at the "Elektrophorese Forum" Munich, 25-27 Oct. 1978. 13. Görg, A., Postel, W., and Westermeier, R.: "Ultradünnschicht-isoelektrische Fokussierung und UltradünnschichtElektrophorese in Polyacrylamidgel auf Folie." GiT Labor-Medizin 2, 32-40 (1979). 14. Fehrnström, H. and Moberg, U.: "SDS and conventional Polyacrylamide gel electrophoresis with LKB 2117 Multiphor." LKB Appi. Note 306 (1977). 15. Görg, A., Postel, W., and Westermeier, R.: "Ultradünnschicht isoelektrische Fokussierung in 0.12 mm Polyacrylamid-Gelen auf Polyesterfolie." Z. Lebensm. Unters. Forsch. 168, 25-28 (1979). 16. Mayer, J.W.: "Simple and rapid methods for drying Polyacrylamide gels after isoelectric focusing." Anal. Biochem. 76, 369-373 (1976).

ULTRATHIN-LAYER ISOELECTRIC FOCUSING IN 50-100 pm POLYACRYLAMIDE GELS ON SILANIZED GLASS PLATES OR POLYESTER FILMS B.J. Radola Institut für Lebensmitteltechnologie und Analytische Chemie, Technische Universität München, D-8050 Freising-Weihenstephan, F.R.G.

Introduction In the commonly used technique of thin-layer isoelectric focusing compact polyacrylamide gels are polymerized on uncoated supporting glass plates with good heat conductive properties. After focusing the gels are transferred to a fixative solution to precipitate the proteins and wash out the carrier ampholytes, a step which in some procedures is combined with staining (1). During fixation and staining the gel slab dissociates from the glass plate and is subjected to mechanical stress on all subsequent visualization steps. This was a limiting factor for using (i) gels with a thickness I g G i , k a p p a i m m u n o g l o b u l i n , b ) t h e p11 h y b r i d o m a , a n d c ) t h e J 1 0 h y b r i d o m a . S a m p l e s w e r e a p p l i e d as 3 ^ul d r o p l e t s . T h e a n o d e is to t h e l e f t .

The

J10

band

clone

in

with

on

antisera clone

The

antibodies

orescence

virus

to

shows

and

that were

hand

other the

against

J10

ficity

the

addition

shows

parental

immunoglobulin the

new

further

band

producer

antigen

cell

(To

be

of

demonstrable

Immunofixation

in

IgM,

of

of

type.

immunoflu-

revealing

the

this

kappa

indirect

tests,

a membrane B95-S

band.

subclasses

is

tested

radioimmunobinding

against

a clearly

P3X63

speci-

Epstein-Barr

published).

125 Amplified

sensitivity

protein

and

Figure

A 4

producing bands with

is

of

shows

from

an

of free

globulin

autoradiograph

cultures. performed

X-ray

sensitivity

bands

the

detection

of

proteins

using

I-

autoradiography

radiolabeled

sensitive

of

The by

of

immunofixation

(LKB

can

now

clonotype,

and

A

and

see, in

not

immunoglobulin immunoglobulin

followed

By

only

addition

chains,

probably

the

exposure

Ultrofilm).

but

immunoglobulin molecules

several

development

protein

film we

of

to

this the

by

overlaying

extremely amplification

major

minor

partially degraded

an

4-5

bands

arising

joined

immuno-

variants

present

ll4 in

the m e d i u m .

#

l liti w

^

%

F i g u r e 5» A u t o r a d i o g r a p h i c d e t e c t i o n u s i n g r a d i o - l a b e l e d p r o t e i n A.

of h y b r i d o m a

clones,

DISCUSSION The d e v e l o p m e n t for

the

has

opened up

polysaccharides. a single

and Milstein

(8,9)

of a

of i m m u n o g l o b u l i n - p r o d u c i n g

a new technology

of a n y m o l e c u l e with

by K o h l e r

isolation

of b i o l o g i c a l

for the interest

These monoclonal

antigenic

serological e.g.

cells

analysis

proteins,

antibodies,

determinant

method

hybrid

which

are the most

react

specific

biological In the

probes

course

of

found, a g a r o s e able

tool

light

system

antibody

these

the

production

stemmed

in vitro,

Since

1968, when Awdeh, Williamson the method

it h a s

gels

to a n a l y z e

been extensively

as w e l l

as i n m o s t

However,

of m a n y

Radola

(16) o v e r c a m e

molecular

large

of n a t i v e

stacles,

s u c h as

cipitation hybridoma

IgM has

sieving give

producing

focusing method elsewhere

detection)

rise

to

the

mo-

sieving.

associated

with

gels,

strength,

Sephadex

displays

simplicity

toxic and

times.

At

polyacrylamide TEMED.

in

(and i n other

the gel

of

immuno

types

of

cross-

sieving

considerably same

more

agarose/poly-

and flexibility and

the

isoelect-

(16), D A T D

minimal

of

parallel

possibilities

(17) a n d m i x e d

detection by immuno-techniques, and washing

paper using

pre-

several

the agarose

in this

ob-

isoelectric

IgM antibodies

to m e t h o d s

gels

by several

Since

consider

m e d i a s u c h as

mechanical

bisacrylamide

(13-15)»

restricts

IEF in granular

(1) o n t h e v a r i o u s

gels. Agarose

the

immunology

to m o l e c u l a r

phenomena.

described

polyacrylamide

staining

severely due

de-

layer

heterogeneity,

in

research

been hampered

IgG, we

acrylamide

avoids

o

human

(12)

in t h i n

for use

by the m e d i u m ,

to b e s u p e r i o r

anti-convection linked

and Askonas

of t h e p r o b l e m s

and aggregation clones

with those detail

many

primary

Sephadex.

Analysis

ric

gel

proteins,

class

our studies

in the

IgG antibody

sieving by performing

particularly

both

of b i o m e d i c a l

the p o l y a c r y l a m i d e

bility

The

from

focusing

developed

areas

valu-

system.

vised

of i s o e l e c t r i c

have

immunoglobulin

and specificity.

the m e t h o d

we

(1) to b e a v e r y

clonality,

(10,11 ) a n d i n t h e m o u s e

polyacrylamide

analysis.

hybridomas

focusing

chain type

for developing

specific

for biochemical

establishing

iso-electric

for analyzing

subclass, reason

available

time,

reagents

and

in

good

sample

shorter

agarose acrylamide,

116 Acknowledgements

This

research

from

the

Cancer Many cal with

was

Division

Institute,

thanks

to

assistance IEF

supported

in part

of

Cause

Cancer

and

Ms

by

the

Marianne

and

to

by

and

Swedish

N01-CP-33316

Prevention, Cancer

Lindström

LKB-Produkter

Contract

for for

National

Society.

excellent

techni-

contribution

reagents.

REFERENCES

1.

R o s i n , A., Ek, K., 28, 1-11 (1979).

2.

R o s e n , A., Ek, K., A m a n , P., a n d V e s t e r b e r g , 0.: P r o t . B i o l . F l u i d s 1979» P e r g a m o n P r e s s (in p r e s s ) (1979)•

3.

V e s t e r b e r g , 0.: Berlin-New York R.F.

and

Aman,

P.:

J.Immunol.

E l e c t r o p h o r e s i s '79. (1979) (in p r e s s ) .

R. : Clin.Chem.

de

Ritchie,

5.

Hunter,

6.

Moar, M., Rosin, ¿ 0 , 219 ( 1 9 7 9 ) .

7.

C r o n v a l l , G., U l y s s e s , S., F i n s t a d , R . C . : J . I m m u n o l . 104, 140 ( 1 9 7 0 ) .

J.

8.

Köhler,

G.

and

Milstein,

C.:

Nature

256 , 495

9.

Köhler, (1976).

G.

and

Milstein,

C.:

Eur. J. I m m u n o l .

10.

Rosin, A., S.i N a t u r e

11.

S t e i n i t z , M. , K l e i n , G. , K o s k i m i e s , N a t u r e 26j?, 4 2 0 ( 1 9 7 7 ).

12.

Awdeh, Nature

13.

Righetti, P.G. 271 (1974).

14.

Acta Ampholinae: S - 1 6 1 26 B r o m m a ,

15.

Vesterberg,

16.

Radola,

17.

B a u m a n , G. a n d 32 ( 1 9 7 6 ) .

and

Smith,

Walter

4.

R.M.

and

Meth.

Greenwood, A.

and

F.C.:

Klein,

Gergely, P., Jondal, 2 6 7 , 52 ( 1 9 7 7 ) .

Z.L., Williamson, 2J_2, 66 ( 1 9 7 7 ) • and

0.:

B.J.:

A.R.,

Drysdale,

(1978) issued Sweden. Int.Lab.

May/June,

Biochpm.Biophys.Acta Chrambach,

A.:

and

Klein, S.,

Askonas,

by

194,

( 1 9 7 6 ).

495

(1962).

J.Immunol.Meth.

M.,

J.W.:

22_, 4 9 7

Nature

G.:

Gruyter

Williams, (1975). 6_, 511 > G. ,

Britton,

Mäkelät B.A.:

J.Chromatogr. LKB-Produkter 61 295,

0.:

98, AB,

(1978). 412(1973).

Analyt.Biochem.70,

IMMUNOPEROXIDASE STAINING AND RADIOIMMUNOBINDING OF HUMAN TUMOR MARKERS SEPARATED BY DIRECT TISSUE AGAROSE ISOELECTRIC FOCUSING

C.A. Saravis, C.G. Cunningham, P.V. Marasco, R.B. Cook, and N. Zamcheck Mallory Institute of Pathology, Harvard Medical School, Boston University School of Medicine, Boston, Mass. 02118; Marine Colloids Div., FMC Corp., Rockland, Me 04841

Introduction Human tumor markers (Zinc Glycinate Marker (1), Carcinoembryonic Antigen (2)) are readily identified and characterized in extracts of small amounts of tissue or c e l l s following direct tissue electrophoretic extraction (3) and separation by agarose isoelectric focusing (4).

After the isoelectric

focusing patterns are fixed, they are reacted with specific anti-tumor marker antisera, then with second antibody either peroxidase conjugated or radiolabeled.

This method offers an opportunity to measure large

molecules (>_ 2 x 106 MW) of biological and potential c l i n i c a l usefulness by direct comparison of amounts of antigen i n malignant, premalignant, and dysplastic tissues.

Material and Methods The agarose (Isogel) and Gelbond (the polyester support material treated on one side for adherence of gel) were from Marine Colloids Division, FMC Corporation', Carrier ampholytes were purchased from Serva Feinbiochemica. Radiolabeled iodine was purchased from New England Nuclear.

Swine anti-

rabbit IgG antiserum, peroxidase conjugated, was purchased from Accurate Chemical & S c i e n t i f i c Corporation. grade available.

© 1980 Walter de Gruyter & Co., Berlin • New York Electrophoresis '79

A l l other reagents were the highest

118 Isoelectric focusing was performed in equipment produced in this laboratory by C.A. Saravis.

However, most available commercial isoelectric fo-

cusing equipment could have been used. Eight ml agarose aliquots, prepared from

1% Isogel in deionized water and

stored at 4°C are placed in a boiling water bath and the agarose gel brought into solution.

The agarose-containing test tubes are transferred

to a water bath or heated block which is maintained at 56°C.

When the

agarose has cooled to 56°C, the ampholyte mixture (pH 3-11) is added until a final concentration of 2.5% has been reached.

A piece of parafilm is

then placed over the end of the test tube and the contents mixed by slowly inverting it several times.

A casting assembly consisting of two glass

plates, a 0.8mm spacer frame, and a 10 x 10 cm piece of 7 mil thick Gelbond is pre-warmed by some convenient means, (IR lamp, oven, etc.). The agarose-ampholyte solution is put into the casting assembly and allowed to gel for 15-20 minutes at room temperature.

The agarose-Gelbond plate

is removed from the casting assembly and stored at room temperature in a humidity chamber for at least 1 hour and preferably no longer than 24 hours before use.

This to obtain reproducible electrophoretic character-

istics. A thin film of water is spread evenly over that portion of the cooling platform to be used. cooling platform. beneath the plate.

The agarose plate is then oriented properly on the

Air bubbles should be excluded from the water layer Prior to its use, the agarose gel plate must be

blotted quickly with a single sheet of Schleicher and Schuell #577 paper. This is done by gently placing a sheet of the S & S paper over the entire surface of the gel followed by its immediate removal.

This will remove

any superficial moisture or condensation on the surface of the gel(which could lead to IEF pattern artifacts). At this point, the platinum electrode wires are placed directly on the gel surface at the ends of the plate. A 2 mil thick Mylar mask containing 2 x 6 mm loading slits is placed on the gel surface excluding air bubbles.

Care should be taken that no

loading slit is closer than 1 cm from the edge of the agarose plate. Fresh frozen human tissue sections 8 microns or greater thick (80,000 or more malignant cells) are placed over the loading slits such that

119 they overlap and drape into the slits making contact with the gel.

The

macromolecules are quantitatively pulled into the agarose by applying 10 mA for 10 minutes.

The power is then shut off, cells removed by

gentle blotting, and the sample mask removed with the tissue sections. The upper limits for power and voltage are then set at 25W and 1500 V for the remainder of the electrofocusing run (usually 15 minutes at 10°C). The plate containing the separated macromolecular proteins are immediately placed in a fixative solution (containing methanol 30%, trichloroacetic acid 5%, and sulphosalicylic acid 3.5%) for 10 mins.

Filter paper

(S&S#577) pre-wet with water is placed on the gel surface, several layers of absorbent toweling placed on top of the filter paper, ampholytes and water removed from the gel (approximately 10 mins), and the agarose dried as a film on the Gel bond.

The fixed proteins can then be stained

for protein using Crowle's stain (5).

After 10 minutes in the stain

solution, the plate is rinsed in acidified water for several minutes, until the background is clear, and then evaluated.

When radiolabeled

samples are used, autoradiography is done to determine microheterogeneity. When immunoperoxidase staining is used, the fixed proteins are successively treated with 3% hydrogen peroxide, 2.28% periodic acid, and 0.02% sodium borohydride to bleach and block acid hematin, endogenous peroxidase, and aldehydes.

The reaction plate is washed with Tris buffer,

0.15M, pH 7.6, and overlaid with normal swine serum, 1:5 dilution, for 30 minutes at room temperature.

The reaction plate is washed with the

Tris buffer, and placed into a dish with wet paper toweling to provide humidity during incubation.

Rabbit anti-tumor marker antiserum (e.g.

6 ml of a 1:25 dilution of whole antiserum) is placed on the gel surface, the dish sealed, and the reaction plate incubated overnight at room temperature.

The reaction plate is then washed with 3 liters of the Tris

buffer for 4 hours or more.

The plate is placed in the dish containing

the wet toweling, and overlaid and incubated overnight at room temperature with swine anti-rabbit IgG antiserum, peroxidase conjugated.

The

next day the reaction plate is washed with 3 liters Tris buffer for 4 hrs. at room temperature, reacted with enzyme substrate, rinsed, and read. Alternatively, the fixed and dried DTIF patterns can be reacted with

120 radiolabeled anti-tumor marker antisera, washed, and autoradiography used to determine tumor marker localization.

Results and Discussion Agarose isoelectric focusing of representative proteins (used as separation monitors) and stained for protein is shown in Figure 1.

Figure 1 - Agarose isoelectric focusing. #1 #2 #3 #4 #5 #6

Separation patterns of

Catalase L-amino acid oxidase Direct tissue isoelectric focusing of R.B.C.'s Malignant ascites from a patient with colon cancer Alpha fetoprotein Isoferritins

One nanogram of radiolabeled CEA was isoelectric focused as described above. graphed.

Following fixing and drying, the agarose plate was autoradioThe presence of several radioactive bands seen in the CEA separa-

tion showed how sensitive this procedure i s

for detecting microhetero-

geneities or contaminants in small amounts of radiolabeled protein. The fixed isoelectric focusing patterns of washed malignant ascites c e l l s , and the cell-free ascites f l u i d from a patient with colon cancer, were reacted with anti-ZGM antiserum, and immunoperoxidase stained (Figure 2).

Figure 2 - Immunoperoxidase staining of i s o e l e c t r i c focused malignant ascites cells and f l u i d s from a patient with colon cancer. #1 Cell-free ascites stained for protein #2 Washed ascites c e l l s , direct application, stained for protein #3 Cell-free ascites reacted with anti-ZGM antiserum, immunoperoxidase stained #4 Washed ascites c e l l s , direct application, reacted with a n t i ZGM antiserum, immunoperoxidase stained #5 Cell-free ascites reacted with anti-ZGM antiserum, immunoperoxidase stained

122 The ZGM in the ascites fluid was found at pH 5.7, while the ZGM's from the malignant ascites cells were at the basic end of the reaction plate (approximately pH8).

This difference in pi's of the cellular ZGM is

being investigated. Radiolabeled rabbit anti-ZGM antiserum gave diffuse autoradiography patterns and had no advantage over the use of unlabeled anti-ZGM antiserum and enzyme-labeled second antibody, and is not recommended for use in this technique. It is important to note that several changes have been made in the original technique (4): a) The Isogel plates now are cast 0.8 mm thick instead of 1.6 mm thick.

Less ampholytes are needed, and higher voltages can be

applied with shorter running times because of the increased cooling efficiency.

The fixing and drying steps of the isoelectric focusing

patterns also are more rapid,

b) The agarose plates now are stored at

room temperature and are not refrigerated.

This eliminated the syneresis

that was sometimes seen during isoelectric focusing when agarose plates were stored at 4°C. In summary, this new technique will

identify, quantitate, and character-

ize specific tumor markers. Supported in part by Grant CA04486 and Contract CB64071 from the National Cancer Institute, National Institutes of Health.

References

1.

Pusztaszeri, G., Saravis, C.A., and Zamcheck, N.:

J. Natl. Cancer

Inst. 56, 275 (1976). 2.

Gold, P., and Freedman, S.O.:

3.

Saravis, C.A., O'Brien, M., and Zamcheck, N.:

J. Exp. Med. 121, 439 (1965).

4.

Saravis, C.A., and Zamcheck, N.:

5.

Crowle, A.J., and Cline, L.J.:

J. Immunol. Methods

29, 97 (1979). J. Immunol. Methods 29, 91 (1979). J. Immunol. Methods 17, 379 (1977).

AGAROSE

AS S U P P O R T I N G

MEDIUM

FOR

SING. DETERMINATION

OF

THE

HUMAN

Gc

(GROUP

SERUM

Mariann

PROTEIN

ANALYTICAL

RECENTLY

ISOELECTRIC FOCU-

DISCOVERED

SPECIFIC

S U B T Y P E S IN

THE

COMPONENT).

Thymann

D e p a r t m e n t of S e r o l o g y , I n s t i t u t e v e r s i t y of C o p e n h a g e n DK-2100 Copenhagen, Denmark

of F o r e n s i c

Medicine,

Uni-

Key w o r d s : Agarose isoelectric focusing Immunofixation print technique - Group Specific Component © 0.5 mM MgCl 2 containing 0.5 % NP 40 and 1 % Trasylol. The lysate was centrifuged at 105 000 x g for 30 min. Unspecific proteins can be largely removed from the supernatant by a pre-precipitation with 20 nl normal goat serum and 200 p.1 10 % S. aureus suspension strain Cowan I (1 h at 4° C) as described by Kessler (5). T-antigen was immunoprecipitated from the supernatant by adding 20 ul hamster SV 40 tumor serum (30 min) and 200 |al S. aureus overnight at 4° C. The immunoprecipitates were washed several times with 0.15 M NaCl, 5 mM EDTA, 50 mM Tris-HCl, pH 7.4 and 1 % sucrose containing 1 % NP 40. Tantigen was eluted by boiling the immunoprecipitates for 3 min in sample buffer (65 mM Tris, pH 7.0, 700 mM 2-mercaptoethanol, 2 % SDS, 0,1 % bromophenoleblue) and run on discontinuous 7 % SDS-gels as described by Lammli (6). After electrophoresis, the gels were frozen and cut into 2 mm

1^3 pieces. T-antigen was eluted from gel slices with 500 |il water, 10 % 232 mercaptoethanol overnight and counted for P by Cherenkov radiation in a j-counter.

Alkylation of SV 40 T-antigen

Lyophilized SDS-PAGE purified T-antigen (containing 5-20 ug protein) was dissolved in 50 ul 0.2 M Tris-HCl, pH 8.8, 0.1 % SDS, 5 mM dithiothreitol by heating for 1 min at 90° C. T-antigen was alkylated by four different ways: the sample was incubated with either iodoacetate (25 mM), iodoacetamide

(25 mM), N-ethylmaleimide (54 mM) or acrylnitrile (60 mM) for 1 h

as described by Riordan and Vallee (7, 8, 9) at 4° C or 37° C. The reactions were stopped by the addition of 2-mercapthoethanol (1.42 M). In order to dissociate SDS from T-antigen, 200 (j.1 15 % NP 40 and 9.5 M urea were added for 12 h at 4° C and then the samples were loaded immediately on IEF gels.

Isoelectric Focusing (IEF) Isoelectric focusing was performed in 3,3 % polyacrylamide flat gels. The polymerization solution containing 2 % (w/v) carrier ampholytes, 9.5 M urea, 2 % NP 40 and 3,3 % acrylamide was deaerated for 2 min and poured between glassplates (1x50x90 mm). After polymerization (40 - 60 min) the gels were placed on a cooling block covered with kerosene to improve the heat transfer. Samples were loaded either in slots cut into pieces of silicone rubber or on Whatman GF-C filter paper placed on various positions on the focusing gel. Electrode strips soaked in 1 M H^PO^ (anode) and 1 M NaOH (cathode) were placed at both ends of the gel. To protect the gel from evaporation it was covered with a polyethylene foil. Gels were run at 20 W (2000 V) for 5 h at 4° C. Prior to staining, the gels were fixed in TCA (20 % w/v) for 60 min. The precipitated carrier ampholyte-NP 40 complex was eluted with a solution of 2-propanol (25 % v/v) and acetic acid (10 % v/v) in water. Proteins were stained with 0,1 % Coomassie brilliant blue R 250 in methanol/acetic acid/water (25/7/68,

lkk v/v/v) for 30 min at 37° C and destained in water/2-propanol/acetic acid (65/25/10 v/v). After drying the gels were exposed to Agfa T 4 X-ray film for autoradiography at - 70° C. The pH gradient was determined at the end of the run by cutting a strip of the gel into 2 mm pieces, eluting with 0.5 ml 10 mM KC1 in deaerated water for 2 h and measuring the pH. For refocusing experiments the gel strip containing the focused T-antigen bands was put on a new flat bed gel near the cathode. The second focusing run was started under the same conditions as described. For molecular weight control focusing gels were run in the second dimension without equilibration on SDS-PAGE (10 %) according to O'Farrell (11).

Results and Discussion

T-antigen was isolated from

32 P-phosphate labeled NP 40 extracts of SV 40

transformed human cells (SV 80) by indirect immunoprecipitation followed by SDS-PAGE. T-antigen migrates on discontinuous SDS gels approximately at the molecular weight of phosphorylase a (94 000). It was eluted from the gel slices, lyophilized and shown to be homogeneous by reelectrophoresis (Fig. 1). Since SDS binds tightly to proteins and influences their charge very strongly, further separation of SDS purified T-antigen by isoelectric focusing required the complete removal of SDS. Earlier reports have shown that SDS can be removed from proteins by forming mixed micelles with NP 40 (10, 11). This treatment does not result in a pi shift, but after removal of SDS much of the material precipitates at the application point (12). SDS was dissociated from purified T-antigen by addition of 15 % NP 40 and 9.5 M urea. T-antigen treated in this way did not enter the focusing gel at any 2-mercaptoethanol concentration but precipitated at the starting point. This precipitation is most likely due to the formation of disulfide bonds, because nucleic acid sequence data have shown that T-antigen is rich in cysteine (13, 14) and therefore, the complete denaturation by SDS possibly exposes several SH-groups. To avoid the formation of disulfide bonds the sulfhydril groups were blocked by alkylation prior to focusing.

Ik5

94 K MM*

68 K

—43 K

40» —25 K

N H S HaT S D S T

Fig. 1

32 P SDST

SDS polyacrylamide gel electrophoresis of immunoprecipitated T-antigen from SV 40 transformed cells (SV 80). NHS: normal hamster serum; H«T: hamster anti T serum; SDS-T: SDS-PAGE purified T-antigen stained with Coomassie; 32 P-SDS-T: 32P-autoradiogram of SDS-PAGE purified T-antigen

Alkylation was performed by four different methods. Fig. 2 shows that after alkylation with either iodoacetamide, iodoacetic acid, N-ethylmaleimide or acrylnitrile T-antigen enters the focusing gel completely. The focusing patterns show a main band at approximately pH 6.5, which is obtained with each of the alkylation methods. Sometimes this band is split into a double band. In refocusing experiments this band has a stable charge and no new bands are formed. Additional bands with higher negative charge can be seen. Some of these bands may arise from alkylation side reactions with amidogroups or from hydrolysis of iodoacetamide, Nethylmaleimide or acrylnitrile to the corresponding acid. These alkylation specific bands were easily identified by refocusing because their charge pattern is stable. Fig. 3 gives a typical example obtained by extensive alkylation of T-antigen with acrylnitrile at pH 9.0. Further possible reasons for the occurrence of highly charged bands can be due to other reagents used for the focusing procedure: (i) Aged urea solutions, for example, contain cyanate/isocyanate which can react with basic aminogroups of T-antigen. It is not likely that carbamylation caused the multiple band patterns, since Tris-buffer and ampholines present

146 pH

a

Fig. 2

b

e

d

Isoelectric focusing patterns of SDS-PAGE purified and alkylated Tantigen a) without alkylation; b) iodoacetamide (25 mM, pH 9); c) iodoacetic acid (25 mM, pH 9) ; d) N-ethylmaleimide (54 mM, pH 9) ; e) acrylnitrile (60 mM, pH 9)

Fig. 3

Artifacts caused by alkylation with acrylnitrile (60 mM, pH 9, 4° C, 12 h).

in both the sample and the gel contain a large excess of aminogroups and can protect T-antigen from carbamylation (15). Varying the urea concentration between 7 and 9.5 M or varying the NP 40 concentration had no influence on the banding pattern, (ii) Heating the T-antigen prior to application to SDS-PAGE can result in deamidation of asparagine and glutamine. Heating between 2-6 min at 90° C had no influence on the banding pattern, (iii) The gel polymerization catalyst ammonium persulfate can produce charge changes by oxidation of sulfhydril groups or by oxidative cleavage of the T-antigen polypeptide chain. Prefocusing the IEF gel to remove the excess of persulfate did not alter the focusing pattern, (iv) Fig. 4 shows that the negative bands donot arise from decomposition products of T-antigen, although direct incubation of purified T-antigen with persulfate did

result in destruction of T-antigen to molecular weights lower than 50 000. (v) Additionally, we analyzed the effects of the sample pH on the focusing pattern. Fig. 5 shows that T-antigen dialyzed against pH 7.0 focuses

pH IE F

pH

1L?

S

I ? i

Ifc» 94 K

68 K

a

Fig. 4

The acidic bands seen in fig. 2 do not arise from T-antigen decomposition. NEM alkylated T-antigen was focused in the first dimension, SDS-PAGE was performed in the second dimension.

Fig. 5

b

pH influence on the banding pattern of NEM alkylated T-antigen a: incubation at pH 9.0 b: incubation at pH 7.0 (12 h)

at pH 6.5. Raising the pH of the sample to pH 9.0 produces more acidic T-antigen species. However, these acidic T-molecules refocus at pH 6.5 and show no stable charge pattern. Therefore these T-molecules do not arise from hydrolytic cleavage of the alkylation reagents used. Also, it seems unlikely that SDS still bound to the T-antigen induces these acidic bands, because neither prolonged equilibrium focusing for 20 000 Vh nor solvent extraction of SDS by amines (triethylamin, tributylamin) affected the focusing patterns significantly.

148 The incubation temperature of the sample had an effect on the banding pattern similar to the pH as seen in fig. 5. Incubation at 4° C resulted in the pH 6.5 focusing band, while a temperature shift to 37° C (6 h) produced the more acidic species which refocused at 4° C into the pH 6.5 band. Temperature shift during refocusing can lead to a new splitting of the bands. The molecular basis for the occurrence of these bands can be either a charge dependent binding of carrier ampholytes or protein-protein interactions, which depend on pH and temperature. To test these possibilities the sample was incubated with hydrophilic carrier ampholytes or bovine serum albumin (BSA). Incubation with various concentrations of carrier ampholytes (2-10 %, pH 3-10) did not influence the banding patterns. A strong binding of carrier ampholytes to protein (16) or polyanions like polyphosphate has been demonstrated by Righetti (17), but all these complexes are dissolved in the presence of 8 M urea. Therefore, it seems unlikely that binding of ampholine to T-antigen causes the multiple band pattern. Only the addition of BSA resulted in a significant shift towards acid pi's and in a banding split during focusing. This indicates that protein-protein interactions may be the cause of the induction of acidic T-antigen species. Hydrophobic domains of the proteins exposed by the unfolding action of urea may be involved in this phenomena.

Conclusions This study shows that SDS-PAGE purified SV 40 T-antigen can be analyzed by IEF. Complete denaturation by SDS leads to the formation of disulfide bridges and crosslinking between the T-antigen polypeptide chains. This causes precipitation at the application point. Crosslinking can be avoided by complete reduction and alkylation of T-antigen. T-antigen alkylated by different reagents focuses at approximately pH 6.5. Additional bands arise dependent on the alkylation procedure used. These alkylation

1^9 specific bands can be easily identified, because their charge pattern is stable during refocusing. Reversible artifacts arise from interaction of T-antigen with other molecules of the solution or from protein-protein interactions. These unstable bands can be identified by refocusing in a second dimension.

References

(1)

Weil, R., Biochim. Biophys. Acta 516, 301 - 387 (1978)

(2)

Tegtmeyer, P., Rundell, K. and Collins, J.K., J. Virol. 2J_, 647 - 657 (1977)

(3)

Griffin, J.D., Spangler, G. and Livingston, D.M., Proc. Natl. Acad. Sci. USA 76, 2610 - 2614 (1979)

(4)

Baumann, E.A. and Hand, R., Proc. Natl. Acad. Sci. USA 76, 3688 - 3692 (1979)

(5)

Kessler, S.W., J. Immunol. 115, 1617 - 1624 (1975)

(6)

Lammli, U.K., Nature 227, 680 - 685 (1970)

(7)

Riordan, J.F. and Vallee, B.L., in Meth. Enzymol., Vol XXV, 449 - 456 eds. Hirs, C.H.W. and Timasheff, S.N., Acad. Press (1972)

(8)

Gurd, F.R.N., in Meth. Enzymol. Vol XXV, 424 - 437, eds. Hirs, C.H.W. and Timasheff, S.N., Acad. Press (1972)

(9)

Seibles, T.S. and Weil, S.L., in Meth. Enzymol. Vol XI, 204 - 206, ed. Hirs, C.H.W.,Acad. Press (1967)

(10)

Ames, G.F. and Nikaido, K. , Biochemistry J_5, 616 - 623 (1976)

(11)

O'Farrell, P.H., J. Biol. Chem. 250, 4007 - 4021 (1975)

(12)

Hamann, A. and Drzenieck, R., J. Chromatogr. 147, 243 - 262 (1978)

(13)

Fiers, W.R., Contreras, R., Haegeman. G., Rogiers, R., Van de Voorde, A., Van Heuverswyn, Van Herreweghe, J., Volckaert, G. and Ysebaert, M., Nature 273, 113 - 120 (1978)

(14)

Reddy, V.B., Thimmappaya, B., Dhar, R., Subramanian, K.N., Zain, B.S., Pan, J., Celma, M.L. and Weissman, S.M., Science 200, 494 502 (1978)

(15)

Vesterberg, O., Protides of the Biological Fluids, 17th Colloq., p. 417 - 422 (1969) Pergamon Press, Oxford

(16)

Frater, R., J. Chromatogr. 50, 469 - 474 (1970)

(17)

Giannazza, E. and Righetti, P.G., Biochim. Biophys. Acta 540, 357 - 364 (1978)

THE ROLE OF SIALIC ACIDS IN THE MICROHETEROGENEITY OF ALPHA^ ACID GLYCOPROTEIN : STUDY BY ISOELECTRIC FOCUSING AND TITRATION CURVES

Philippe Arnaud, E. Gianazza, P.G. Righetti and H.H. Fudenberg Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina, Charleston, S.C., USA, and Department of Biochemistry, University of Milano, Italy

Introduction Alpha^ acid glycoprotein (A^AGP, Orosomucoid) represents one of the most acidic proteins present in human serum. Its serum concentration varies between 60-140 mg % in normal individuals. Although it is a well-known acute phase reactant protein and detailed information is available concerning its protein and carbohydrate structure, as yet no physiological role has been demonstrated, for this protein. For a recent review, see Schmid (1). During the course of isolation and purification of alpha^ antitrypsin (2) we noticed that, following preparative isoelectric focusing of albumin-depleted serum in a pH gradient 2.5 - 6, the first protein peak present at the anode corresponded to pure A^AGP. We report here on this technique of purification and the results obtained in studies of the microheterogeneity of A AGP, following removal of sialic acid.

e 1980 Walter de Gruyter & Co., Berlin Electrophoresis '79

New York

152

Material and Methods A. Subjects and Samples Blood samples (50 ml) were obtained from healthy volunteers. After clotting and centrifugation at + 4°C at 2,500 rpm during 20 mn, the serum was collected and used as follows :

B. Techniques Cibaovon blue Chromatography

(3) : a serum sample (12 ml) was

layered on the top of a Cibacron blue linked to Sepharose (Blue Sepharose, Pharmacia, Uppsala) column (25 x 500 mm) and equilibrated with sodium phosphate buffer, 0.05 M, pH 7.4. Following elution of the unbound proteins at room temperature with the same buffer, bound albumin was desorbed using 0.2 M Ammonium Thiocyanate and re-equilibrated with the starting buffer. The unbound fractions were collected, dialyzed overnight at + 4°C with stirring, against 1 % Glycine buffer countaining 2 mM Calcium lactate and 0.1 M 2-mercapto-ethanol. The protein solution was then concentrated under pressure to a final volume of 5 ml. Preparative

Isoelectric

Vesterberg and Svensson column

focusing was performed according to (4), using a LKB isoelectric focusing

(LKB 8101, 110 ml) in a sucrose gradient obtained by

diluting 3/4th of the carrier ampholytes to 42 ml and dissolving 28 g sucrose

(final volume : 60 ml) for the dense solu-

tion. The light solution consisted of the remainder of ampholytes diluted to 60 ml with distilled water. The total ampholyte concentration

(w/v) was 2 %. The gradient was obtained

using a gradient mixer (LKB 2101). Ampholytes consisted on an equal mixture of LKB 1809.111

(pH 3.5 - 5) and LKB 1809.116

(pH 4-6). The electrode solutions were : cathode

(at the top)

153 1.0 M sodium hydroxyde, anode

(at the bottom) 1.0 M phosphoric

acid. The column was 2/3rd filled, then, the sample solution diluted with the dense solution

(v : v), was layered on top of

the gradient. Then, the column was filled with the remainder of the gradient and the cathode mixture overlayered at the top. The isoelectric focusing separation was conducted for 16 hours at + 4°C at constant voltage

(1 600 V). After completion of

the run, the column was emptied by aspiration from the top and 2.0 ml fractions were collected. O.D. reading at 280 nm and pH measurement at room temperature using a combination electrode and a digital pH meter were performed on each fraction. Electrophoresis

and Immunological

electrophoresis

(5), Agarose electrophoresis

acrylamide gel electrophoresis trophoresis

control. Polyacrylamide gel (6), SDS poly-

(7) , fused rocket Immunoelec-

(8), Ouchterlony's double immunodiffusion

Immunoelectrophoresis

(9) and

(10) were performed according to stan-

dard techniques, using antisera against whole human serum and antisera against specific human proteins, obtained from Meloy. Removal of Sialic acids. Agarose-linked neuraminidase

(inso-

luble neuraminidase, C. Perfringens SIGMA Chemicals Co, St Louis, USA) was equilibrated with acetate buffer, 0.1 M, containing 0.1 % Ca CI2' P H 5.0, and packed in a column 5 x 100 mm. The protein fractions corresponding to A^AGP were pooled, concentrated and dialyzed against the acetate buffer, and an aliquot

(0.5 ml, approximately 20 mg/ml) was applied on the

top of the column. After elution with the acetate buffer, fractions corresponding

to the proteins as determined by absor-

bance at 280 nm, were pooled, concentrated to the initial volume and recycled on the neuraminidase column.

15^ Analytical

isoelectric

viously described

focusing.

This was performed as pre-

(11), except that thin gels (0.3 mm) were

obtained using parafilm layers as gasket. The final volume of the gel solution was 12 ml. Several pH gradients

(final ampho-

lyte concentration 2 % w/v) were obtained by using several commercial available ampholytes

(LKB 1809.111, pH 3.5 - 5,

LKB 1809.116, pH 4-6, Pharmalyte pH 2.5 - 5, Servalyte pH 3-5) in view to improve the separation of A^AGP isotypes. The gels were run at + 4°C, using : 2.2 watts (450 volts and 6.0 mA) . pH measurement was obtained by cutting slices of the gel (20 x 5 mm) and eluting them in 2.0 ml double distilled deionized water. The gel stain was a colloidal suspension of Coomassie Blue G 250 in 12 % trichloracetic acid and in 1.0 N sulfuric acid. Protein

titration

curves. This was done on A^AGP before and

after neuraminidase treatment, according to Righetti (12) and the pH gradient in the gel was determined as above. Measurement

of Sialic acid

release.

Sialic acid measure-

ment was performed according to the technique of Reinhold A^AGP

(13)

(before and after neuraminidase treatment) was dialyzed

overnight against distilled water and freeze-dried and 0.5 mg were dissolved in anhydrous methanol containing 500 mM HC1. Removal of sialic acid was performed by mild acid hydrolysis at 65°C for 1.5 hour. The solvent was then evaporated under a stream of nitrogen, and N-acetylation of the free amino groups was achieved by suspension of the dry residue in 0.1 ml Pyridine and 0.1 ml acetic anhydride. After immediate evaporation of the sample to dryness, the simultaneous partial O-acetylation was reversed by refluxing the sample in 1.0 ml methanolHC1 for 1 hour. Then, derivatization was performed by adding 3 parts of Tri-SYL reagent plus 10 parts of solvent to the sample for 15 mn at room temperature. After evaporation, the

155 sample was extracted with Hexane and the trimethyl silyl derivatives analyzed by gas liquid chromatography on a OV 17 column. Meso inositol, used as internal standard, was added to the samples before freeze drying, and sialic acid used for calibration was derivatized by the same technique.

Results and discussion a) Preparative isoelectric focusing in the pH gradient used provided an easy way to obtain purified A^AGP from albumindepleted serum obtained from individual subjects. The elution profile and the pH gradient obtained are indicated on Fig. 1 A. The protein was found to be homogeneous by several criteria including polyacrylamide gel electrophoresis, agarose gel electrophoresis and SDS-polyacrylamide gel electrophoresis. The latter indicated a m.w. approximately 40,000, in good agreement with previous results (1). Absence of detectable contamination by other serum proteins was assessed by Ouchterlony1s double immunodiffusion and Immunoelectrophoresis using standard antiserum and antisera directed towards prealbumin, albumin, alpha^-antitrypsin, transferrin, ceruloplasmin and alpha^-antichymotrypsin. Furthermore, fused rocket Immunoelectrophoresis performed on the elution fraction collected from the isoelectric focusing column indicated the exact location of the A ^ G P containing fractions using a specific antiserum, and further demonstrated the absence of detectable contaminants in these fractions using a total antihuman antiserum (Fig. 1 B) . In addition, analytical isoelectric focusing in a pH gradient 2 . 5 - 5 revealed that purified A^AGP exhibits microheterogeneity (Fig. 2). This microheterogeneity consists in a series of at least seven isotypes with an isoelectric point between 3.4 and 3.8. Similar heterogeneity has been

156

.

O D. READING AT

280 nm .

. . p H MEASURED AT

22*C

O.Q 280 n n i i

A

/

,J \

J 30

F1

g-

m

1 A

:

9-

50

\

/

60

70

80

tub« number

Elution profile from preparative isoelectric focusing.

X W

Fl

40

V

1 B

0

:

«

&

*

II

W

51

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Fused rocket immunoelectrophoretic analysis of fractions following preparative isoelectric focusing. LEFT : total antihuman antiserum, RIGHT : specific antiserum against A.AGP.

h

157

1

2

3 4 5 6

7

8 9

10

11

fl BjBP

Fig. 2 : Microheterogeneity of A^AGP purified from individual subjects.

Purified protein

Normal serum

Fig. 3 : Comparison of the microheterogeneity of purified A,AGP with that of A,AGP from serum.

158 reported by Schmid and Binette (14) when pooled native human A^AGP was analyzed on starch gel electrophoresis at pH 2.9 or when rat A^AGP was analyzed on isoelectric focusing (15). In addition, this microheterogeneity does not appear to be related to the purification procedures, for normal serum presents an

identical microheterogeneity when submitted

to isoelectric focusing in the same pH range (Fig. 3). b) Desialylation of AjAGP (Fig. 4). Desialylation of the pure protein was performed by recycling the preparation on an agarose-linked neuraminidase column at a pH corresponding to the optimal activity of the enzyme. The isotypes reached a new isoelectric point between 4.3 and 4.7 pH units. It is interesting to note that the pH shift following removal of sialic acid occured in the present experimental conditions after one step and that additional exposure to neuraminidase in the same conditions did not lead to further shift or modify the isotypic pattern (Fig. 5). The results suggest that complete desialylation occured during the first run through the column and that a charge difference of approx. 0.9 pH unit resulted from sialic acid removal. This is in contrast with the shift to an isoelectric point of 5.4 reported by Schmid etal (16), but in good agreement with results obtained in our laboratory for similar sialoglycoproteins such as alpha^-antitrypsin (17) . c) Sialic acid measurement and titration curves. To verify that complete desialylation had occured following exposure of A^AGP to neuraminidase, determination of sialic acid was performed before and after desialylation. Several precautions were taken, including the use of an internal standard (mesoinositol) added to each preparation before lyophyllization. Results on the native material indicate that 7.9 (+0.5) sialic acid residues/mole of protein were

159

OD. 280 nmi 0.3

first cycle A

0.2.

0.1.

second cycle third cycle

J *

Il

V //

A

*-+****£—

Fraction number

Fig. 4 : Recycling of purified A AGP on agarose-linked neuraminidase column. For details, see text.

pH

€

III

Fig. 5 : From top to b o t t o m : native A ^ G P , A-^AGP after 1, 2 and 3 cycles of desialylation. The pH gradient measured in the gel is reported on the picture. Anode at the left.

l6o

released upon mild hydrolysis (mean of three determinations on different preparations), assuming a m.w. 40,000 for the A^AGP molecule. Measurement of sialic acid on the material recycled on the neuraminidase column indicated that more than 90 % (within the limits of sensitivity of the technique) were released. This was calculated by comparison with the mesoinositol standard and represents the mean of three different experiments. According to these results, each sialic acid should be responsible for a net charge difference of approx. 0.1 pH unit, assuming an identical effect of each sialic acid on the charge of the molecule. Titration curves performed on the native material (Fig.6 A) indicate that the microheterogeneity of A^AGP did not disappear when acidic dissociating groups were titrated to the non-charged form. It is important to note that the microheterogeneity of the protein was minimal between pH 6 and 8, suggesting that the radicals involved are titrated in this pH zone. The pattern of the titration curve of asialo-Aj^AGP is indicated on Fig. 6 B. It demonstrates the persistence of the microheterogeneity which is present over the entire pH range generated. In summary, our results strongly suggest that the microheterogeneity of A^AGP is not dependent upon an uneven sialylation of the isotypes of the protein. Other mechanisms are involved in the charge difference of the molecular variants of the protein. This could be due to post-transcriptional modification such as acetylation, deamidation or modification of sugar chains. In favor of the latter, Fournet et al. (18) have recently demonstrated that each glycosylation site of A^AGP (from a pool of blood donors) possesses carbohydrate units with different structure. Finally, A^AGP microheterogeneity could be due to point amino acid mutations which would affect the net charge of each isotype. It is striking to note that

l6l

Fig. 6 : Titration curves (12) of A. AGP in a pH gradient 3.5 - 10. On the top (A), native A ± AGP, on the bottom, (B), A, AGP after desialylation. First dimension (pH gradient) : anode at the left. Second dimension (electrophoresis), anode at the top.

162

Schmid et al. (19) demonstrated the existence, within the A^AGP molecule, of at least 2 2 amino acid mutations. Some of them could account for A^AGP polymorphism ( i ), which can also be demonstrated by isoelectric focusing (20), but some others could be responsible for the microheterogeneity. Structural studies of single isotypes will be necessary to demonstrate the basis of this microheterogeneity.

Acknowledgments This work was supported by grants USPHS HD 09938 CA 25746. We thank Dr R.C. Allen for fruitful discussions. This is publication n°

from the Department of Basic and Clinical

Immunology and Microbiology, Medical University of South Carolina.

References 1. Schmid, K., in : The Plasma Proteins (F.W. Putman, Ed.), Academic Press, New York, p. 183 (1976). 2. Arnaud, P., Galbraith, R.M., Fudenberg, H.H. (in preparation) . 3. Pannell, R., Travis, J., Bowen J., Tewksbury, Johnson D. : Biochem. J. , 157, 301-306 (1976). 4. Vesterberg, O., Svensson, H. : Acta chem. scand., 20, 620-627 (1966). 5. Chrambach, A., Reisfeld, R.A., Wyckoff, M., Zaccari, J. : Anal. Biochem., 20, 150-156 (1967). 6. Laureil, C.B. : Anal. Biochem., 15, 45-49 (1966). 7. Weber, K., Osborne, M. : J. biol. Chem., 44, 4406 (1969). 8. Axelssen, N.H., in : Manual of quantitative electrophoresis Methods and applications (N.H. Axelssen, J. Kroll and G. Weeke, Eds), Universitatsforlagot, Oslo, Norway, p. 69 (1973) . 9. Ouchterlony, 0., in : Progress in Allergy (P. Kallos, Ed.) Karger, Basel, 5, p. 1 (1958).

163 10. Grabar, P., Williams, C.A. : Biochim. biophys. Acta, 10, 193-196 (1953) . 11. Arnaud, P., Chapuis-Cellier, C., Creyssel, R. : C.R. Soc. Biol., 168, 58-64 (1974) . 12. Righetti, P.G., Krishnamoorthy, R., Gianazza, E., Labie, D. : J. Chromatogr., 166, 455-460 (1978). 13. Reinhold, V.N. : Meth. enzymol., 25, 244-249 (1972). 14. Schmid, K., Binette, J.P. : Nature, 190, 630-631 (1961). 15. Gordon, A.H., Dykes, P.J. : Biochem. J., 130, 95-103 (1972). 16. Schmid, K., Polis, A., Hünziker, K., Fricke, R., Yayoshi, M. : Biochem. J., 104, 361-37 (1967). 17. Arnaud, P., Wilson, G.B., Allen, R.C., Fudenberg, H.H. : (submitted for publication). 18. Fournet, B., Montreuil, J., Strecker, G., Dorland, L., Haverkamp, J., Vliegenthart, F.G., Binette, J.P., Schmid, K. : Biochemistry, 17, 5206-5214 (1978). 19. Schmid, K., Kaufmann, H., Isemura, S., Bauer, F., Emvra, J., Motoyama, T., Ishiguro, M., Nanno, S. : Biochemistry, 12, 2711-2724 (1973) . 20. Arnaud, P., Gianazza, E., Fudenberg H.H. : (in preparation) .

THE EFFECT OF ELECTRODE SOLUTIONS ON THE pH DRIFT IN THIN-LAYER ISOELECTRIC FOCUSING

H. Delincee I n s t i t u t für Biochemie, Bundesforschungsanstalt für Ernährung, D-7500 Karlsruhe, Federal Republic of Germany

Summary The cathodic d r i f t of pH gradients hinders the estimation of i s o e l e c t r i c points of proteins with very low m o b i l i t i e s . In contrast to focusing in polyacrylamide gel tubes, in which s t a b i l i z a t i o n of the pH gradient has been achieved by regulating the anolyte pH, t h i s approach was not as successful in horizontal t h i n - l a y e r i s o e l e c t r i c focusing in e i t h e r granulated g e l s , polyacrylamide gel slabs or u l t r a t h i n - l a y e r s . A reduction in the cathodic d r i f t was not observed by varying the electrode s o l u t i o n s from s t r o n g l y a c i d i c and basic e l e c t r o l y t e s to weaker e l e c t r o l y t e s as e.g. amino acids i s o e l e c t r i c at the terminal values of the pH gradient. Independent of the type of c a r r i e r ampholytes employed, Ampholine, Serval y t or Pharmalyte, i n s t a b i l i t y was p r o g r e s s i v e l y observed by increasing focusing time. The b i d i r e c t i o n a l d r i f t and the s l i g h t differences in d r i f t obtained by varying the granulated gels indicate that electroendosmosis

is

involved. C0£ interference a l s o seems to play a role in the cathodic pH d r i f t . By the use of s u i t a b l e amino acids as separators, the pH d r i f t could be delayed, enabling the determination of i s o e l e c t r i c points of very slowly migrating proteins.

Introduction The cathodic d r i f t of pH gradients i s a hitherto unexplained phenomenon in i s o e l e c t r i c focusing. I t i s a great hindrance when the protein of

© 1980 Walter de Gruyter & Co., Berlin • New York Electrophoresis '79

166 interest has a high isoelectric point and does not approach its steadystate ("equilibrium") before decay of the pH gradient occurs, and the protein is lost by migration into the catholyte. This problem was e.g. encountered with horseradish peroxidase, the predominant isoenzyme of which required an excessively long focusing time to attain the steady-state, due to the reduced mobility near its isoelectric point (1). Several explanations of the pH drift in isoelectric focusing have been advanced (2-9), some possible mechanisms involved are: i) electrophoretic migration of isoelectric carrier ampholytes, ii) electroendosmosis, iii) interaction between carrier ampholytes themselves or between ampholytes and proteins, iv) CO2 interference. The pH drift has also been observed when carrier ampholytes were replaced by buffers (10,11) thus chemical interactions or modifications of carrier ampholytes cannot be the only explanation. Stabilization of the pH gradient has been described in buffer focusing (12) as well as in focusing employing narrow pH range Ampholine (13) by the use of appropriate electrode solutions.

Materials and Methods Horseradish peroxidase with an absorbance ratio 403/280 mm of 0.6 was purchased from Boehringer (Mannheim, FRG) and fractionated into individual isoenzymes (14). The pH marker proteins cytochrome c, ribonuclease, sperm whale and horse myoglobin, conalbumin, bovine serum albumin were from Serva (Heidelberg, FRG), and amyloglucosidase was from Merck (Darmstadt, FRG).

Carrier ampholytes "Ampholine" were from LKB (Bromma, Sweden), "Servalyt" were from Serva (Heidelberg, FRG) and "Pharmalyte" from Pharmacia (Uppsala, Sweden). Sephadex gels were from Pharmacia (Uppsala, Sweden), Ultrodex from LKB (Bromma, Sweden) and Bio-Gels from Bio-Rad Labs (Richmond, Calif.). Acrylamide and N,N-methylenebisacrylamide were from Serva (Heidelberg, FRG). Thin-layer isoelectric focusing in granulated gels was performed essentially as described previously (15,1). The separation distance was 15 cm, per-

167 mitting a focusing time of 2 h at 200 V followed by 2 h at 600 V to attain the steady-state of the pH marker proteins. Thin-layer isoelectric focusing in continuously polymerized polyacrylamide gels (16) was performed using ready-prepared PAG-plates from LKB (Bromma, Sweden). Ultrathin-layer isoelectric focusing (17,18) in polyacrylamide gels (7 % T, 2.5 % C) was carried out on 10 x 20 cm glass plates, the separation distance between the electrode strips being 7 cm.

Results 1. Strong or weak electrolytes. When thin-layer isoelectric focusing was carried out in granulated gels using Sephadex G-75 Superfine containing 1 % pH 3.5 - 10 Ampholine, and the platinum electrodes were placed directly on paper strips wetted with electrolytes on top of the gel, and the strongly acidic and basic electrode solutions were substituted with amino acids isoelectric at the terminal values of the pH gradient, practically no influence on the pH drift was observed. All pH marker proteins except the most acidic amyloglucosidase were displaced to the cathode, whereas amyloglucosidase migrated to the anode with prolonged focusing time (2 h at 200 V followed by either 6 or 16 h at 600 V). Electrolytes tested were: a) 1 % sulfuric acid (pH 1.0) - 2 % ethylenediamine (pH 12.0), b) 0.01 M glutamic acid (pi 3.2) - 0.01 M lysine (pi 9.7), c) 0.05 M glutamic acid - 0.05 M lysine, d) 0.1 M glutamic acid - 0.1 M lysine and e) 0.01 M p-aminobenzoic acid (pi 3.6) - 0.01 M arginine (pi 10.8). Using the latter electrolytes (e), the pH in the electrolyte paper strips was measured in experiments lasting up to 8 hours. In the anolyte strip the pH during the run was about 3.5 and at the cathode about 11.6. Renewal of the strips every 20 minutes neither changed the anolyte pH, it being constant about 3.5, nor influenced the pH drift. In another experiment electrolyte vessels (250 ml) were used to prevent a depletion of the electrolytes. The platinum electrodes were placed in the vessels and the contact

168 to the gel was established with paper wicks. To achieve an equal field strength as with electrolyte strips the potential drop across the paper wicks was corrected. Again the anolyte pH was about 3.5 during the run, but the pH drift towards the cathode was markedly increased. If the first electrolytes (a) were used, the pH at the anodic strip or paper wick from the vessel (1 % sulfuric acid), remained about 1, but the pH drift was similar to that observed with the weaker electrolytes.

2. Influence of different carrier ampholytes. When the Ampholine were substituted by Servalyt, focusing in Sephadex G-75 Superfine containing 1 % pH 2-11 Servalyt again showed the pH drift with increasing time, essentially independent on the use of strong or weak electrolytes. The pH changes in the electrolyte paper strips were similar as with Ampholine. The results with 2 % pH 3-10 Pharmalyte were analogous to those with Ampholine and Servalyt (the use of 2 % Pharmalyte was necessary to obtain regular patterns; with Ampholine or Servalyt 1 % was adequate).

3. Effect of varying the granulated gel. In these experiments Sephadex G-75 and G-200 both Superfine, Sephadex IEF, Ultrodex and Bio-Gel P-60 were used together with Ampholine carrier ampholytes. Although more or less steep pH gradients were obtained with these gels, prolonged focusing showed the pH drift in all these gels. With Sephadex IEF also Servalyt and Pharmalyte were tested using different electrode solutions: a) 1 % H 2 S 0 4 - 2 % ethylenediamine; f) 0.1 M H 2 S 0 4 - 1.0 M ethylenediamine (19); g) 0.05 M aspartic acid - 1.0 M ethylenediamine (18); c) 0.05 M glutamic acid - 0.05 M lysine; e) 0.01 M p-aminobenzoic acid - 0.01 M arginine. For all types of carrier ampholytes, a bidirectional pH drift was observed. Small differences were noticeable between the use of strong or weak electrolytes, the pH drift being somewhat increased using weak electrolytes. The least pH drift was found with Pharmalyte using strong electrolytes. This is in contrast with the experiments in Sephadex G-75 Superfine in which a marked pH drift occurred already after a moderate focusing time (8 h).

4. Polyacrylamide gel slabs instead of granulated gels. A few experiments

169 with focusing in PAG-plates containing pH 3.5 - 9.5 Ampholine also showed the pH drift with strong as well as with weak electrolytes. Ultrathinlayer isoelectric focusing in polyacrylamide gels on silanized glass plates using wide range pH 3.5 - 10 Ampholine in a concentration of 2 % yielded similar results. Irrespective of the electrode solutions employed: a) 1 % H 2 S 0 4 - 2 % ethylenediamine; h) 1.0 M H 3 P 0 4 - 1.0 M NaOH (16); g) 0.05 M aspartic acid - 1.0 M ethylenediamine (18); i) 0.05 M aspartic acid - 1.0 M NaOH (19); e) 0.01 M p-aminobenzoic acid - 0.01 M arginine, the bidirectional pH drift was observed. Amyloglucosidase and bovine serum albumin migrated to the anode with increasing focusing time, whereas the other proteins moved to the cathode. After 17 hrs of focusing cytochrome c and ribonuclease were lost in the catholyte. Drastic differences in the patterns with the varying electrode solutions were not observed. The experiment using 2 % pH 2 - 11 Servalyt or 2 % pH 3 - 10 Pharmalyte gave analogous results.

5. Ultrathin-layer isoelectric focusing in narrow pH range ampholytes. Electrofocusing of hemoglobin was performed in pH gradients, containing either 2 % pH 6 - 8 Ampholine, 2 % pH 6 - 8 Servalyt or 2 % 5 - 8 Pharmalyte. The experiment was carried out in analogy to that of Davies (4), the hemoglobin being applied at three different positions: in the middle and at 15 mm from the anodic and cathodic electrode strips, respectively. The electrode solutions were either strong electrolytes: 1 % H^SO^ - 2 % ethylenediamine, or the anolyte as amino acid: 0.05 glutamic acid - 1.0 M ethylenediamine (19) or both anolyte and catholyte as amino acids: 0.01 M glycine - 0.01 M histidine (13). After a certain time, the hemoglobin zones reached the same position, thus the steady-state had been attained. Although the time needed to reach the same position was slightly different for the varying types of carrier ampholytes and electrode solutions, this occurred within maximal 3 hrs of focusing. Prolonged focusing showed a pH drift to the cathode as already described by Davies (4). In about 25 hrs the hemoglobin zones were within a distance of less than 0.5 cm of the cathode. A stabilization of the pH gradient was not observed.

6. CO,; interference with the pH drift. Carbon dioxide interferes with the

170 separation process and subsequent pi measurements in horizontal

thin-layer

isoelectric focusing in compact and granulated gels (1). If thin-layer focusing was carried out in a CC^-free atmosphere, a slight delaying effect on the cathodic pH drift was observed.

7. Addition of amino acids to the gel. In addition to the carrier ampholytes, 0.05 % glutamic acid and 0.05 % lysine were added to the gel, to act as terminal constituents. With glutamic acid and lysine as electrode solutions, the so formed "pH-cage" was thought to prevent the pH drift. This was, however, not the case. A similar pH drift as without addition of the amino acids was observed. To delay the entrance of the basic proteins into the catholyte, the addition of basic amino acids as separators (20) to the gel was tested. In a concentration of 0.25 % each, lysine and arginine were successful to repress the basic proteins from the cathode. Thus with all types of carrier ampholytes, in granulated as well as in polyacrylamide gels, cytochrome c was still in front of the cathode after the longest focusing time of 18 hrs.

Discussion The instability of the pH gradient with time in isoelectric focusing (8) seems to have been resolved for focusing in polyacrylamide gel rods, in which a steady-state pH gradient within a narrow pH range (pH 6 - 8 )

could

be maintained for at least as long as a week by the use of appropriate electrode solutions, in particular by the proper choice of the anolyte pH (12,13). Using horizontal thin-layer isoelectric focusing, however, this approach does not seem to be as successful. With neither granulated gels, nor polyacrylamide gel slabs nor ultrathin-layers was a reduction of the pH drift observed when the strongly acidic and basic electrolytes were substituted with amino acids isoelectric at the terminal values of the pH gradient. For example, using Sephadex G-75 Superfine containing broad pH range carrier ampholytes, Ampholine pH 3.5 - 10 or Servalyt pH 2 - 11 or Pharmalyte pH 3 - 10, already after about 6 hours of focusing the most basic test protein, cytochrome c, had migrated into the catholyte. In the

171 case of horseradish peroxidase, however, at least 18 hours of focusing at our conditions were required for the predominant basic isoenzyme to attain the steady-state. But not only a cathodic drift was observed; the protein with the lowest isoelectric point in our test mixture, amyloglucosidase, migrated to the anode. This bidirectional drift indicates that electroendosmosis is one of the mechanisms in pH gradient instability. Variation of the granulated gels also showed that electroendosmosis is involved in pH drift, but the effects were rather small. A noticeable exception was the use of Sephadex IEF containing Pharmalyte which showed a greatly reduced cathodic drift. Another remedy to reduce the cathodic drift is focusing in a C0 2 -free atmosphere. A further advantage of CC^ exclusion is the more correct estimation of the isoelectric point in the alkaline region. With all types of carrier ampholytes and in granulated gels as well as in polyacrylamide gels the addition of separators to the gel, e.g. basic amino acids to delay the cathodic drift, seems to be a panacea for the pH drift. By this means, the entrance of proteins with high isoelectric points into the catholyte could be delayed, thus enabling the measurement of isoelectric points after prolonged focusing.

References 1. Delincèe, H., Radola, B.J.: Anal. Biochem. 90, 609-623 (1978). 2. Chrambach, A., Doerr, P., Finlayson, G.R., Miles, L.E.M., Sherins, R., Rodbard, D.: Ann. N.Y. Acad. Sci. 209, 44-64 (1973). 3. Haglund, H. in Isoelectric Focusing (Arbuthnott, J.P. and Beeley, J.A., eds.) Butterworths, London, pp. 3-22 (1975). 4. Davies, H. in Isoelectric Focusing (Arbuthnott, J.P. and Beeley, J.A., eds.) Butterworths, London, pp. 97-113 (1975). 5. Baumann, G., Chrambach, A. in Progress in Isoelectric Focusing and Isotachophoresis (Righetti, P.G., ed.) North-Holland, Amsterdam, pp. 1323 (1975). 6. Fawcett, J.S. in Progress in Isoelectric Focusing and Isotachophoresis (Righetti, P.G., ed.) North-Holland, Amsterdam, pp. 25-37 (1975). 7. Fawcett, J.S. in Electrofocusing and Isotachophoresis (Radola, B.J. and Graesslin, D., eds.) de Gruyter, Berlin, pp. 59-69 (1977). 8. Rilbe, H. in Electrofocusing and Isotachophoresis (Radola, B.J. and Graesslin, D., eds.) de Gruyter, Berlin, pp. 35-50 (1977).

172 9. Gianazza, E., Astorri, C., Righetti, P.G.: J. Chromatogr. 171, 161-169 (1979). 10. Nguyen, N.Y., Chrambach, A.: Anal. Biochem. 79, 462-469 (1977). 11. Pollack, S.: Biochem. Biophys. Res. Commun. 87, 1252-1255 (1979). 12. Nguyen, N.Y., Chrambach, A.: Anal. Biochem. 82, 54-62 (1977). 13. Nguyen, N.Y., Chrambach, A.: Anal. Biochem. 82, 226-235 (1977). 14. Delincee, H., Radola, B.J.: Eur. J. Biochem. 52, 321-330 (1975). 15. Radola, B.J.: Biochim. Biophys. Acta 295, 412-428 (1973). 16. Ampholine PAGplate, Instructions, LKB (1974). 17. Görg, A., Postel, W., Westermeier, R.: Anal. Biochem. 89, 60-70 (1978). 18. Radola, B.J., de la Vigne, U.: Desaga Report, pp. 16-18 (1979). 19. Pharmalyte, Instructions, Pharmacia (1979). 20. Caspers, M.L., Posey, Y., Brown, R.K.: Anal. Biochem. 79, 166-180 (1977). —

QUANTITATIVE IMMUNOELECTROPHORESIS. A S U R V E Y A N D SOME A P P L I C A T I O N S .

T.C.B0g-Hansen,

Irena

Lorenc-Kubis

The P r o t e i n L a b o r a t o r y , U n i v e r s i t y 34, DK 2200 C o p e n h a g e n N , D E N M A R K ") P e r m a n e n t a d r e s s : D e p a r t m e n t Wroclaw, Wroclaw, POLAND

1.

and of

O.J.Bjerrum Copenhagen,

of B i o c h e m i s t r y ,

Sigurdsgade

University

of

INTRODUCTION

The p u r p o s e of this review is to p o i n t out some potentialities of new q u a n t i t a t i v e i m m u n o e l e c t r o p h o r e t i c m e t h o d s that have b e e n developed within recent years for studies of molecular properties of proteins. It is p o s s i b l e to o b t a i n v a l u a b l e information without prior purification of proteins: Electrophoretic migration velocity and microheterogeneity, i s o e l e c t r i c p o i n t , c o n c e n t r a t i o n in relative units, stability, degradation, molecular weight, lipoprotein and glycoprotein character, tissue specificity, subcellular localization, and Also information about complex-formation with other proteins. b i o l o g i c a l a c t i v i t y c a n be o b t a i n e d , e.g. e n z y m a t i c or r e c e p t o r activity. The methods are also a means for analyzing f r a c t i o n a t i o n p r o c e d u r e s in such d e t a i l e d ways which was not possible earlier. Three manuals have been published recently giving all the technical d e t a i l s of the p r e s e n t s t a t e of the art [4,6,59]. An u p - t o - d a t e a c c o u n t is being p r e p a r e d at this moment [5]. The reader is referred to these m a n u a l s for the t e c h n i c a l d e t a i l s and m a n y applications of the methods. The literature was covered extensively up to 1975 in a review by V e r b r u g g e n [7], and A x e l s e n [3] and O w e n and Smyth discussed extensively the a p p l i c a t i o n s of the m e t h o d s for m i c r o b i o l o g i c a l p u r p o s e s [66] as Bjerrum and coworkers did for membrane proteins [9,11]. Lowenstein has covered the field of a l l e r g e n r e s e a r c h [60]. Reviews of application of general immunological methods (immunodiffusion and immunoelectrophoretic analysis ad m o d u m G r a b a r [43]) o n v a r i o u s s u b j e c t s have a p p e a r e d by G r a b a r [42], by Uriel (on e n z y m e s ) [77-80], by D a u s s a n t (on p l a n t p r o t e i n s ) [37]. A s h o r t i n t r o d u c t o r y review appeared in G e r m a n in an e a r l i e r v o l u m e of this s e r i e s of p r o c e e d i n g s [21]. 2. The

GENERAL REMARKS ABOUT QUANTITATIVE quantitative

immunoelectrophoresis

1980 Walter de Gruyter & Co., Berlin • New York Electrophoresis '79

IMMUNOELECTROPHORESIS is

based

upon

17'i electrophoretic m i g r a t i o n of p r o t e i n s (antigens) in a g a r o s e gel containing antibodies. In this respect there is a great contrast to the w i d e l y used t r a d i t i o n a l i m m u n o e l e c t r o p h o r e t i c analysis ad modum Grabar [43] which is essentially electrophoresis f o l l o w e d by a step of i m m u n o d i f f u s i o n . Because of this d i f f e r e n c e there a p p e a r s to be four main features of quantitative Immunoelectrophoresis: 1. The r e s o l u t i o n is very good and based upon specific recognition of individual p r o t e i n s by their c o r r e s p o n d i n g antibodies allowing detailled analysis of individual p r o t e i n s in crude p r o t e i n e x t r a c t s or m i x t u r e s . 2. The biological activity is retained after immunoprecipitation even in the fixed position in the m a c r o m o l e c u l a r n e t w o r k in the supporting medium. This allows f u r t h e r c h a r a c t e r i z a t i o n of the p r o t e i n s . 3. The a r e a (or r o u g h l y the h e i g h t ) of the i m m u n o p r e c i p i t a te is proportional to the a m o u n t of a n t i g e n a p p l i e d a n d inversely proportional to the antibody concentration a l l o w i n g q u a n t i f i c a t i o n of the p r o t e i n s . 4. The m e d i u m is a g a r o s e gel the large p o r e s of w h i c h a l l o w analysis of heterogeneous large protein c o m p l e x e s (e.g. membrane proteins). M o r e o v e r , a g a r o s e gel is e a s y to h a n d l e a l l o w i n g d e s i g h of c o m p o s i t e g e l s . In q u a n t i t a t i v e I m m u n o e l e c t r o p h o r e s i s the c o n d i t i o n s are chosen so that in a v e r a g e the a n t i b o d y m o l e c u l e s w i l l not m o v e in the gel w h e r e a s p r o t e i n s w i t h o t h e r e l e c t r o p h o r e t i c p r o p e r t i e s will migrate during the electrophoresis. Under such c o n d i t i o n s , p r o t e i n s and t h e i r c o r r e s p o n d i n g antibodies interact to form complexes which increase in s i z e d u r i n g the e l e c t r o p h o r e s i s . F i n a l l y the i n t e r a c t i o n b e t w e e n p r o t e i n m o l e c u l e s and antibody molecules result in f o r m a t i o n of a large m o l e c u l a r n e t w o r k , a p r e c i p i t a t e t h a t is i m m o v a b l e by the e l e c t r i c f i e l d in the gel. T h e a r e a e n c l o s e d by a p r e c i p i t a t e is d i r e c t l y proportional to the amount of protein and inversely proportional to the corresponding antibody concentration in the gel. Therefore m i g r a t i o n in a gel c o n t a i n i n g the c o r r e s p o n d i n g a n t i b o d i e s o p e n s p o s s i b i l i t i e s for q u a n t i f i c a t i o n of p r o t e i n s . T h e m o s t r e m a r k a b l e f a c e t is that a s i n g l e i n d i v i d u a l p r o t e i n in a complex mixture with other proteins may be measured i n d i v i d u a l l y , for i n s t a n c e in o n e - d i m e n s i o n a l rocket Immunoelectrophoresis with monospecific a n t i b o d i e s or in t w o - d i m e n sional crossed Immunoelectrophoresis with oligospecific antibodies. In c r o s s e d I m m u n o e l e c t r o p h o r e s i s each p r o t e i n h a s its characteristic position, shape of precipitate and precipitate m o r p h o l o g y and in such a p a t t e r n the q u a n t i f i c a t i o n c a n be p e r f o r m e d by use of p l a n i m e t r y of the the a r e a b e l o w the individual precipitates.

175 A n o t h e r n o t a b l e f e a t u r e of c r o s s e d I m m u n o e l e c t r o p h o r e s i s is that proteins can be c o m p a r e d w i t h r e s p e c t to their i m m u n o c h e m i c a l i d e n t i t y or their p a r t i a l i d e n t i t y . However, certain conditions must be met as l i s t e d in the f u n d a m e n t a l laws of q u a n t i t a t i v e i m m u n o e l e c t r o p h o r e s i s [11] : F I R S T LAW: A n t i g e n and a n t i b o d y m u s t veloci ty.

have

different

migration

S E C O N D LAW: For quantification, the standard and the test antigen must h a v e the same p h y s i c o - c h e m i c a l and i m m u n o c h e m i c a l structures and must exist in the same environment. The immunoprecipitation must take place at a uniform antibody concentration. T H I R D LAW: I d e n t i t y r e a c t i o n s require "unity" in s p a c e and of the i n v o l v e d a n t i g e n - a n t i b o d y r e a c t i o n s .

time

In c o m p a r i s o n w i t h o t h e r t y p e s of e l e c t r o p h o r e s i s , quantitative advantages and some d i s a d v a n immunoelectrophoresis has some tages. Compared with the traditional immunoelectrophoretic analysis ad m o d u m G r a b a r [43] as it is g e n e r a l l y used, c r o s s e d i m m u n o e l e c t r o p h o r e s i s has b e t t e r r e s o l u t i o n and is more rapid. In m a n y cases r e s u l t s w i t h these m e t h o d s may be o b t a i n e d in a few h o u r s or o v e r n i g h t . S o m e m e a s u r e m e n t s of p r o t e i n s by r o c k e t immunoelectrophoresis only take 3-4 hrs. None of the q u a n t i t a t i v e m o d i f i c a t i o n s r e q u i r e the t i m e - c o n s u m i n g diffusion step. It is also possible to p e r f o r m w a s h i n g and s t a i n i n g w i t h i n an h o u r b e c a u s e the gel is p r e s s e d to remove liquid and s o l u b l e p r o t e i n s [6]. In addition to this, the basic methods (rocket immunoelectrophoresis, crossed immunoelectrophoresis, and line i m m u n o e l e c t r o p h o r e s i s ) h a v e now b e e n f u r t h e r d e v e l o p e d so t h a t it is now p o s s i b l e to c h a r a c t e r i z e p r o t e i n s w i t h r e s p e c t to t h e i r molecular parameters without prior purification. 3.

RABBIT

ANTIBODIES

A n a b s o l u t e r e q u i r e m e n t for the use of these methods, is the a v a i l a b i l i t y of a n t i b o d i e s a g a i n s t the p r o t e i n or p r o t e i n s w h i c h are g o i n g to be s t u d i e d by t h e s e m e t h o d s . A n i m p o r t a n t p o i n t to notice is that the k i n d of i m m u n o c h e m i c a l work d i s c u s s e d h e r e c o u l d be d i v i d e d into two s e p a r a t e p a r t s that m a y be r e g a r d e d as totally separate problems: 1. How to obtain the desired a n t i b o d i e s , and 2. H o w to use the antibodies for analysis. This p a p e r m a i n l y d e a l s w i t h the s e c o n d a s p e c t . H o w e v e r , a few p o i n t s on a n t i b o d y - p r o d u c t i o n w i l l be m a d e h e r e . 3.1 I m m u n i z a t i o n of

rabbits.

U s u a l l y r a b b i t s are chosen as the experimental animals production of antibodies. Generally rabbits provide

for good

176 a n t i b o d i e s and are v e r y easy to keep. W e find it e s s e n t i a l to use m u l t i v a l e n t a n t i b o d i e s in the start of the studies of an u n k n o w n protein system. Thus the a n t i b o d i e s u s e d in m o s t of our s t u d i e s are o b t a i n e d f r o m r a b b i t s immunized over a long p e r i o d - over more t h a n a y e a r . W e like to k e e p the a n i m a l s for a long time, since the antibody titer (the concentration of specific i m m u n o g l o b u l i n s in the blood) i n c r e a s e s d u r i n g the c o u r s e of i m m u n i z a t i o n . W h e n w e s t a r t the p r o d u c t i o n of a n t i b o d i e s a g a i n s t a new c o m p l e x protein s a m p l e w e p e r f e r to use s e v e r a l r a b b i t s (at least 3) in o r d e r to e l i m i n a t e i n d i v i d u a l d i f f e r e n c e s in the antibody response. For an ordinary crossed immunoelectrophoretic experim e n t is u s u a l l y r e q u i r e d b e t w e e n 0.3 and 3 ml of a n t i s e r u m , and we recommend that a large pool of a n t i s e r u m (>0.5 litre) is c o l l e c t e d b e f o r e the a n i m a l s are k i l l e d if it is n e c e s s a r y . In the f i r s t approach, crude protein extracts or partially fractionated p r o t e i n s may be u s e d as i m m u n o g e n in d o s e s b e t w e e n 50 and 500 ug per i n j e c t i o n . W i t h pure i m m u n o g e n s as little as 25 ug protein per kg b o d y w e i g h t m a y be a d m i n i s t e r e d per i n jection. T h e a n i m a l s m a y be u s e d w h e n they are three months old. T h e y are i n j e c t e d s u b c u t a n e o u s l y and b l e e d r e g u l a r l y every 2-4 w e e k s a c c o r d i n g to the s c h e m e of H a r b o e and I n g i l d [45]. W h e n the f i r s t p o o l of a n t i b o d i e s is ready for u s e , we use this for comparison with the f o l l o w i n g b l e e d i n g s , and w e e s t a b l i s h the p r o t e i n p a t t e r n . For our e x p e r i m e n t s w e choose a protein extract that gives a g o o d r e a c t i o n w i t h the a n t i b o d i e s . The p r o t e i n s a p p e a r r e p r o d u c i b l y in the p a t t e r n and in this way we establish a r e f e r e n c e s y s t e m or r e f e r e n c e p a t t e r n for our p r o t e i n s , w h e r e each p r o t e i n is c h a r a c t e r i z e d by its s p e c i f i c l o c a tion and precipitation peculiarities (see b e l o w ) . N o w it is easy - by c o m p a r i s o n - to s t u d y the i n d i v i d u a l proteins, their distribution and occurrence etc. as w e l l as new antibody preparations. Normally the rabbits produce a well-balanced antibody in response to the antigen mixture. If this is not the case a w e l l - b a l a n c e d p a t t e r n m a y be o b t a i n e d by mixing antisera from d i f f e r e n t r a b b i t s i m m u n i z e d w i t h v a r i o u s p r o t e i n f r a c t i o n s [16]. In c o n t r a s t to c r o s s e d I m m u n o e l e c t r o p h o r e s i s where we usually use multivalent antibodies w e use m o n o s p e c i f i c a n t i b o d i e s for rocket Immunoelectrophoresis. T h e p r o d u c t i o n of such antibody preparations raises special questions. A n i n t e r e s t i n g use of the q u a n t i t a t i v e I m m u n o e l e c t r o p h o r e s i s m e t h o d s is the preparative approach to obtain a p r o t e i n for p r o d u c t i o n of s p e c i f i c a n t i b o d i e s by c u t t i n g o u t the s e p a r a t e d and immunoprecipitated protein directly from the a g a r o s e gel. The agarose-enclosed p r e c i p i t a t e is u s e d d i r e c t l y as i m m u n o g e n for i m m u n i z a t i o n (see for instance [51]. However, in some cases w h e r e w e h a v e a specific staining procedure for a protein we can use

177 multispecific antibodies, for instance histochemical reaction will stain only p r e c i p i t a t e [32]. 3.2 P u r i f i c a t i o n of rabbit

for e n z y m e s w h e r e the the specific enzyme

immunoglobulins.

Fractionation of the antiserum in o r d e r to obtain the immunoglobulin fraction is u s u a l l y r e q u i r e d , since the s u r p l u s of s e r u m p r o t e i n s in u n f r a c t i o n a t e d a n t i s e r u m o f t e n gives rise to an undesirable h e a v y b a c k g r o u n d s t a i n i n g of the g e l s w h i c h m a k e s it d i f f i c u l t to d e t e c t the m i n o r p r o t e i n p r e c i p i t a t e s . T h e i m m u n o g l o b u l i n f r a c t i o n of the a n t i s e r u m m a y be i s o l a t e d by precipitation with 1.75 M ammonium sulfate with subsequent removal of l i p o p r o t e i n s by dialysis at pH 5.0 followed by ion-exchange chromatography with QAE-sephadex at pH 5.0 as described by Harboe and Ingild [45]. This fractionation involves that the a n t i b o d i e s are m u c h more c o n c e n t r a t e d in the final p r e p a r a t i o n t h a n in the s e r u m . T h e r e f o r e a larger amount of a n t i b o d i e s can be a p p l i e d to an e x p e r i m e n t . 3.3 S t a b i l i t y of

antibodies.

The p u r i f i e d r a b b i t a n t i b o d i e s are h i g h l y stable at 4 d e g r e e s C. T h e loss in a n t i b o d y a c t i v i t y is less t h a n 2% per y e a r [45]. To a v o i d b a c t e r i a l c o n t a m i n a t i o n 15 mM s o d i u m azide m a y be a d d e d . 3.4 Some l i m i t a t i o n s of the

method.

T h e d i s a d v a n t a g e s and l i m i t a t i o n s of the m e t h o d s are connected with the antibodies. U s i n g t h e s e m e t h o d s w e c a n only i n v e s t i g a t e the p r o t e i n s t o w a r d s w h i c h w e have a n t i b o d i e s , and w e must realize that the a n t i b o d i e s are b i o l o g i c a l p r o d u c t s w i t h g r e a t variation. 4 SOME T E C H N I C A L

DETAILS

The medium used for quantitative Immunoelectrophoresis is agarose since it allows a n t i b o d i e s and p r o t e i n s to be m e l t e d into the g e l . U s u a l l y a g a r o s e is p r e p a r e d as a 1 % s o l u t i o n in TRIS-veronal buffer pH 8.6 by h e a t i n g . Due to the v a r i a b i l i t y in the e l e c t r o e n d o s m o t i c p r o p e r t i e s of v a r i o u s agarose batches it is r e c o m m e n d e d t h a t m i g r a t i o n of the a n t i b o d i e s is c h e c k e d in a pilot experiment. The consumption of antibodies in the r e d u c e d if the a n t i b o d i e s have a immunoelectrophoresis can be slightly cathodic migration. The gel - with or without antibodies or proteins is cast on g l a s s p l a s t s on a level table. T h e e l e c t r o p h o r e s i s is p e r f o r m e d in a L a u r e l l apparatus with a c o o l i n g s u r f a c e , and c o n n e c t i o n to the e l e c t r o d e v e s s e l s is s e c u r e d by 5-8 l a y e r s of f i l t e r p a p e r . The c u r r e n t is u s u a l l y between 2 and 15 V o l t / c m . T h e g e l s m a y be h a n d l e d c o n v e n i e n t l y w i t h s c a l p e l s and long razor b l a d e s . H o l e s are p u n c h e d w i t h an automatic suction device of W e e k e and T h o m s e n [82], a h o l e

178 p u n c h , and s a m p l e s are c o n v e n i e n t l y a p p l i e d w i t h g r e a t accuracy with a double constriction pipet. A f t e r e l e c t r o p h o r e s i s , the g e l s are p r e s s e d u n d e r f i l t e r p a p e r for 15 m i n , s o a k e d in water for 30 m i n , t h e n p r e s s e d for a n o t h e r 15 m i n , d r y e d in a s t r e a m of hot or c o l d air (from a h a i r d r y e r ) and s t a i n e d . The technical d e t a i l s are d e s c r i b e d c a r e f u l l y by A x e l s e n et al. [6]. 5 THE METHODS In t h i s p a r t w e w i l l d e s c r i b e some of the m o s t t i o n s of q u a n t i t a t i v e i m m u n o e l e c t r o p h o r e s i s . 5.1 R O C K E T I M M U N O E L E C T R O P H O R E S I S

used

modifica-

[58]

R o c k e t i m m u n o e l e c t r o p h r o e s i s is a m e t h o d in w h i c h proteins are e l e c t r o p h o r e s e d in one d i m e n s i o n in gel c o n t a i n i n g m o n o - s p e c i f i c antibodies. In the e l e c t r i c f i e l d the p r o t e i n s m i g r a t e through the gel until the reaction w i t h the a n t i b o d i e s r e s u l t s in a large immobile molecular network, a precipitate without mobility. If a mixture of proteins is a n a l y z e d , only the s p e c i f i c p r o t e i n is p r e c i p i t a t e d w i t h its specific antibodies. Unrelated proteins will pass nearly unhindered t h r o u g h the p r e c i p i t a t e (small a m o u n t s of u n r e l a t e d p r o t e i n may stick to the p r e c i p i t a t e , see b e l o w s e c t i o n 6.1 on e n z y m e s ) . The p r e c i p i t a t e d e v e l o p e d h e r e h a s the f o r m of a rocket. The d i s t a n c e from the a p p l i c a t i o n w e l l to the tip of the rocket is p r o p o r t i o n a l to the a m o u n t of a n t i g e n a p p l i e d . The unknown samples are compared w i t h s t a n d a r d s a m p l e s w i t h k n o w n a m o u n t s of the s p e c i f i c p r o t e i n - o f t e n d e f i n e d in a r b i t r a r y u n i t s - o b s e r v i n g the s e c o n d law of immunoelectrophoresis, see above. A s t a n d a r d curve is p l o t t e d of the h e i g h t of the r o c k e t s as a function of the standard antigen concentration and quantitation is t h e n p e r f o r m e d by interpolation. This method has been extensively used in clinical chemistry for routine q u a n t i t a t i o n of a long l i s t of individual human serum proteins, urinary proteins and cerebros p i n a l f l u i d s (for a r e v i e w , cf. V e r b r u g g e n [81]). 5.2 F U S E D R O C K E T I M M U N O E L E C T R O P H O R E S I S

[74]

T h i s m e t h o d is d e v e l o p e d for c a r e f u l m o n i t o r i n g of f r a c t i o n a t i o n experiments. The method is a modification of the rocket i m m u n o e l e c t r o p h o r e s i s and c o m p r i s e s a d i f f u s i o n step prior to electrophoresis. T w o g e l s are u s e d : one w i t h o u t a n t i b o d i e s and one w i t h a n t i b o d i e s . In the a n t i b o d y - f r e e g e l , a row of holes are m a d e for s m a l l a l i q u o t s of e a c h f r a c t i o n and a f t e r a b o u t one h o u r of d i f f u s i o n , the e l e c t r o p h o r e s i s is p e r f o r m e d into the antibody-containing gel. As a result of the d i f f u s i o n a n d electrophoresis each protein will appear as a continuous precipitation line instead of separated rockets. T h u s the i m m u n o c h e m i c a l e l u t i o n p r o f i l e of each individual protein is provided. T h e f u s e d r o c k e t i m m u n o e l e c t r o p h o r e s i s w i l l show the fractions in w h i c h a protein is eluted and the relative

179 (quantitative) distribution of the p r o t e i n s in the f r a c t i o n s , A l s o the distribution of the other proteins including the c o n t a m i n a t i n g p r o t e i n s is s h o w n . T h e f u s e d r o c k e t I m m u n o e l e c t r o p h o r e s i s is u s e d in a d d i t i o n to UV absorption and activity measurements to obtain maximal i n f o r m a t i o n a b o u t the p r o t e i n s o b t a i n e d in the steps of the fractionation. T h i s m e t h o d is u s e f u l w h e n w o r k i n g w i t h m i x t u r e s of proteins - especially when working with multicomponent systems like b i o l o g i c a l f l u i d s or t i s s u e e x t r a c t s . The m e t h o d a l l o w s s p e c i f i c a n a l y s i s for i n d i v i d u a l p r o t e i n s i r r e s p e c t i v e of other proteins and disregarding the p r e s e n c e of i n t e r f e r i n g UV-absorbing components like nucleotides or nucleic acids, detergents or A m p h o l i n e s . In one set of e x p e r i m e n t s w e u s e d fused rocket immunoelectrophoresis after filtration of irradiated p r o t e i n s a m p l e s in o r d e r to d e t e c t size c h a n g e s , and w e w e r e able to s h o w that p r o t e i n s a g g r e g a t e w i t h c o v a l e n t b o n d s after irradiation with mega-rad doses [J.Maffei and T.C. B 0 g - H a n s e n , in p r e p a r a t i o n ] . 5.3 C R O S S E D

IMMUNOELECTROPHORESIS

[34,57]

C r o s s e d i m m u n o e l e c t r o p h o r e s i s is a two-dimensional method in which a mixture of antigens is s e p a r a t e d by simple zone e l e c t r o p h o r e s i s f o l l o w e d by the s e c o n d d i m e n s i o n e l e c t r o p h o r e s i s into a n t i b o d y - c o n t a i n i n g g e l . It is a m e t h o d by w h i c h each p r o t e i n is i d e n t i f i e d in the p a t t e r n by its s p e c i f i c location and p r e c i p i t a t e p e c u l a r i t i e s as s t a i n i n g i n t e n s i t y , size and f o r m of p r e c i p i t a t e and p r e c i p i t a t e m o r p h o l o g y . Crossed immunoelectrophoresis is w e l l s u i t e d as an i n v e n t o r y of p r o t e i n s in m i x t u r e s containing many proteins. Thus Axelsen could demonstrate about 80 d i s t i n c t a n t i g e n s in C a n d i d a a l b i c a n s u s i n g s t a n d a r d i z e d e x t r a c t i o n p r o c e d u r e s and s t a n d a r d i z e d a n t i b o d i e s [3]. There are two slightly different ways to perform crossed immunoelectrophoresis: A semiquantitative method (crossed i m m u n o e l e c t r o p h r o e s i s ad m o d u m L a u r e l l [57]) and a quantitative method (crossed immunoelectrophroesis ad modum Clarke and F r e e m a n [34]). T h e f o r m e r m e t h o d is u s e d by Laurell and his c o w o r k e r s for s t u d i e s of p r o t e i n c o m p l e x e s , p r o t e i n d e g r a d a t i o n , and d e m o n s t r a t i o n of a b n o r m a l p r o t e i n s [59]. W e p r e f e r to use c r o s s e d i m m u n o e l e c t r o p h o r e s i s ad modum Clarke a n d F r e e m a n b e c a u s e it is q u a n t i t a t i v e and v e r y easy to p e r f o r m . E a c h i n d i v i d u a l p r o t e i n r e a c t s w i t h its s p e c i f i c antibodies to form a characteristic precipitate. W h e n this m e t h o d is u s e d for q u a n t i t a t i v e p u r p o s e s the area e n c l o s e d by each precipitate is m e a s u r e d and e x p r e s s e d r e l a t i v e l y to the area of the p r o t e i n s in the s t a n d a r d . For analysis of molecular interactions it is possible to incorporate ligand in the f i r s t d i m e n s i o n g e l . C h a n g e s in the precipitation pattern will indicate specific interaction (see section 6.2 below on crossed hydrophobic interaction immunoelectrophoresis). We have described lectin interaction

l8o Immunoelectrophoresis w h e r e l e c t i n is i n c o r p o r a t e d in the f i r s t dimension gel as a very sensitive way to identify and with r e s p e c t to e.g. microheterocharacterize glycoproteins g e n e o u s forms (see the accompanying paper [28]). Presumably this is the most sensitive a n a l y t i c a l m e t h o d for s t u d i e s of specifically interacting macromolecular systems. 5.4 T A N D E M C R O S S E D

IMMUNOELECTROPHORESIS

[53]

T a n d e m c r o s s e d I m m u n o e l e c t r o p h o r e s i s can be u s e d to study the relation of two p r o t e i n m i x t u r e s , w h e t h e r they c o n t a i n the same p r o t e i n s or d i f f e r e n t p r o t e i n s . A l s o the relative c o n c e n t r a t i o n of proteins can be studied. The two p r o t e i n s a m p l e s are run t o g e t h e r in the f i r s t d i m e n s i o n e l e c t r o p h o r e s i s from two wells s p a c e d some mm a p a r t . I d e n t i c a l p r o t e i n s w i l l a p p e a r as d o u b b l e p e a k s in the p a t t e r n , the relative h i g h t of the p e a k s r e f l e c t i n g the relative a m o u n t s of i n d i v i d u a l p r o t e i n s in the s a m p l e s . 5.5 L I N E I M M U N O E L E C T R O P H O R E S I S

[54]

T h i s m e t h o d is similar to rocket immunoelectrophoresis. It consists of only one e l e c t r o p h o r e s i s into a n t i b o d y - c o n t a i n i n g g e l , but the a n t i g e n s a m p l e s are m e l t e d into gel s l a b s next to each other and next to the antibody-containing gel. In m u l t i c o m p o n e n t s y s t e m s the result after electrophoresis is a spectrum of precipitin bands showing identity and partial i d e n t i t y b e t w e e n the i n d i v i d u a l c o m p o n e n t s of the s a m p l e s (as in tandem crossed immunoelectrophoresis). Quantitative information c a n be o b t a i n e d in the same way as for the other methods, b e c a u s e the p o s i t i o n of the p r e c i p i t a t e is d i r e c t l y p r o p o r t i o n a l to the a m o u n t of p r o t e i n a n a l y z e d . It is also p o s s i b l e to c o m p a r e with this m e t h o d . By p l a c i n g c o n t a i n d i f f e r e n t a n t i s e r a and it is p o s s i b l e to compare s p e c i f i c a n t i b o d i e s in the two 5.6 C R O S S E D

different antibody preparations b e s i d e s each o t h e r two g e l s w h i c h a n a l y s i n g o n l y one a n t i g e n s a m p l e the c o n t e n t and c o n c e n t r a t i o n of a n t i s e r a [54].

IMMUNOELECTROPHORESIS WITH INTERMEDIATE GEL

[75]

T h e i n t e r m e d i a t e gel m e t h o d is a m o d i f i c a t i o n of the crossed immunoelectrophoresis. The intermediate gel is interposed b e t w e e n the f i r s t d i m e n s i o n and the s e c o n d d i m e n s i o n gel and m a y contain antiserum or proteins. When protein samples are i n c l u d e d in the intermediate gel, the method is sometimes r e f e r r e d to as c r o s s e d line i m m u n o e l e c t r o p h o r e s i s . If the i n t e r m e d i a t e gel c o n t a i n s a s e r u m w i t h a n t i b o d i e s to some of the a n t i g e n s a p p l i e d in the w e l l it is p o s s i b l e to i d e n t i f y and q u a n t i f y these a n t i b o d i e s in terms of the s p e c i f i c i t i e s and titres of the reference antiserum. In this w a y S v e n d s e n and A x e l s e n i d e n t i f i e d and q u a n t i f i e d a n t i b o d i e s in p a t i e n t serum a g a i n s t m i c r o b i a l i n f e c t i o n s w i t h C a n d i d a a l b i c a n s [75].

l8i W e use the i n t e r m e d i a t e gel technique for identification of s p e c i f i c a n t i g e n s by s p e c i f i c a n t i b o d i e s in the i n t e r m e d i a t e gel a n d as line c r o s s e d i m m u n o e l e c t r o p h o r e s i s for t e s t of absorbed a n t i b o d i e s as w e l l as for s p e c i f i c i d e n t i f i c a t i o n of a n t i g e n s as an a l t e r n a t i v e to t a n d e m crossed immunoelectrophoresis. Also macromolecular c o m p l e x e s m a y be s t u d i e d by the i n t e r m e d i a t e gel technique. We have used this method in combination with affinity electrophoresis for i d e n t i f i c a t i o n of m o l e c u l e s w i t h specific molecular characteristics (for instance binding to lectins), for prediction of f r a c t i o n a t i o n e x p e r i m e n t s [26,27, 29], see also the a c c o m p a n y i n g p a p e r [28], and for d e t e c t i o n of circulating complexes between bacterial lipopolysaccharides (endotoxin) and l i p o p r o t e i n s [28a]. 6 SOME B I O C H E M I C A L 6.1

APPLICATIONS

ENZYMES

M a n y b i o l o g i c a l l y i m p o r t a n t p r o t e i n s as e n z y m e s are present in so s m a l l q u a n t i t i e s that they are not s t a i n a b l e w i t h the u s u a l protein stain. B u t this d o e s not e x c l u d e such components from study by m e a n s of q u a n t i t a t i v e i m m u n o e l e c t r o p h o r e s i s , as one of the o u t s t a n d i n g a d v a n t a g e s of these m e t h o d s is the r e t e n t i o n of the biological activity after immunoprecipitation. The o n l y r e q u i r e m e n t s are t h a t there are p r e c i p i t a t i n g a n t i b o d i e s in the antibody p r e p a r a t i o n and t h a t the e n z y m e can be b r o u g h t to f o r m a precipitate. We found for enzymes that the precipitating limit is around 100 pg, see Fig. 1. H o w e v e r , this d o e s not a l l w a y s m e a n that a r e g u l a r , q u a n t i f i a b l e p r e c i p i t a t e is formed [68] . The high sensitivity shows that there is a great p o t e n t i a l for e n z y m e a n a l y s i s by q u a n t i t a t i v e immunoelectrophoresis. N o r m a l l y e n z y m e s are a c t i v e after precipitation, which means that h i s t o c h e m i c a l c o l o u r r e a c t i o n s may be u s e d , e i t h e r d i r e c t l y or s l i g h t l y m o d i f i e d , for s p e c i f i c s t a i n i n g of immunoprecipitated enzyme. In F i g . 1 is s h o w n the c h o l i n e s t e r a s e in n o r m a l

1 jfi oOoC? a. D

6l

C-

F I G U R E 1. Rocket immunoelectrophoresis of s e r u m c h o l i n e s t e r a s e . a: 100 pg c h o l i n e s t e r a s e . b: 500 pg c h o l i n e s t e r a s e . c: 5000 pg c h o l i n e s t e r a s e . The e l e c t r o p h o r e s i s was performed with diluted normal human serum (100 pcj cholinesterase represents about 3-10 molecules). Antibodies were specific anticholinesterase (immunoglobulin fraction 0.01 pl/cm2). E l e c t r o p h o r e s i s for 18 h at 2 V / c m . Stained histochemically w i t h n a p h t h y l a c e t a t e and F a s t R e d T R salt [31] .

182 human serum stained with the histochemical method for esterases [32], Therefore enzymes can be defined and characterized with respect to substrate specificity, inhibitors and activators, in addition to other parameters in quantitative Immunoelectrophoresis - even individual isoenzymes can be identified and quantified specifically (see below). Many enzymes have been studied by quantitative immunoelectrophoresis as summarized in [32,66]. It appears that co-precipitation of mixed immunocomplexes (coprecipitation and entrapment) and dye-binding are problems in identification and quantitation of enzymes by immunoelectrophoretic methods. Other problems may occur when a new enzyme system is studied. Often failure to detect enzyme precipitates may be due to absence of antibodies or to inhibition of the enzyme by the antibodies. The inhibition may be total or partial, but also activation may be expected, since antibody-binding is known to lead to activation in some cases [40,79]. It is unlikely that antigenic determinants should be found with rabbit antibodies in the active site of mammalian enzymes, since the active site may be considered to be evolutionarily conservative. The presence of antigenic determinants in the neighbourhood of the active centre could result in steric inhibition even for small substrate molecules, but probably the most dominating antigenic determinants are other parts of the enzyme molecule, for statistical reasons alone. Furthermore, since precipitates in immunoelectrophoresis are formed in excess of antigen, only few antigenic determinants have reacted when the enzyme is precipitated. Generally, inhibition is rare if the substrate is small, but is to be expected if the substrate is another macromolecule. On the other hand, failure to detect an enzyme activity could have several causes unrelated to the antibody-binding as for instance denaturation, degradation, inhibition, genetically determined inactivation, or silent genes. But the lack of activity does not exclude such phenomena from study. If such invisible immunoprecipitates are expected to be present, they may be visualized and quantified indirectly by addition of known amounts of active enzyme. One frequent problem occurs when the enzyme cannot be identified as an individual component. In some experiments enzyme activity appears rather as a smear. When the plate is counterstained for proteins it appears that the enzyme activity is associated with major protein precipitates. We tend to interprete such patterns as the result of unspecific coprecipitation of enzyme with other proteins, presumably as a result of lack of specific antibodies against the enzyme molecule. It is necessary for some studies of enzymes by this approach to secure that the enzyme appears as an individual precipitate. Several tests are described and discussed in [32].

183 Isoenzymes S i m u l t a n e o u s m e a s u r e m e n t s of i s o e n z y m e s c a n n o t be o b t a i n e d with other methods, unless the i s o e n z y m e s are t o t a l l y s e p a r a t e d or c a n be i n h i b i t e d i n d i v i d u a l l y . L a n z e r o t t i and G u l l i n o [56] h a v e shown that single radial i m m u n o d i f f u s i o n (SRD, "Mancini t e c h n i q u e " ) is a r e l a t i v e l y s i m p l e y e t h i g h l y a c c u r a t e w a y for q u a n titation of e n z y m e s d u r i n g a b i o l o g i c a l p r o c e s s . Nevertheless, s i n g l e radial i m m u n o d i f f u s i o n is not w e l l s u i t e d for estimation of e n z y m e s w i t h the s a m e s u b s t r a t e s p e c i f i c i t y , i.e. i s o e n z y m e s . B e f o r e q u a n t i t a t i v e d e t e r m i n a t i o n , i s o e n z y m e s m u s t be s e p a r a t e d , and this is m o s t c o n v e n i e n t l y p e r f o r m e d as a n a l y t i c a l e l e c t r o phoresis. If only two isoenzymes are present in an enzyme e x t r a c t they m a y be m e a s u r e d i n d e p e n d e n t l y by rocket I m m u n o e l e c t r o p h o r e s i s (as D a u s s a n t and c o w o r k e r s d i d for two auxin oxidases in D a t u r a s t r a m o n i u m [38]). H o w e v e r , this r e q u i r e s t h a t o n l y two i s o e n z y m e s are p r e s e n t and that One migrates towards the c a t h o d e and the o t h e r t o w a r d s the a n o d e . S i n c e it h a s b e e n s h o w n that i m m u n o e l e c t r o p h o r e s i s c a n be p e r f o r m e d at any d e s i r e d pH w i t h i n the range of pH 4.5 to 8.6 [18] this m e t h o d c o u l d be a g e n e r a l m e t h o d for q u a n t i t a t i v e d e t e r m i n a t i o n of two isoenzymes simultaneously. H o w e v e r , this i n v o l v e s c o n t r o l l e d c a r b a m y l a t i o n of h i g h l y p u r i f i e d a n t i b o d i e s r e s u l t i n g in immobile antibodies at the pH were one i s o e n z y m e has c a t h o d i c m i g r a t i o n a n d the o t h e r i s o e n z y m e has a n o d i c m i g r a t i o n . On the other hand by crossed immunoelectrophoresis it w a s p o s s i b l e to m e a s u r e the contents of two alpha-amylases in barley seeds during germination with great ease and we c o u l d d i s t i n g u i s h b e t w e e n a c t i v a t o r c o n t r o l a n d s y n t h e s i s and d e g r a d a t i o n [31]. 6.2 M E M B R A N E

PROTEINS

I m m u n o e l e c t r o p h o r e t i c a n a l y s i s of m e m b r a n e p r o t e i n s is a field of increasing importance. This is due to the s c a r c i t y of methods available for analysis of insoluble proteins in a functionally active state. At p r e s e n t the s o l u b i l i z a t i o n of m e m b r a n e p r o t e i n s for the i n v e s t i g a t i o n of f u n c t i o n a l p r o p e r t i e s is achieved b e s t w i t h n o n - i o n i c d e t e r g e n t s , since the m e m b r a n e p r o t e i n s are t h e n m a i n t a i n e d at c o n d i t i o n s w h i c h m i m i c those in the m e m b r a n e [8,46,48]. A d d i t i o n of a s u r p l u s of d e t e r g e n t to a biological membrane results in a re-arrangement of the amphiphilic molecules present in the s y s t e m . With non-ionic d e t e r g e n t , s o l u b i l i z a t i o n of the m e m b r a n e p r i n c i p a l l y a p p e a r s to result from a replacement of lipid molecules by d e t e r g e n t molecules. The membrane lipids, as w e l l as the amphiphilic "integral" or "intrinsic" membrane proteins [70] become incorporated into the dominating, water-soluble detergent m i c e l l e s , r e s u l t i n g in m e m b r a n e d i s i n t e g r a t i o n . Protein-protein i n t e r a c t i o n s are u s u a l l y not a f f e c t e d d u r i n g this process, and proteins are s e l d o m l y d e n a t u r e d . D e t e r g e n t - b i n d i n g is c o n f i n e d to the apolar surfaces of membrane proteins; hydrophilic "peripheral" membrane proteins do not s i g n i f i c a n t l y bind s u c h m i l d d e t e r g e n t s [46,76].

184 From these considerations, it follows that the presence of non-ionic d e t e r g e n t s in a p r o t e i n s o l u t i o n n e e d s not a f f e c t the h y d r o p h i l i c m o l e c u l a r r e g i o n s of p r o t e i n s . The a n t i g e n i c s i t e s , being primarily confined to such h y d r o p h i l i c s u r f a c e s r e m a i n a v a i l a b l e for i n t e r a c t i o n w i t h s p e c i f i c a n t i b o d i e s . T h i s is the basis for performing immunochemical analyses of p r o t e i n s in d e t e r g e n t s o l u t i o n [9]. S i n c e the s o l u b i l i t y of a m p h i p h i l i c m e m b r a n e p r o t e i n s is based on the presence of detergent it is e s s e n t i a l to i n c o r p o r a t e d e t e r g e n t in the a g a r o s e gel during the immunoelectrophoretic analysis. Otherwise the procedures follow those for conventional Immunoelectrophoresis. The concentration of non-ionic detergent in the gel s h o u l d be above the c r i t i c a l m i c e l l a r c o n c e n t r a t i o n as o t h e r w i s e aggregation of "integral" membrane proteins will occur. However, incorporation of different non-ionic detergents in the gel may change the precipitation pattern [13] . The d i f f e r e n t d i s s o c i a t i v e e f f e c t of v a r i o u s d e t e r g e n t s on p r o t e i n c o m p l e x e s may be responsible for changes in the n u m b e r of p r e c i p i t a t e s , and v a r i a t i o n s in e l e c t r o p h o r e t i c m i g r a t i o n may be due to d i f f e r e n c e s in s i z e of the different detergent micelles b o u n d to the p r o t e i n s [46]. T h e b o u n d m i c e l l e also r e d u c e s the a v e r a g e c h a r g e d e n s i t y of the protein. Q u a n t i t a t i v e i m m u n o e l e c t r o p h o r s i s of p r o t e i n s in the p r e s e n c e of ionic detergents is p o s s i b l e [9,13,14,50] but is h a m p e r e d by several factors: First, the denaturation of the proteins, structure and i n h i b i t i o n of resulting in loss of antigenic antibody-binding. S e c o n d , the a d d i t i o n a l charge conferred to the p r o t e i n s , r e s u l t i n g in a s i m i l a r f i r s t d i m e n s i o n m i g r a t i o n . T h i r d , the c h a r g e c o n f e r r e d to the antibodies, resulting in e l e c t r o p h o r e t i c r e m o v a l of the a n t i b o d i e s out of the p l a t e . T h e s e u n d e s i r a b l e e f f e c t s of ionic d e t e r g e n t s can be partially counteracted by introduction of n o n - i o n i c d e t e r g e n t into the g e l s [14,35]. P r o t e i n - b o u n d ionic d e t e r g e n t is t h e n r e p l a c e d by non-ionic detergent during the Immunoelectrophoresis with concomitant antigenic renaturation. In this w a y it is possible to p e r f o r m S D S - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s for the f i r s t d i m e n s i o n and directly combine it w i t h a second dimension i m m u n o e l e c t r o p h o r e s i s [33,35]. Neutral zwitterionic detergents carry both positively and negatively charged groups but their net charge in the pH i n t e r v a l of 3 - 1 1 is essentially zero. Examples of neutral z w i t t e r i o n i c d e t e r g e n t s are: E m p i g e n BB [2] and the s u l f o b e t a i n e s e r i e s [41] . T h e s e d e t e r g e n t s do not a f f e c t the e l e c t r o p h o r e t i c m i g r a t i o n of a n t i g e n s and a n t i b o d i e s though they have d e n a t u r i n g e f f e c t s [13]. M e m b r a n e p r o t e i n s have b e e n s o l u b i l i z e d from a long list of cells and analyzed by q u a n t i t a t i v e i m m u n o e l e c t r o p h o r e s i s , see [9] for a review. Furthermore, recent applications of these

185 methods have proven that certain molecular properties of m e m b r a n e p r o t e i n s c a n now be a p p r o a c h e d . Thus membrane proteins have b e e n c h a r a c t e r i z e d w i t h r e s p e c t to c a r b o h y d r a t e [15,19,44] sialic acid [15] residual lipid [22], enzyme activities [36,64-66] and ligand i n t e r a c t i o n s [19,61]. The quantitative a s p e c t of the i m m u n o e l e c t r o p h o r e t i c m e t h o d s has b e e n a p p l i e d for determination of the distribution of membrane antigens in d i f f e r e n t s u b c e l l u l a r f r a c t i o n s [23,24], for membrane protein determination in p a t h o l o g i c a l t i s s u e s [20,67] and e v e n in h u m a n b i o p s i e s [72]. H e t e r o g e n e i t y and c r o s s - r e a c t i o n s of m e m b r a n e p r o t e i n s have b e e n investigated in n u m e r o u s s y s t e m s , e x e m p l i f i e d in the s t u d i e s of the h u m a n e r y t h r o c y t e m e m b r a n e [14,15], t h r o m b o c y t e s [44], b r u s h border [36,71,72], rat s y n a p t o s o m a l m e m b r a n e s [25], rat h e p a t o cyte m e m b r a n e s [22], the m e m b r a n e s of b a c t e r i a [ 4 9 , 5 0 , 6 4 , 6 5 , 7 3 ] , yeast [39] and v i r u s [63], S t u d i e s on the o r i e n t a t i o n of m e m -

sj

1

F I G U R E 2. A u t o r a d i o g r a p h i c a l d e m o n s t r a t i o n of T r i t o n - b i n d i n g to bovine milk fat globule membrane proteins. Crossed immunoelectrophoresis of 50 pg Triton-solubilized membrane m a t e r i a l [62] in p r e s e n c e of 0.22% (v/v) T r i t o n X - 1 0 0 and 0 . 0 2 5 % 125 I-label led T r i t o n X - 1 0 0 . A. Autoradiograph. B. The same p l a t e a f t e r s t a i n i n g w i t h C o o m a s s i e B r i l l i a n t B l u e . O n l y one of the m a j o r p r e c i p i t a t e s (no. 2) e x h i b i t s r e a d i a c t i v e label but is h e t e r o g e n e o u s with respect to detergent-binding (arrow). X i n d i c a t e s a new p r e c i p i t a t e not s e e n on the s t a i n e d plate. F i r s t and s e c o n d d i m e n s i o n e l e c t r o p h o r e s i s w e r e 60 min at 10 V / c m and 18 h at 2 V / c m , r e s p e c t i v e l y . Anti-membrane antibodies 5 pl/cm2. E x p o s u r e time for a u t o r a d i a g r a p h y : 10 d. T h e bar r e p r e s e n t s 1 cm.

186 b r a n e p r o t e i n s by m e a n s of quantitative immunoelectrophoresis have been described [65] . T h u s , t h e m e t h o d s r e p r e s e n t a l t e r n a t i v e w a y s for c o n d u c t i n g s u c h studies, as they also allow d e t e c t i o n of p r o t e i n s s p a n n i n g t h e m e m b r a n e [9]. Especially quantitative immunoelectrophoresis has proven to useful in the s t u d y of m e m b r a n e p r o t e i n s for d e m o n s t r a t i o n d e t e r g e n t - b i n d i n g as i n d i c a t i o n of a n a m p h i p h i l i c s t r u c t u r e .

be of

U p o n r e m o v a l of n o n - i o n i c d e t e r g e n t in the gel, we see for intrinsic m e m b r a n e p r o t e i n s in c r o s s e d i m m u n o e l e c t r o p h o r e s i s a n i n c r e a s e in e l e c t r o p h o r e t i c m o b i l i t y a n d a c h a n g e of p r e c i p i t a t e morphology. Therefore differences in precipitation with and without detergent may indicate detergent-binding, but by no means it is final proof. Direct demonstration of detergent-binding can be carried out by performing the i m m u n o e l e c t r o p h o r e s i s in p r e s e n c e of r a d i o a c t i v e d e t e r g e n t [12]. Precipitates containing amphiphilic proteins will appear more intensively labelled upon autoradiagraphy than the background,

Tx

TX + D O C

TX+CTAB

F I G U R E 3. C h a r g e - s h i f t c r o s s e d i m m u n o e l e c t r o p h o r e s i s of 40 pg Triton-solubi1ized bovine milk fat g l o b u l e m e m b r a n e p r o t e i n s . F i r s t d i m e n s i o n e l e c t r o p h o r e s i s w a s p e r f o r m e d in: A. 0 . 5 % (v/v) T r i t o n X - 1 0 0 , B. 0 . 5 % (v/v) T r i t o n X - 1 0 0 p l u s 0 . 2 % (w/v) d e o x y c h o l a t e , a n d C. 0 . 5 % (v/v) T r i t o n X - 1 0 0 p l u s 0 . 0 1 2 5 % c e t y l t r i m e t h y l a m m o n i um bromide. Hemoglobin migration was 20 mm. Second dimension immunoelectrophoresis was performed in gels containing 0.5% T r i t o n a n d 4 p l / c m 2 of a n t i - m e m b r a n e a n t i b o d i e s . Otherwise as for F i g . 2. U s i n g t h e m e d i a n of e a c h p r e c i p i t a t e t h e f o l l o w i n g c h a r g e - s h i f t s w e r e o b s e r v e d : P r o t e i n n o 1: + 1 0 mm and -7 mm; protein no 2: + 1 5 m m a n d - 7 m m ; p r o t e i n no 3: + 1 2 m m a n d - 8 m m ; p r o t e i n no 4: + 1 m m a n d - 1 m m .

187 due to s e l e c t i v e b i n d i n g of d e t e r g e n t . Recent applications are found in [8,11,71]. Fig. 2 shows the t e c h n i q u e a p p l i e d to bovine milk fat globule membrane proteins [62] employing 125 I-label led Triton X-100 [12]. Only one of the major p r e c i p i t a t e s (no 2) e x h i b i t s r a d i o a c t i v i t y . Charge-shift

crossed Immunoelectrophoresis

[7]

C h a r g e - s h i f t c r o s s e d I m m u n o e l e c t r o p h o r e s i s is an indirect way for identification of amphiphilic proteins [47]. Three different first dimension electrophoreses are performed in p r e s e n c e of: 1. The n o n - i o n i c d e t e r g e n t T r i t o n X - 1 0 0 , 2. T r i t o n X - 1 0 0 p l u s the a n i o n i c d e t e r g e n t d e o x y c h o l a t e , and 3. T r i t o n X - 1 0 0 plus the cationic detergent N-cety1-N,N,Ntrimethyl ammonium bromide. The second dimension gels contain Triton X-100. When two detergents are present t o g e t h e r they w i l l f o r m m i x e d m i c e l l e s and if one d e t e r g e n t is charged the mixed micelle will be a c h a r g e d m i c e l l e w i l l c h a n g e the charged. Binding of such e l e c t r o p h o r e t i c p r o p e r t i e s of a m p h i p h i l i c p r o t e i n s to r e s u l t in another position than t h a t in the r e f e r e n c e p a t t e r n w i t h o n l y Triton X-100. Some hydrophilic proteins may bind either a p o s i t i v e l y or a n e g a t i v e l y c h a r g e d d e t e r g e n t t h r o u g h ionic b o n d s and show a u n i - d i r e c t i o n a l s h i f t . The amphiphilic proteins bind both negatively and positively charged micelles and show bi-directional shifts. Fig. 3 shows the results of such analyses on solubilized proteins of bovine milk fat globule membranes. As outlined a b o v e t h r e e d e t e r g e n t s y s t e m s are shown. Migration distances are r e l a t e d to the h y d r o p h i l i c m a r k e r p r o t e i n , h e m o g l o b i n , w h i c h in all t h r e e s y s t e m s m i g r a t e a d e t e r m i n e d d i s t a n c e e.g. 20 mm. On basis of experience with a large range of a m p h i p h i l i c m e m b r a n e p r o t e i n s b i - d i r e c t i o n a l c h a r g e s h i f t s of m o r e t h a n 5 m m under these c o n d i t i o n s is i n d i c a t i v e of d e t e r g e n t - b i n d i n g [7]. T h u s p r o t e i n s nos 1, 2 and 3 show significant bi-directional shifts. Examples of recent a p p l i c a t i o n s are f o u n d in [1,8,17, 44,51,71] Crossed hydrophobic

interaction

immunoelectrophoresis

[10]

In crossed hydrophobic interaction immunoelectrophoresis membrane proteins are electrophoresed into an agarose gel c o n t a i n i n g a h y d r o p h o b i c m a t r i x (e.g. h e x y l or p h e n y l groups). In absence of detergent and at low ionic s t r e n g t h (I < 0.05) only amphiphilic molecules interact with the matrix. Watersoluble, hydrophilic molecules do not i n t e r a c t w i t h the g e l s [10] and a p p e a r in the p a t t e r n at the n o r m a l p o s i t i o n as in the reference experiment without hydrophobic matrix included. B o u n d a m p h i p h i l i c m o l e c u l e s can be l i b e r a t e d by i n c o r p o r a t i o n of n o n - i o n i c d e t e r g e n t in the s e c o n d d i m e n s i o n gel and p r e c i p i t a t e d in the antibody-containing gel. The electroendosmotic flow b r i n g s the d e t e r g e n t m o l e c u l e s into c o n t a c t w i t h the h y d r o p h o b i c

188 matrix in the first dimension gel to liberate the bound proteins [10]. Fig. 4 shows the technique applied to bovine milk fat globule membrane proteins. All designated proteins are bound to the phenyl-Sepharose matrix incorporated in the first dimension gel except for some protein material marked 4? which appear to be unretarded. Note the faster migration velocity and the aggregation of the proteins due to the omission of detergent from the first dimension gel. as a mixture of an In case the membrane protein is present amphiphilic and a cleaved hydrophilic form it is possible to differentiate and quantify the amount of the two types by crossed hydrophobic interaction immunoelectrophoresis [10,11, 17]. Other examples of recent applications are found in [44,69] . Discussion The choice of immunoelectrophoretic method for demonstration of amphiphilic properties of a membrane protein will depend on the antigen-antibody system. Each of the three methods mentioned above have their individual drawbacks. Thus with radioactive detergents only major precipitates can be identified and with charged detergents the precipitation pattern can be so drastically changed that it becomes very difficult to identify a particular precipitate [1]. Furthermore, denaturing can take

A

B

4?

FIGURE 4. Crossed hydrophobic interaction immunoelectrophoresis of 10 pg Triton-solubilized bovine milk fat globule membrane proteins. A. Phenyl-Sepharose is incorporated (33%, Pharmacia Fine Chemicals) in the first dimension gel. B. The corresponding control experiment without phenyl-Sepharose. First dimension electrophoresis was performed in detergent-free gel. Second dimension in gel with 1% (v/v) Triton X-100 and 3 p l / c m 2 of anti-membrane antibodies. Otherwise as for Fig.2. All proteins except 4? are bound to the hydrophobic matrix.

189 p l a c e in p r e s e n c e of cetyl trimethyl ammonium bromide. With hydrophobic interaction immunoelectrophoresis detergent is excluded during the interaction which may give rise to unspecific precipitation. Therefore, the demonstration of a m p h i p h i l i c p r o p e r t i e s is s a f e r if two or three of the methods are used t o g e t h e r . T h u s p r o t e i n 2 w e r e found to be a m p h i p h i l i c in all t h r e e s y s t e m s ; p r o t e i n 1 and p r o t e i n 3 in two s y s t e m s and p r o t e i n 4 m a y be in one of the s y s t e m s . 7

CONCLUSION

B a s e d u p o n the r e t e n t i o n of biological activity of proteins during quantitative Immunoelectrophoresis, we find that t h e s e m e t h o d s o f f e r g o o d p o s s i b i l i t i e s for molecular and biological description of p r o t e i n s . E s p e c i a l l y we find that i n c o r p o r a t i o n of s p e c i f i c a l l y reacting c o m p o n e n t s in the gels, e.g. in an intermediate gel or in the f i r s t d i m e n s i o n gel in c r o s s e d Imm u n o e l e c t r o p h o r e s i s , is a p o w e r f u l t e c h n i q u e for determination of m o l e c u l a r c h a r a c t e r i s t i c s of p r o t e i n s . ACKNOWLEDGEMENTS. M s . K i r s t e n N . J e n s e n , Pia J e n s e n , and Maritechnical assistance. anne Thomsen are thanked for their S u p p o r t is g r a t e f u l l y a c k n o w l e d g e d f r o m "Kong C h r i s t i a n d e n X's Fond".

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

REFERENCES: Alexander, A.G. and K e n n y , G . E . (1978). Infect.Immun. 20, 8 6 1 - 8 6 3 . Allen, J.C. and H u m p h r i e s , C. (1975). FEBS Lett. 57, 1 5 8 - 1 6 2 . Axelsen, N.H. (1973). Infect.Immun. 7, 9 4 9 - 9 6 0 . Axelsen, N.H. (1975). Quantitative Immunoelectrophoresis. N e w D e v e l o p m e n t s and A p p l i c a t i o n s . Universitetsforlaget, Oslo. Scand.J.Immunol. 4, S u p p l . 2. Axelsen, N.H. (1980). A M a n u a l of I m m u n o p r e c i p i t a t i o n in Gel. Scand.J. Immunol., Suppl. 10 (in p r e s s ) . A x e l s e n , N . H . , K r 0 l l , J. and W e e k e , B. (1973). Scand. J.Immunol. 2, S u p p l . 1. B h a k d i , S . , B h a k d i - L e h n e n , B. and B j e r r u m , O . J . (1977). B i o c h i m . B i o p h y s . A c t a 470, 3 5 - 4 4 . B h a k d i , S . , B j e r r u m , O . J . , B h a k d i - L e h n e n , B. anad Tranum-Jensen, J. (1978). J.Immunol. 121, 2 5 2 6 . Bjerrum, O.J. (1977). B i o c h i m . B i o p h y s . A c t a 472, 1 3 5 - 1 9 5 . Bjerrum, O.J. (1978). Anal. Biochem. 90, 3 3 1 - 3 4 8 . Bjerrum, O.J. (1980). Scand.J.Immunol., Suppl. 10 (in p r e s s ) . Bjerrum, O.J. and B h a k d i , S. (1977). FEBS L e t t . 81, 1 5 1 - 1 5 6 . Bjerrum, O.J. and B h a k d i , S. (1980). Scand.J.Immunol. Suppl. 10. B j e r r u m , O . J . , B h a k d i , S., B 0 g - H a n s e n , T . C . , K n u f e r m a n n , H.

190 and Wallach, D.F.H. (1975). Biochim.Biophys.Acta 406, 489-504. 15. Bjerrum, O.J. and Befg-Hansen, T.C. (1975). Biochim.Biophys.Acta 455, 66-89. 16. Bjerrum, O.J. and B0g-Hansen, T.C. (1976). In Biochemical Analysis of Membranes (Maddy, A.H., ed.) 378-425, Chapman and Hall, London. 17. Bjerrum, O.J., Helle, K. and Bock, E. (1979). Biochem.J. 181, 231-237. 18. Bjerrum, O.J., Ingild, A. and L0wenstein, H. (1974). Immunochemistry 11, 797-802. 19. Bjerrum, O.J., Ramlau, J., Bock, E. and B0g-Hansen, T.C. (1980). In S.J. Jacob and P.Cuatrecasas (eds.): Techniques for Membrane Receptor Characterization and Purification. Chapman and Hall, London. 20. Blanchet, J.P. (1976). Develop.Biol. 48, 411-420. 21. Blirup, S. (1979). In B.J.Radola (ed.): Elektrophorese Forum, Technische Universität, München, 1978, pp 15-27. 22. Blomberg, F. and Raftell, M. (1974). Eur.J.Biochem. 49, 21-30. 23. Bock, E. and Hamburger, H. (1976). Brain Res. 112, 329-335. 24. Bock, E., Jtfrgensen, O.S., Dittmann, L. and Eng, L.F. (1975). J.Neurochem. 25, 867-870. 25. Bock, E., J0rgensen, O.S. and Morris, S.J. (1974). J.Neurochem. 22, 10-13. 26. Bfsg-Hansen, T.C. (1973). Anal.Biochem. 56, 480-488. 27. Biag-Hansen, T.C. (1979). In Egly, J.M. (ed.): Proceedings of the Third International Symposium of Affinity Chromatography and Molecular Interaction, Inserm Symposia Series, Paris, in press. 28. B0g-Hansen, T.C. These proceedings. 28a. Btfg-Hansen, T.C. and Back, U. (1980). In Eaker, D. and Wadstrom, T. (eds.): Natural Toxins, Invited Papers and Workshops from the 6th International Symposium on Animal, Plant and Microbial Toxins, Uppsala, August, 1979. Pergamon Press, Oxford, in press. 29. Bjag-Hansen, T.C., Bjerrum, O.J. and Ramlau, J. (1975). Scand.J.Immunol.4, Suppl.2 141-147. 30. Bpg-Hansen, T.C. and Brogren, C.H. (1975). Scand.J.Immunol.4, Suppl. 2,135-139. 31. Bjag-Hansen, T.C. and Daussant, J. (1974). Anal.Biochem. 61, 52.2. - 527. 32. Brogren, C.H. and B«jg-Hansen, T.C. (1975) Scand.J.Immunol.4, Suppl. 2, 37-51. 33. Chua, N.H. and Blomberg, F. (1979). J.Biol.Chem. 254, 469-472. 34. Clarke, H.G.M and Freeman, T. (1968). Clin. Sei. 35, 403-413. 35. Converse, C.A. and Papermaster, D.S. (1975). Science 189, 469-472. 36. Danielsen, E.M., Sjöström, H., Noren, 0. and Dabelsteen, E. (1977). Biochim.Biophys.Acta 494, 332-342. 37. Daussant, J., Lauriere, C., Carfantan, N. and Skakoun, A.

191

38. 39.

40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65.

(1977) in Smith, H. (ed.): Regulation of Enzyme Synthesis and Activity in Higher Plants. Phytochemical Society Symposium Series No. 14. Academic Press, London. Daussant, J., Roussaux, J. and Manigault, P. (1971). FEBS Lett. 14, 245-250. Gerlach, J.H., Bjerrum, O.J., Rank, G.H. and B0g-Hansen, T.C. (1980). In H.Peeters (ed.): Protides of the Biological Fluids, Proc. XXVII Colloquium, Vol. 27, Pergamon Press, Oxford (in press). Goedde, H.W., Doenicke, A. and Altland, K. (1967). PseudoCholinesterasen. Pharmakogenetik-Biochemie-Klinik. Springer, Berlin, 1967. Goenne, P. and Ernst, R. (1978). Anal.Biochem. 87, 28-38. Grabar, P. (1975) J.Immunol.Methods 7, 305-326. Grabar, P. and Williams, C.A. (1953). Biochim.Biophys.Acta 10, 193. Hagen, I, Bjerrum, O.J., and Solum, N.O. (1979). Eur.J.Biochem. 99, 9-22. Harboe, N. and Ingild, A. (1973). Scand.J.Immunol. 2, Suppl. 1, 161-164. Helenius, A and Simons, K. (1977). Biochim.Biophys.Acta 415, 26-29. Helenius, A and Simons, K. (1977). Proc.Natl.Acad.Sei. USA 74, 529-532. and Chrambach, A (1978). Hjelmeland, L.M., Nebert, D.W. In Catsimpoolas, N.N. (ed.): Electrophoresis '78, Elsevier, North Holland, Amsterdam, 1978, pp 29-56. Johansson, K.E., Blomquist, I. and Hjerten, S. (1975). J.Biol.Chem. 250, 2463-2469. Johansson, K.E and Wroblewski, M. (1978). J.Bact. 136, 324-330. J0rgensen, O.S. (1977). FEBS Lett. 79, 42-44. Koch, C. and Nielsen, H.E. (1975). Scand.J.Immunol. 4, Suppl 2, 121-124. Kr0ll, J. (1973). Scand.J.Immunol.2, Suppl. 1, 57-59. Krtfll, J. (1973). Scand.J.Immunol.2, Suppl. 1, 61-67. Kr«Sll, J. (1976). J. Immunol .Methods 13, 333-339. Lanzerotti, R.H. and Gullino, P.M. (1972). Anal.Biochem. 50, 344. Laurell, C.B. (1965). Anal. Biochem. 10, 358-361. Laurell, C.B. (1966). Anal. Biochem. 15, 45-52. Laurell, C.-B. (1972). Electroimmuno Assay. Scand.J.Clin.Lab.Invest. 29, Suppl. 124. L0wenstein, H. (1978). Prog.Allergy 25, 1-62. Mattsson, C., Heilbronn, E., Ramlau, J. and Bock, E. (1979). J.Neurochem. 32, 301-311. Nielsen, C.S. and Bjerrum, O.J. (1977). Biochim.Biophys.Acta 466, 496-509. Norrild, B., Bjerrum, O.J., Ludwig, H. and Vestergaard, B.F. (1978). Virology 87, 307-316. Owen, P. and Kabach, H.R. (1979). Biochemistry 18, 1413-1421. Owen, P. and Kabach, H.R. (1979).

66. 67. 68. 69.

70. 71. 72. 73. 74. 75. 76. 77. 78.

79. 80.

81. 82. 83.

B i o c h e m i s t r y 18, 1 4 2 2 - 1 4 2 6 . O w e n , P. and S m y t h , C . J . (1977). P p . 1 4 7 - 2 0 2 in S a l t o n , M.R.J. (ed.) I m m u n o c h e m i s t r y of E n z y m e s and t h e i r Antibodies. W i l e y , N e w Y o r k , 1977. R a f t e l l , M . , B e r z i n s , K. and B l o m b e r g , F. (1977). Arch.Biochem.Biophys. 181, 534. R a m l a u , J. and B j e r r u m , O . J . (1977). Scand.J.Immunol. 6, 867. R a m l a u , J. and B o c k , E. (1979). In Egly, J . M . (ed.): P r o c e e d i n g s of the T h i r d I n t e r n a t i o n a l S y m p o s i u m of A f f i n i t y C h r o m a t o g r a p h y and M o l e c u l a r I n t e r a c t i o n , I n s e r m S y m p o s i a S e r i e s , P a r i s , in p r e s s . Singer, S.J. and N i c o l s o n , G . L . (1972). S c i e n c e , 175, 7 2 0 - 7 3 1 . S j o s t r o m , M . , N o r e n , 0., J e p p e s e n , L., S t a u n , M . , S v e n s s o n , B. and C h r i s t e n s e n , L. (1978). Eur.J.Biochem. 88, 503-511. S k o v b j e r g , H., S j o s t r o m , H . , N o r e n , 0. and H 0 y e r , G . (1979). C l i n . C h i m . A c t a 92, 3 1 5 - 3 2 2 . and O w e n , P. S m y t h , C . J . , S i e g e l , J., S a l t o n , M . R . J . (1978). J.Bact. 133, 306. Svendsen, P.J. (1973). S c a n d . J . I m m u n o l . , 2, S u p p l . 1, 6 9 - 7 0 . Svendsen, P.J. and A x e l s e n , N . H . (1972). J . I m u n o l . M e t h o d s 1, 1 6 9 - 1 7 6 . T a n f o r d , C. and R e y n o l d s , J. (1976). B i o c h i m . B i o p h y s . A c t a 457, 1 3 3 - 1 7 0 . Uriel,J. (1963). Ann.N.Y.Acad.Sci. 103, 956. U r i e l , J. (1964). P p 30-57 in G r a b a r , P. and B u r t i n , P. (eds.) I m m u n o - E l e c t r o p h o r e t i c A n a l y s i s . A p p l i c a t i o n s to Human Biological Fluids. Elsevier, Amsterdam. (English e d i t i o n of A n a l y s e i m m u n o - e l e c t r o p h o r e t i q u e . M a s s o n et C i e , P a r i s , 1960). Uriel.J. (1967). Pp. 1 8 1 - 1 9 6 in C i n a d e r , B. (ed) A n t i b o d i e s to B i o l o g i c a l l y A c t i v e M o l e c u l e s . Pergamon, O x f o r d , 1967 Uriel, J. (1971). Pp. 2 9 4 - 3 2 1 in W i l l i a m s , C . A . and Chase, W.W. (eds.) M e t h o d s in I m m u n o l o g y and Immunochemistry, Vol.3. R e a c t i o n s of A n t i b o d i e s w i t h Soluble Antigens. Academic Press, New York, London. V e r b r u g g e n , R. (1975). Clin.Chem. 21, 5 - 4 3 . W e e k e , B. and T h o m s e n , J . P . (1968). Scand.J.Clin.Lab.Invest. 22, 1 6 5 - 1 6 6 . Whiteside, T.L. and S a l t o n , M . R . J . (1970). B i o c h e m i s t r y 9, 3 0 3 4 - 3 0 4 0 .

AFFINITY ELECTROPHORESIS. QUANTIFICATION AND OF GLYCOPROTEINS WITH LECTINS.

CHARACTERIZATION

T.C.B0g-Hansen T h e P r o t e i n L a b o r a t o r y , U n i v e r s i t y of C o p e n h a g e n , S i g u r d s g a d e 34, DK 2200 C o p e n h a g e n N , D e n m a r k .

INTRODUCTION It is w e l l - d o c u m e n t e d that glycoconjugates are modified in diseased states. Thus cancer cells are preferentially are agglutinated by lectins [17] and serum glycoproteins It a p p e a r s g l y c o s y l a t e d d i f f e r e n t l y in c a n c e r p a t i e n t s [21,33]. e s s e n t i a l to be able to s t u d y in d e t a i l the i n d i v i d u a l m o l e c u l e s which are changed during the disease. Quantitative i m m u n o e l e c t r o p h o r e s i s c o m p r i s e s a s e r i e s of m e t h o d s that allow s t u d i e s of i n d i v i d u a l m o l e c u l e s . One of the major a d v a n t a g e s of these m e t h o d s is that i n f o r m a t i o n on m o l e c u l a r p a r a m e t e r s c a n be o b t a i n e d w i t h o u t p r i o r p u r i f i c a t i o n , cf. the a c c o m p a n y i n g p a p e r on q u a n t i t a t i v e i m m u n o e l e c t r o p h o r e s i s [13] . For d e t a i l e d study of m o l e c u l a r parameters, I have described various types of a f f i n i t y e l e c t r o p h o r e s e s w i t h lectins [5-12, 14-15], Affinity electrophoresis is based on the reaction between interacting components during electrophoresis. An early s y s t e m a t i c study w a s c o n d u c t e d by N a k a m u r a and coworkers [26], Later the term a f f i n i t y e l e c t r o p h o r e s i s w a s i n t r o d u c e d in [5] and [22]. Applications of the principle are counter immunoelectrophoresis, rocket immunoelectrophoresis (electro i m m u n o a s s a y ) and c r o s s e d i m m u n o e l e c t r o p h o r e s i s . A short review of affinity electrophoresis w a s g i v e n recently by H o r e j s i and c o w o r k e r s [23] . T h e c o m b i n a t i o n s of a f f i n i t y e l e c t r o p h o r e s i s with lectins and q u a n t i t a t i v e i m m u n o e l e c t r o p h o r e s i s d e s c r i b e d here w e r e d e v e l o p e d for i d e n t i f i c a t i o n a n d c h a r a c t e r i z a t i o n of g l y c o p r o t e i n s and as "table top" analytical methods for p r e d i c t i o n of p r e p a r a t i v e s e p a r a t i o n s [5,6,15]. The basic methodology of quantitative immunoelectrophoresis in agarose gel is outlined in the a c c o m p a n y i n g p a p e r [13]. Interactions during electrophoresis have been the m a i n o b j e c t of my s t u d i e s , b e c a u s e c o n s i d e r a b l e information can be obtained in this way. Moreover, characterization of e l e c t r o p h o r e t i c a l l y s e p a r a t e d g l y c o p r o t e i n s and g l y c o p e p t i d e s by b i n d i n g of l e c t i n s , as for instance with FITC-labelled lectin, enzyme-labelled lectin, or antibodylabelled lectin, have been developed elsewhere [1,2,35,36]. Recently a review a p p e a r e d w i t h lists of p r o t e i n s p u r i f i e d or c h a r a c t e r i z e d by m e a n s of i n t e r a c t i o n w i t h lectins [20].

© 1980 Walter de Gruyter & Co., Berlin • New York Electrophoresis '79

19^

FIGURE 1. Quantification of glycoprotein bands in SDS-electrophoresis. Combined SDS-polyacrylamide gel e l e c t r o p h o r e s i s and l e c t i n a f f i n i t y e l e c t r o p h o r e s i s . Individual P o l y a c r y l a m i d e gel lanes w e r e p l a c e d on top of flat a g a r o s e g e l s c o n t a i n i n g 5% T r i t o n X - 1 0 0 (lower p a r t of F i g s . , d a r k e s t area). The position of the a c r y l a m i d e gel is i n d i c a t e d by the l i g h t s t r i p (1+ i n d i c a t e s the a n o d e ) . The upper g e l s contain con A (Pharmacia Fine Chemicals, Uppsala). 2+ i n d i c a t e s the s e c o n d dimension electrophoresis. la. 1 mg h u m a n s e r u m g l y c o p r o t e i n , 2 . 2 - 1 0 " M con A. lb. 1 mg h u m a n s e r u m g l y c o p r o t e i n , 1 . 5 - 1 0 _M con A. lc. 0.7 mg h u m a n s e r u m g l y c o p r o t e i n , 1 . 8 - 1 0 M con A. Id. 1 mg h u m a n s e r u m g l y c o p r o t e i n , 1 . 8 - 1 0 " M con A. T h e bar r e p r e s e n t s one cm.

195 It is the o b j e c t of this c o m m u n i c a t i o n to give some results quantification and characterization of glycoproteins e l e c t r o p h o r e s i s w i t h lectins. QUANTIFICATION

on by

OF GLYCOPROTEIN

O n e - d i m e n s i o n a l and t w o - d i m e n s i o n a l e l e c t r o p h o r e t i c systems in agarose with lectins have been developed as methods for quantification of denatured glycoproteins [9] and for quantification and characterization of microbial and other c a r b o h y d r a t e s [27,29-31]. Here this m e t h o d is d e v e l o p e d f u r t h e r for quantification of glycoproteins separated in SDS-polyacry1amide. Fig. 1 shows how SDS-polyacrylamide gel electrophoresis of denatured glycoproteins can be combined with lectin affinity electrophoresis. The protein sample was con A-binding glycoproteins from normal human serum purified by a f f i n i t y treated c h r o m a t o g r a p h y on con A - S e p h a r o s e . The samples were with S D S b e f o r e a p p l i c a t i o n to the S D S - p o l y a c r y l a m i d e gel s l a b s (0.1 M T R I S , 0.22 M a c e t a t e , 0.02 M E D T A , pH 7 . 4 , 0.1% SDS, 5% acrylamide). After separation in the SDS gel, lanes w e r e cut and s o a k e d for 15 m i n in the a g a r o s e gel buffer (TRIS-veronal, pH 8.6) or s t o r e d f r o z e n . The second dimension electrophoresis was performed in agarose with non-ionic detergent Triton X-100, see Fig. 1. A 1% a g a r o s e gel c o n t a i n i n g 5% T r i t o n w a s c a s t and the p o l y a c r y l a m i d e About gel lane w a s p l a c e d on the s u r f a c e of the a g a r o s e gel. one cm f r o m the p o l y a c r y l a m i d e gel a lectin-containing agarose gel with a lower c o n c e n t r a t i o n of n o n - i o n i c d e t e r g e n t w a s c a s t (1% a g a r o s e w i t h 1% Triton). The second dimension electrop h o r e s i s into the l e c t i n - c o n t a i n i n g gel took p l a c e o v e r - n i g h t at 2 V / c m , and the r e s u l t i n g p a t t e r n s are s e e n in F i g . 1. D u r i n g the s e c o n d e l e c t r o p h o r e s i s p r e c i p i t a t e s develop between glycoproteins and lectin. The present experiments show precipitates between serum glycoproteins and con A. The precipitate formation is dependent upon the amount of g l y c o p r o t e i n and the a m o u n t of l e c t i n a p p l i e d . The p l a t e s s h o w n h e r e are w i t h a b o u t 1 mg g l y c o p r o t e i n and 0 . 6 - 1 . 2 mM con A w h i c h w e r e found to be c o n v e n i e n t a m o u n t s for precipitate formation and detection w i t h these g e l s . T h e r e are q u a n t i t a t i v e a s p e c t s of the m e t h o d , since the rocket h e i g h t of the precipitates is di rectly r e l a t e d to the a m o u n t of a p p l i e d g l y c o p r o t e i n . It is r e m a r k a b l e that the SDS in the acrylamide gel does not disturb the binding to the lectin. T h i s is p r e s u m a b l y due to the n e u t r a l i z i n g e f f e c t of Triton by inclusion of SDS into m i c e l l e s , so t h a t the l e c t i n is not e x p o s e d to free S D S . O n the o t h e r h a n d l e c t i n s are known to be stable and reactive in presence of d e t e r g e n t , see for i n s t a n c e [4]. Earlier we showed t h a t d e n a t u r a t i o n of the g l y c o p r o t e i n h a s little e f f e c t u p o n the

196 TABLE 1. A list of reactions seen in modified crossed Immunoelectrophoresis with lectin incorporated in the first lectin dimension gel or in the intermediate gel, either as free or as immobilized lectin.

Reaction Appearance of affinity precipitate Disappearance of precipitate

Interpretation Some or all molecules contain two or more binding sites to the lectin All molecules have affinity for the lectin

Binding to immobilized lectin

May be observed with glycoprotein enzymes or radioactively labeled glycoproteins .

4. Shift of position in electrophoretic pattern

Binding to the lectin during the 1st dimension electrophoresis

5. Appearance of multi-peak precipitates, e.g. 2-peak precipitate

Reveals various molecular forms with different affinity to the lectin. Seen with free and insolubilized lectin in the 1st dimension

6. Decrease of precipitate size

Only part of the molecules have binding sites or the binding is weak

7. Increase of precipitate size 8. Change in precipitate profile 9. Change in precipitate morphology

Binding of lectin leads to stearic hindrance of antibody-binding As 5, but may also be seen with lectin in the intermediate gel

10. Reactions of 'partial identity'

As 5 and 7, or cross-linking of different glycoproteins through one molecule of lectin (the lectin mediates the 'partial identity')

As 7, or interrupted precipitation, i.e. violation of the 'law of unity of time and space' + )

Reactions seen for various glycoproteins with lectins (mainly con A). With immobilized lectin, reactions 1, 7 and 10 have not been seen. Modified from (15). + )Proposed as 'the first law of quantitative Immunoelectrophoresis' (0.J.Bjerrum, cf. (13)).

197

n

n

c

d

ptl

Ptl

e

A FIGURE 2. Characterization of microheterogeneous glycoprotein. Lectin interaction immunoelectrophoresis of orosomucoid (alpha-1 acid glycoprotein) in human serum with con A and WGA in thin layers of agarose gel. The amount of serum was for all experiments 2 jjl, the con A concentration was 0 . 8 5 - 1 0 - 5 M, the WGA concentration was 1.7-10 ~ 5 M (both corresponding to a binding site concentration of 3 . 4 - 1 0 - 5 M). Further technical details in [6,8,15].

198 b i n d i n g to l e c t i n (con A) whether denaturation was performed with detergent, heat or acid-treatment and that the precipitation in affinity precipitates was quantitative i r r e s p e c t i v e of the d e g r e e of d e n a t u r a t i o n [9]. LECTIN INTERACTION

IMMUNOELECTROPHORESIS

W h e n l e c t i n is i n c o r p o r a t e d in the f i r s t dimension gel during crossed immunoelectrophoresis, characteristic reactions occur between glycoproteins and lectins. Characteristics of experiments with lectin incorporated in crossed i m m u n o e l e c t r o p h o r e s i s , e.g. in the f i r s t d i m e n s i o n gel ore in an i n t e r m e d i a t e gel are listed in T A B L E 1. T h e m a i n f e a t u r e is the r e t a r d a t i o n d u r i n g the f i r s t d i m e n s i o n electrophoresis and formation of an affinity precipitate (Fig. 2, a r r o w ) . The r e t a r d a t i o n is r e l a t e d to the s t r e n g t h of the binding between glycoprotein and the l e c t i n , and the d i s s o c i a t i o n c o n s t a n t m a y be c a l c u l a t e d f r o m such experiments [16]. Thus the various coherent peaks of an i n d i v i d u a l g l y c o p r o t e i n r e p r e s e n t v a r i o u s m i c r o h e t e r o g e n e o u s forms w i t h d i f f e r e n t b i n d i n g a f f i n i t y to the lectin. Under n o r m a l c o n d i t i o n s each s e r u m g l y c o p r o t e i n has a c h a r a c t e r i s t i c r e t a r d a t i o n and a c h a r a c t e r i s t i c p r o f i l e . Normal and pathological serum w a s a n a l y z e d w i t h d i f f e r e n t l e c t i n s in v a r i o u s a m o u n t s and the r e s u l t i n g b i n d i n g w a s s t u d i e d . Fig. 2a and b shows normal human serum orosomucoid (alpha-l-acid g l y c o p r o t e i n ) w i t h con A and W G A in the f i r s t d i m e n s i o n (total s e r u m w a s a n a l y z e d w i t h s p e c i f i c a n t i s e r u m in the second dimension gel to give the p r e c i p i t a t e of the i n d i v i d u a l protein). In F i g . 2c, d, e and f is s h o w n the corresponding pattern of the same s e r u m p r o t e i n in 2 acute p h a s e p a t i e n t s . The a c u t e p h a s e is c h a r a c t e r i z e d by i n c r e a s e s of the so-called "acute phase proteins" of w h i c h orosomucoid is a p r o m i n e n t e x a m p l e and the p a t t e r n is s t r i k i n g in its d i f f e r e n c e s to n o r m a l serum. dimension, W h e n o r o s o m u c o i d is a n a l y z e d w i t h con A in the first 3 c o h e r e n t p e a k s are s e e n : 01, 02 and 03. 01 d o e s not i n t e r a c t w i t h con A (01 is at the o r i g i n a l p o s i t i o n of s e r u m orosomucoid without con A ) . 02 binds s l i g h t l y to con A . 03 (the minor c o m p o n e n t ) h a s a m o d e r a t e , s t r o n g e r b i n d i n g to con A. In n o r m a l serum the p e a k s of 01 and 02 have the same s i z e , i n d i c a t i n g the same c o n c e n t r a t i o n (Fig.2a), but u n d e r pathological conditions the r e l a t i v e h i g h t of 01 and 02 is c h a n g e d (Fig. 2c and e). In one a c u t e p h a s e p a t i e n t 01 is h i g h e s t , in the other patient 02 is highest, the o t h e r c o m p o n e n t remaining at the n o r m a l level. A l s o the m i n o r c o m p o n e n t , 03, is i n c r e a s e d . In a g g r e e m e n t w i t h the d i f f e r e n c e s in b i n d i n g s p e c i f i c i t y of con A (mannose) and wheat germ agglutinin, WGA (N-acetylated glucosamine, sialic acids) the pattern of orosomucoid is completely d i f f e r e n t w i t h W G A c o m p a r e d w i t h con A (Fig. 2b, d, and f). The n o r m a l p a t t e r n (Fig. 2b) shows a double-peaked

199 precipitate close to the application hole (note also the a f f i n i t y p r e c i p i t a t e in the f i r s t d i m e n s i o n gel as w i t h con A). T h o u g h o r o s o m u c o i d w a s d i f f e r e n t in the two p a t i e n t s w i t h con A , there is a close similarity between the patterns with WGA. However, the profile is t o t a l l y d i f f e r e n t from that of n o r m a l serum with much greater amounts of the slightly retarded fractions. In separate experiments (not shown) it w a s f o u n d that the i n c r e a s e d a m o u n t of reacting protein results in a slightly s h i f t e d p o s i t i o n of i d e n t i c a l p e a k s . T h u s the f a s t e s t moving component in all experiments represents the same molecular s p e c i e s of o r o s o m u c o i d , as the i n t e r m e d i a t e c o m p o n e n t represents another differing form and the slowest migrating c o m p o n e n t r e p r e s e n t s a third form. T h e c o n c l u s i o n of these experiments is that there is less binding of acute p h a s e o r o s o m u c o i d to W G A , p o s s i b l y c a u s e d by a d i m i n i s h e d c o n t e n t of s i a l i c a c i d s in the outer parts of the carbohydrate chains. The r e l a t i o n of these m i c r o h e t e r o g e n e o u s components of orosomucoid to the seven bands seen in electrofocusing (cf. P.Arnaud, E.Gianazza, P . G . R i g h e t t i and H . H . F u d e n b e r g , these p r o c e e d i n g s ) is not clear. W h e n we use m u l t i s p e c i f i c a n t i s e r u m for d e t e c t i o n of the c h a n g e d r e t a r d a t i o n p r o f i l e s we o b s e r v e a m u l t i t u d e of c h a n g e s for other proteins during pathological conditions. Thus we find characteristic changes during treatment with estrogen-like drugs and during pregnancy [34] . Possibly the changes in binding-patterns reflect changes in the biosynthetic m o d i f i c a t i o n of the g l y c o p r o t e i n s or the m e t a b o l i c t u r n o v e r (the secretion, the i n t e r c h a n g e b e t w e e n the m i c r o h e t e r o g e n e o u s forms or the c a t a b o l i s m ) d u r i n g the acute p h a s e . DISCUSSION For c l o s e r u n d e r s t a n d i n g of many biological phenomena it is essential to o b t a i n a g r e a t e r k n o w l e d g e of the f u n c t i o n s of the carbohydrate in m a c r o m o l e c u l a r glycoconjugates. It m a y be surprising that little is k n o w n a b o u t the f u n c t i o n of the m a j o r plasma glycoprotein, orosomucoid, H o w e v e r , it may be r e l a t e d to the changes in c a r b o h y d r a t e . R e c e n t l y C o s t e l l o et al. showed t h a t d e s i a l i s a t i o n of o r o s o m u c o i d is a s s o c i a t e d with increased inhibitory activity towards platelet a g g r e g a t i o n [19a]. The methods described here were developed for quantification and closer molecular c h a r a c t e r i z a t i o n of g l y c o p r o t e i n s w i t h o u t the n e e d for p u r i f i c a t i o n . The m e t h o d s h o w n in Fig. 1 o f f e r s an opportunity to measure the amounts of lectin-binding glycoprotein present in individual protein bands in SDS electrophoresis. T h e m e t h o d is e s s e n t i a l l y s i m i l a r to a m e t h o d described by Bjerrum for immunological quantification of i n d i v i d u a l p r o t e i n s s e p a r a t e d in SDS e l e c t r o p h o r e s i s [3]. Other s i m i l a r a p p r o a c h e s for i m m u n o l o g i c a l e s t i m a t i o n of S D S - s e p a r a t e d proteins have appeared [18,19]. An important detail is inclusion of a great amount of non-ionic detergent for

200 n e u t r a l i z a t i o n of the S D S . The m a j o r p r o b l e m is the recovery of p r o t e i n , w h i c h is low, i.e. the p r o t e i n is not electrophoresed out of the Polyacrylamide ge 1 in full y i e l d . P r e s u m a b l y the r e c o v e r y c o u l d be i n c r e a s e d by u se of ultrathin Polyacrylamide gels. The modified crossed Immunoelectrophoresis with lectin in the first dimension gel (Fig. 2) is one of the m o s t s e n s i t i v e m e t h o d s for d e t e c t i o n and c h a r a c t e r i z a t i o n of m i c r o h e t e r o g e n e o u s forms of i n d i v i d u a l g l y c o p r o t e i n s - e v e n d i s s o c i a t i o n c o n s t a n t s of glycoprotein and lectin complexes may be calculated (T.C.B0g-Hansen and K . T a k e o , in p r e p a r a t i o n ) . This method may be used d i r e c t l y w i t h s e r u m or plasma, since the pattern is identical, i r r e s p e c t i v e of the use of p l a s m a , s e r u m or p u r i f i e d p r o t e i n [7]. For an i n d i c a t i o n of the p o s s i b i l i t i e s of lectin characterization of glycoproteins, the reader is r e f e r r e d to M u r a k a w a , O s u n k o y a and R o s t e n b e r g ' s s t u d i e s of changes in the lectin-binding properties of serum proteins during numerous d i s e a s e s [25,28,33]. Also changes relating to post-synthetic processes may be assessed in this w a y . A p p l y i n g this m e t h o d of lymphocytes from chronic HLA-alloantigens in m e m b r a n e s l y m p h a t i c l e u k e m i a p a t i e n t s have b e e n found to vary w i t h r e s p e c t to lectin-binding (T.Plesner and O.J.Bjerrum, Copenhagen, personal communication) and the lentil reactivity of a l p h a - f e t o p r o t e i n has b e e n found to d e c r e a s e a l m o s t l i n e a r l y in normal p r e g n a n c i e s w i t h the g e s t a t i o n a l age f r o m 15 to 35 w e e k s (J.P.Kerckaert, Lille, personal communication). The crossed Immunoelectrophoresis with lectin in the first dimension gel m a y be g e n e r a l i z e d for other i n t e r a c t i n g s y s t e m s . T h u s R a m l a u and Bock d i s c u s s the g e n e r a l a p p l i c a b i l i t y of free and m a t r i x - b o u n d l i g a n d s in c r o s s e d i m m u n o - e l e c t r o p h o r e s i s f r o m the v i e w p o i n t of p r o t e i n separation and conclude from their e x p e r i m e n t s that the g e n e r a l m e t h o d may be u s e d as a s m a l l s c a l e a n a l y t i c a l m e t h o d for p r e d i c t i o n of s e p a r a t i o n e x p e r i m e n t s [32]. In this r e l a t i o n it m a y be n o t e w o r t h y that w i t h lectins there is g o o d c o r r e l a t i o n b e t w e e n the analytical experiments and preparative affinity chromatography experiments [5,15,24]. The m e t h o d a p p l i e d to s t u d i e s of r e c e p t o r s is t r e a t e d generally in [4] . M a i n a d v a n t a g e s of the a n a l y t i c a l a p p r o a c h a p p e a r s m a y be o u t l i n e d in the f o l l o w i n g p o i n t s : 1. M a c r o m o l e c u l a r i n t e r a c t i o n s may be s t u d i e d in a c o n v e n i e n t manner. 2. P u r i f i c a t i o n or s e p a r a t i o n of i n t e r a c t i n g c o m p o n e n t s is not requi red. 3. A large n u m b e r of i n t e r a c t i n g p r o t e i n s may be s t u d i e d simultaneously. 4. T h e r e s u l t s of p r e p a r a t i v e a f f i n i t y s e p a r a t i o n s c a n be predicted. 5. T h e a n a l y t i c a l p r i n c i p l e may be g e n e r a l i z e d to o t h e r interacting systems.

201 Conclusion. A f f i n i t y e l e c t r o p h o r e s i s w i t h lectins may be used for identification and q u a n t i f i c a t i o n of l e c t i n - b i n d i n g g l y c o p r o t e i n s , irrespective of their denatured or native state, and for investigation of the different microheterogeneous forms of glycoproteins. ACKNOWLEDGEMENTS. Ms. Pia J e n s e n is t h a n k e d for her excellent t e c h n i c a l a s s i s t a n c e and the D a n i s h M e d i c a l R e s e a r c h C o u n c i l for support. REFERENCES Allen, R.C., Spicer, S.S. and Z e h r , D. (1976) J.Hi stochern.Cytochem. 24, 908-914 . 2. B i t t i g e r , H. and S c h n e b l i , H . P . (1976). Concanavalin A as a T o o l . Wiley, London. 3. B j e r r u m , O . J . (1980) S c a n d . J . I m m u n o l . Suppl. 10 (in press). 4. B j e r r u m , O . J . , R a m l a u , J . , B o c k , E. and Bjig-Hansen, T . C . (1979) in S . J . J a c o b s and P . C u a t r e c a s a s (eds.) T e c h n i q u e s for M e m b r a n e R e c e p t o r C h a r a c t e r i z a t i o n and P u r i f i c a t i o n , C h a p m a n and H a l l , L o n d o n (in p r e s s ) . 5. B 0 g - H a n s e n , T . C . (1973) A n a l . B i o c h e m . 56, 4 8 0 - 4 8 8 . 6. B0g-Hansen, T.C. (1979) in J . M . E g l y (ed.) P r o c e e d i n g s of the T h i r d I n t e r n a t i o n a l S y m p o s i u m on A f f i n i t y C h r o m a t o g r a p h y and M o l e c u l a r I n t e r a c t i o n s , I n s e r m S y m p o s i a S e r i e s , P a r i s (in p r e s s ) . 7. B0g-Hansen, T.C. (1980) in H . P e e t e r s (ed.) P r o t i d e s of the B i o l o g i c a l F l u i d s , P r o c e e d i n g s of the X X V I I C o l l o q u i u m , V o l 27, P e r g a m o n P r e s s , O x f o r d (in p r e s s ) . (1980) S c a n d . J . I m m u n o l . Suppl. 10 (in 8. B 0 g - H a n s e n , T . C . press). 9. B0g-Hansen, T.C., Bjerrum, O.J. and B r o g r e n , C . H . (1978) Anal.Biochem. 81, 7 8 - 8 7 . 10. B0g-Hansen, T.C., Bjerrum, O.J. and R a m l a u , J. (1975) Scand.J.Immunol. 4, S u p p l . 2, 1 4 1 - 1 4 7 . 11. B0g-Hansen, T.C. and B r o g r e n , C . H . (1975) Scand.J.Immunol. 4, S u p p l . 2, 1 3 5 - 1 3 9 . 12. B0g-Hansen, T.C. Brogren, C.H. and M c M u r r o u g h , I. (1974) J . I n s t . B r e w i n g 80, 4 4 3 - 4 4 6 . 13. B 0 g - H a n s e n , T . C . , L o r e n c - K u b i s , I. and B j e r r u m , O . J . These proceedings. 14. B0g-Hansen, T.C. and N o r d , M. (1974) J . B i o l . E d u c . 8, 167-173. 15. B 0 g - H a n s e n , T . C . , P r a h l , P. and L p w e n s t e i n , H. (1978) J . I m m u n o l . M e t h o d s 22, 2 9 3 - 3 0 7 . 16. B0g-Hansen, T.C. and T a k e o , K. (in p r e p a r a t i o n ) . 17. Burger, M.M. (1973) F e d . P r o c . 32, 9 1 - 1 0 1 . 18. C h u a , N . H . and B l o m b e r g , F. (1979) J . B i o l . C h e m . 254, 215-223. 19. C o n v e r s e , C . A . and P a p e r m a s t e r , D . S . (1975) S c i e n c e 189, 469-472. 19a. C o s t e l l o , M . , F i e d e l , B . A . and G e w ü r z , H. (1979) N a t u r e 1.

20 2 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.

33. 34. 35. 36.

281, 677-678. Dulaney, J.T. (1979) Mol.Cell.Biochem. 21, 43-62. Gralnick, H.R., Givelber, H. and Abrams, E. (1978) New Engl.J.Med. 299, 221-226. Horejsi, V. and Kocourek, J. (1974) Biochim.Biophys.Acta 336, 338-343. Horejsi, V., Ticha, M. and Kocourek, J. (1979) TIBS 4, 1, N6-N7. Kerckaret, J.P., Bayard, B. and Biserte, G. (1979) Biochim.Biophys.Acta 576, 99-108. Murakawa, S. and Nakamura, S. (1963) Bull.Yamaguchi Medical School 10, 11-29. Nakamura, S (1966) Cross Electrophoresis. Its Principle and Applications. Igaku Shoin, Tokyo and Elsevier, Amsterdam. Oppenheim, J.D., Owen, P., Nachbar, M.S., Colledge, K. and Kapian, H.S. (1977) Immunol.Commun. 6, 167-181. Osunkoya, B.O. and Williams, A.I.O. (1971) CIin.Exp.Immunol. 8, 205. Owen, P., Oppenheim, J.D., Nachbar, M.S. and Kessler, R.E. (1977) Anal.Biochem. 80, 446-457. Owen, P. and Salton, M.R.J. (1975) Proc.Natl.Acad.Sei. U.S.A. 72, 3711-3715. Owen, P. and Salton, M.R.J. (1976) Anal.Biochem. 73, 20-26. Ramlau, J. and Bock, E. (1979) in J.M.Egly (ed.) Proceedings of the Third International Symposium on Affinity Chromatography and Molecular Interaction, Inserm Symposia Series, Paris (in press). Rostenberg, I., Guizar-Vazquez, J., Suarez, P., Rico, R., Nungaray, L. and Dominguez, C. (1978) J.Natl.Cancer Inst. 60, 83-87. Wells, C., Cooper, E. and Bpg-Hansen, T.C. in preparation. 74, West, C.M. and McMahon, D (1977) J.Cell.Biol. 264-273. Wood, J.G. and Sarinana, F.O. (1975) Anal.Biochem. 69, 320-322.

RECENT ADVANCES IN MICROELECTROPHORESIS

V. Neuhoff Max-Planck-Institut für experimentelle Medizin, Forschungsstelle Neurochemie, Hermann-Rein-Str. 3, 3400 Göttingen, FRG

Introduction The profit inheritent in micromethods is still not fully recognized. This is mostly due to the prejudices of the experimenter about any method which has been reduced to microscale. However, there is ample literature available (for review see 1, 2) clearly demonstrating that micromethods have at least the same resolving power as the corresponding macroscale procedures. Furthermore, there is no doubt that any technical competent experimenter can easily learn to use micromethods. The necessary equipment is inexpensive, as is the price for the reagents. Another advantage lies in the fact that several analysis can only be performed in microscale, e.g., enzyme kinetics of separated isoenzymes. However, the major advantage of micromethods is their speed, approximate an order of magnitude faster than the equivalent macroprocedure. In the following paper some refinements of previously described microelectrophoretic methods are described, as well as some new developments in this field. A new method for protein determination will also be briefly described, which has certain advantages over existing procedures.

© 1980 Walter de Gruyter 8. Co., Berlin • New York Electrophoresis '79

204 Separation of enzyme variants and isoenzymes Fractionation of isoenzymes, e.g. of lactate dehydrogenase (LDH) can be performed in polyacrylamide microgels by isoelectric focusing (3,4) and disc electrophoresis (5). In principle isoelectric focusing (IEF) is the optimal separation procedure for isoenzymes, since the molecules are separated according to their isoelectric points and isoenzymes have almost identical molecular weights, but different isoelectric points. The use of microgels for separation followed by incubation in a suitable tetrazolium assay allows the quantitative measurement of the enzyme activity of each isoenzyme (4,5). In spite of the highly reproducible results obtainable with micro-IEF of LDHisoenzymes it was observed in comparison with results obtained with other methods the staining of the most anodic isoenzymes (LHD^ and LDI^) is unexpectedly weak. Gustke and Neuhoff (6) have shown that this inactivation is due to the acid denaturation of the enzyme protein at its isoelectric point and is not dependent on the presence of Ampholine. The electrophoretic separation of isoenzymes in 20% homogenous polyacrylamide microgels (5) is not very successful since the isoenzymes are not separated well enough for optical evaluation. Therefore the method of Diez and Lumbrano (7,8) was adapted to the microscale (6) using the same gel composition (T = 5,5%, C = 2,6%; Tris-Cl, pH 8.9 as gel buffer) and with a 13 mM Tris/glycine (pH 8.4) electrode buffer. An excellent separation is obtainable within 10 to 12 min. at 120 V. The LDH-isoenzyme patterns obtained with this method compare favourable with the results reported in the literature, although in this case the most cathodic isoenzyme LDHj. is partially inactivated. These observations clearly demonstrate that a critical analysis of the methods used for the separation of isoenzymes is necessary before an isoenzyme pattern can be accepted as the correct pattern.

205

The tetrazolium assay is most suitable for demonstrating different dehydrogenase isoenzymes (1,3,5,9) after separation in microgels. Gustke and Neuhoff (6) have shown, that in the presence of 0.05% Triton X-100 in the microgels the sensitivity of the assay can be increased two- to four-fold. This low concentration of Triton X-100 has no effect on the isoenzyme separation and also not on the background staining during incubation in the tetrazolium assay mixture, whilst higher concentrations of Triton X-100 (0.1 - 0.5%) cause a relatively intensive background staining which interfere with the densitometric evaluation. This increase in sensitivity is not due to a direct effect on the isoenzymes but most likely to a better electron transfer from the reduced coenzyme to the tetrazolium salt as is described (10,11) for colloids, like albumin, agar or agarose. It was shown earlier (5) that it is possible to successfully separate both the isoenzymes of glucose-6-phosphate dehydrogenase and perform enzyme kinetic measurements in microgels. However, for the fractionation a method originally designed for the separation of brain proteins (12) was used which cannot be considered to be optimal for the separation of glucose6-phosphate dehydrogenase variants. This is indicated by the fairly long electrophoresis time of 2 hs which was necessary to achieve a complete separation of the enzyme variants from man and mouse. The application of the procedure to other fractionation problems was not successful, for instance the fractionation of the enzyme from human erythrocytes was poor, due to overlap with the hemoglobin zone and the fact that only a diffuse enzyme band was formed which was not suitable for quantitative analysis. Gustke and Neuhoff (13) have improved the fractionation of glucose-6-phosphate dehydrogenase variants on the microscale, using either 10% gels cast in 10 JJ.1 capillaries, or 1-35% micro gradient gels followed by the tetrazolium assay to specifically stain the enzyme variants (for technical de~ tails see 13).

206 A clear separation of the enzyme variants from a mixture of kidney extracts from cat, guinea pig and rat was achieved by disc electrophoresis in 25 min. The resolution achieved in this time is sufficient for quantitative densitometric determinations. Even shorter runs may be possible for qualitative analyses. Similar fractionations were achieved with extracts from other organs, for example liver and brain. The quality of fractionation is strictly dependent on the concentration of sucrose in the sample. At high concentrations the bands are very sharp, but narrowly spaced and they move only slowly (Fig. 1). Therefore, sucrose concentrations of 2 0-25% in the sample are recommended for qualita-

© Fig. 1: Separation of a mixture of cat and rat kidney extract on 10% micro disc gels. The sample contained equal amounts of extract but different sucrose concentrations of 10% (a), 15% (b) and 20% (c). The time of separation and staining was identical for all three gels.

© tive analysis, for instance in genetic experiments. However, for quantitative determinations a wide spacing of the bands is advantageous and the sample should contain 10% sucrose, although the bands are more diffuse under these conditions. Cat and rat glucose-6-phosphate dehydrogenase can also be separated on a gradient gel. Although a clear resolution of both variants into well separated sharp bands is achieved on the gradient, the bands are too narrowly spaced for exact quantitative evaluation. Attempts to use flatter gradients did not give satisfactory results.

207 The fractionation of glucose-6-phosphate dehydrogenase from hemolysates of erythrocytes is not possible on normal disc gels, due to the high concentration of hemoglobin, which under the conditions which were optimal for the fractionation of the dehydrogenase interfered with their separation. However, sharp enzyme bands were achieved on the 1-35% acrylamide gradient gels. The rat enzyme separates into two major bands and a third component migrating just cathodical of the faster main band. Human erythrocyte glucose-6-phosphate dehydrogenase shows one main band which is well separated from the hemoglobin zone and a more slowly migrating, somewhat diffuse and weakly staining second component. The staining intensity of the erythrocyte enzyme bands on gradient gels is strictly linear with time for at least 30 min. and this confirms the possibility of making quantitative enzyme activity measurements in microgels. In spite of the wide use polyacrylamide gel electrophoresis for the separation of alkaline phosphatase

(14,15,16,17,18)

the results have not been satisfactory. Neither quantitative analysis of enzyme variants in gels, nor micro procedures were available until Gustke and Neuhoff (19) have shown that the multiple forms of alkaline phosphatase can successfully be separated in 1-20% continuous polyacrylamide gradient micro gels. Using a special assay mixture it is also possible to study the enzyme kinetics of the phosphatases in microgels like as with dehydrogenases. As an enzyme source butanol extracts of several rat tissues were used, which were prepared following the procedure of Gosh and Fishman (20). Weighted samples of the freshly excised tissues were homogenized with 0.05 M Tris-Cl buffer, pH 8.6 to give 10-20% homogenates. 0.4 volumes of icecold butanol were added dropwise to the magnetically stirred homogenate and stirring was continued for 30 min., followed by centrifugation for 30 min. at 50,000 g. The aqueous phase was dialysed overnight against 100 vol of Tris-buffer and spun for 30 min. at 100,000 g. Appropriate dilutions of the

208 final supernatant were used directly for electrophoresis. The whole procedure was carried out at 4°C. Gradient gels with acrylamide concentrations increasing continuously from 1-20% were used for electrophoresis. The gels were prepared (0.35 mM Tris-sulfate, pH 7.2 as gel buffer) in * (R) 10 p.1 capillaries (Blauband intraEND , Brand) essentially as described earlier (21). (For details see 19). The addition of Triton X-100 to give a final concentration of 0.5% proved to sharpen the alkaline phosphatase bands considerably, confirming the findings of Fishman (17). Triton gels occasionally tend to slip out of the capillaries. Therefore, extensive cleaning of the capillaries as described (1) is not advisable, although impurities may thus be left at the inner wall of the capillaries which sometimes hinder a steady stream of fluid during production of the gradients. This can be overcome by filling the capillaries completely with the ammonium peroxodisulfate solution and draining them again onto smooth tissue prior to use. The butanol extracts have to be appropriately diluted with water and a solution of 40% sucrose in 1:10 diluted gel buffer to give a constant final concentration in the sample of 20% sucrose and 1:20 diluted gel buffer. Of this sample solution usually 0.95 - 2.5 p.1 were applied on top of the gel. This high dilution is essential in the generation of sharp bands of alkaline phosphatase. A 95 mM Tris buffer adjusted to pH 8.8 with crystalline boric acid was used as the electrode buffer in preference to a Tris-glycine system, since glycine inhibits alkaline phosphatase (14). Electrophoresis was usually performed at a constant voltage of 100 V for two hours. This relative long running time is necessary to achieve a sufficient separation of the slowly migrating bands. Since bromophenol red traveled through the gel in less than one hour, no dye-marker was used to mark the end of a run. Attempts to reduce the running time by increasing the voltage lead to considerable tailing and broadening of the bands.

209

The enzyme bands are located in the gel by specific staining with the a-naphtylphosphate Fast Blue VB-system at alkaline pH (14). Since the solutions containing the dye are rather unstable, a postcoupling technique for staining of weakly active samples is advised (14), which includes preincubation of the gel with the substrate solution for a sufficient time and subsequent coupling to the diazonium salt. Such a procedure is not possible in kinetic experiments. The instability of the staining mixture is one of the major problems in obtaining quantitative measurements. As could be anticipated from the structure of the molecule the dye is readily soluble in either acid media, or organic solvents. Since in this case an alkaline pH is required. Various organic solvents were added to the stock solutions in an attempt to increase the solubility of the dye. Butanol proved to be the most appropriate, whereas other solvents such as acetone, chloroform and methanol, which all solubilized the dye alone very well, were either incompatible with the whole mixture or inhibited the enzyme. The following stock solutions have proved to be suitable: Solution A: 20 mg a-naphtylphosphate in 20 ml Tris-Cl buffer (50 mM, pH 9.5) containing 250 ul 2% MgCl 2 . Solution B: 100 mg Fast Blue VB salt is dissolved as completely as possible in 10 ml I^O containing 0.5 ml butanol. This solution is filtered through ordinary filter paper and stored in the dark. Stock solution B is stable for several months if stored in the dark at 4°C. Although some brown material gradually precipitates from the solution this does not influence the results. The actual staining solution is less stable upon prolonged standing, but if kept in the dark at 4°C it can safely be used for up to 24 hs. Thus it is possible to use the simultaneous coupling method which is essential in kinetic measurements even for very weakly active bands which have to be incubated for several hours. The assay mixture is prepared

210

by mixing 1 ml A with 250 )il B just prior to use. The gels are pushed out from the capillaries with water pressure (1) into 1 ml of Tris-buffer containing 50 nl butanol. 0.5 ml solution A+B are added and the gels are incubated in the dark at room temperature. The gels are than transferred to 7.5% acetic acid to stop the reaction and destain the gels. The gels are than scanned with a ZK4 disc attachment for PMQ spectralphotometers (Zeiss) equipped with a special cuvette for micro gels as described by Zimmer and Neuhoff (22).

rat

human

Fig. 2: Separation and enzyme assay of rat liver and human serum alkaline phosphatase (for details see text). The two main peaks on the densitometric tracing corresponding to rat liver and probably to human liver isoenzyme, whereas the additional peaks, marked with arrows, in the 80 min. tracing all belong to the serum. The increase in optical density was constant up to 80 min. incubation as shown by the plots of the main peak areas against time on the right side of the figure. The dotted lines indicate the standard deviations of the observations.

211

In figure 2 the results of an experiment with a mixture of human serum and rat liver extract is shown. Human serum was mixed with an equal volume of a diluted rat liver extract. 2.5 jj.1 of this mixture corresponding to 31 jig fresh liver and 1.25 |jl of undiluted serum were electrophoresed on 6 gels under identical conditions and subsequently incubated for the indicated times. The gels were scanned and the peak areas of the two main bands were plotted against time. Both enzymes exhibited a linear relationship between time and peak area for up to 80 min. of incubation, the correlation coefficient 'r' being 0.996 and 0.992 for the rat liver and the serum band respectively. The slopes of the regression line, representing the increase in optical density, can be taken as a measure of enzyme activity. With increasing time of incubation several more bands appear (marked with arrows), all of which are serum components since pure liver extract showed only one band under these conditions. This experiment demonstrates the possibillity of simultanously determining the enzyme activity of several enzyme variants in the same gel, which is one of the main advantages of this method.

Fig. 3: Enzyme kinetics in microgels with different dilutions of rat liver extract (for details see text). The slopes of the calculated regression lines, representing enzyme activity, were replotted against the respective amounts of wet tissue as shown in the inset.

8-34 pg r = 0-994

5-56 pg r=0-999

A

4-17 M9

r=0-984 3 13 tig r=0997 2-78 pg r=0-989

o 0

5

10

15

incubation (min)

212

Figure 3 presents the kinetics of different amounts of enzyme. 2.5

of various dilutions of rat liver extract, correspond-

ing to 2.8, 3.2, 4.7, 5.6, 8.4 and 12.5 p.g of fresh tissue, were subjected to electrophoresis, using 6-7 gels for each dilution. The correlation between peak areas and incubation time is linear for each dilution tested. The inset in figure 3 shows a replot of the slopes, which represent the reaction rates, against the corresponding quantity of tissue. Again a good linear correlation is observed (r = 0.994). The regression line can be used as calibration curve for the quantitative determination of enzyme activity since the quantity of tissue can easily be transferred into units of enzyme activity by measuring the activity of the extract used.

Microelectrophoresis in combination with immunonreaction and glycoprotein staining Neuhoff and Mesecke (23) have described a method for direct immunological identification of single protein components after fractionation of a protein mixture in polyacrylamide microgels. The gels are transferred after gel electrophoresis into agarose layers containing suitable antisera, monospecific, as well as polyvalent antisera can be used. The formation of the immunoprecipitates can be observed within approximately 1 h. Immunoprecipitates are also formed in the presence of SDS, or other detergents, thus allowing immunoreactions to be performed with water insoluble proteins. The SDS concentration must not higher than 0.1%. Excess of SDS can be precipitated with KC1. Staining of the proteins with Amidoblack, or Coomassie Blue partially inhibits the immunoreaction, while dansylation of proteins has no effect. The influence of different detergents e.g. sodium dodecylsulfate, Triton X-100, Brij 99, NP-40 and urea, as well as different reducing agents e.g. mercaptoethanol, dithiothreitol, thioglycolic acid, on two-dimensional microdiffusion was

213

also studied. When suitable concentrations of these compounds were used, the formation of immunocomplexes was observed within approximately 1 h. This technique can also be applied to immunoreactions with water-insoluble proteins dissolved in detergents. Lane et al. (24) have described a technique which allows one to label and quantitate glycoproteins. Small amounts of protein from biological samples (0.5 - 2.5 jig for mixtures and less for individual proteins) are separated by sodium dodecylsulfate gel electrophoresis on 1-30 percent polyacrylamide gradient microgels. The gels are stained with Coomassie Brilliant Blue R 2 50 to evaluate relative migration, or fixed in isopropanol-acetic acid and stained with FITC-labelled Concanavalin A. The microgels are then scanned using a fluorescence microscope coupled to a computer. Glycoproteins on the order of 5-30 nanograms protein (or 1-5 nanograms carbohydrate) can be detected without difficulty. This technique may prove valuable in evaluating glycoproteins when only limited material is available.

Microslab gel electrophoresis Polyacrylamide slab gel electrophoresis is one of the most favoured separation methods due to its inheritant advantage of allowing direct comparison between different samples separated under identical conditions. Pohling and Neuhoff (25) have transferred this method to the micro scale since a macroprocedure is not suitable when only limited amounts of material are available for analysis. Normal object slides are cut into pieces of 30 x 25 mm and carefully cleaned. Spacers as combs are cut from plastic foil (0.1 or 0.2 mm in thickness) and the gel cassette sealed with 2% agarose or Haward glass wax (1). To prepare homogenous gels the chamber is filled to a height of 1-2 mm with 60% sucrose and overlayered with a suitable gel mixture. After poly-

merisation the stacking gel solution is added and the comb with teeth of 1 mm or less carefully inserted into the gel sandwich. The preparation of micro gradient slab gels depends on the slope of the gradient, which one like to obtain. In order to generate a gradient of acrylamide concentration, capillary attraction can be used in the same way as described by Riichel et al. (21), for the preparation of gratient gels in capillaries. First the persulphate solution is allowed to rise to a certain hight, followed by the concentrated acrylamide/bis acrylamide mixture. In this case the gradient, in contrast to that formed in capillaries, is not linear, but follows a saturation curve of logarithmic type. To produce a linear gradient the chamber has first to be filled with the concentrated acrylamide solution followed by the peroxodisulfate solution and than immediately turned upside down and fixed in a vertical position prior to polymerisation. Finally a stacking gel and comb are added only to form suitable chambers for the samples. To avoid the formation of artefacts during electrophoresis the sample application has to be performed very carefully. Therefore, the small chambers are first filled with electrode buffer under which the samples, which contain 8-10% glycerine, are layered. Fig. 4 illustrates the fractionation of a protein mixture in a micro gradient slab gel. All other techniques of separation and staining, e.g., IEF, isoenzyme activity tests etc. can be performed in these slab gels, as in normal micro gels(1). Fig. 4: SDS-electrophoresis in micro slab gradient gels (original size) a. 1-35% acrylamide, b. 1-30% acrylamide. Gels were loaded with ovalbumin (1,6), chymotrypsinogene (2,7), cytochrome c (3,8), bovine serum albumin (5) and a mixture of these marker proteins (4). 50 ng of each protein was applied to the gel and after separation stained with Coomassie Blue.

215 Automatic gel evaluation and a new method for protein determination which is independent of external

The evaluation of gel-electrophoresis

influences

is preferentially

per-

formed by photometry and there are densitometers and photometers commercially available for this purpose. With regard to the simplicity of data aguisition and signal processing the procedure is normally restricted to a linear scan across the sample and neglects the problems of disorted or

inhomogeneous

bands. But in order to achive high accuracy the absorption has to be homogenous within the measuring area and the bands have to be measured in their full extent. Both requirements can be met simultaneously by scanning even one-dimensional gels in two dimensions using a small measuring area and

calculating

the absorption of each band by two-dimensional integration of the measured values. For the evaluation of two-dimensional

gels

this procedure is the most appropriate method of evaluation. The main problems in the two-dimensional evaluation of gel-electrophoreses lie in the number of measuring values which have to be acquired, stored, and processed and in the automatic recognition of bands and their extent for correct

integration.

Measuring an area of 40 mm by 30 mm with a step size of 0.1 mm results in 120,000 measuring values. The evaluation requires methods of digital signal processing and pattern recognition which run for several minutes even on modern mini-computers. Zimmer et al.

(26,27,28) developed a fluorometric evaluation of

two-dimensional thin-layer-chromatograms

in microscale

(40 mm x

30 mm) which is now routinely used in our laboratory. They use a microscope photometer with a mechanical scanning stage

(step

size 0.1 mm, 200 steps per s) which is controlled by a laboratory computer PDP-12

(Digital Equipment Corp.). Besides the

differences in measuring fluorescence and absorption the problems are similar in both the evaluation of chromatograms and

216

gel-electrophoreses. The main difference is that in gradient gels there are finer structures (higher spatial frequencies) than in chromatograms of the same size. Therefore the technique developed for chromatograms is applicable only to homogenous gels. For gradient gels there is a loss of spatial resolution: bands less than 0.6 mm apart are not yet separated automatically. But this disadvantage will be overcome by the development of a signal modell which is better suited to the structure of bands in gelelectrophoreses. Neuhoff et al. (29) recently have described a method for protein determination which in principle is a quantitative "spotanalysis". 0.5 - 5 |il of a protein solution in the concentration range between 0.01 - 10 mg/ml is used for one determination. The sample is taken up by capillary attraction in a 0.5, 1, 2 or 5 |il capillary and transferred to a cellulose acetate strip. By dipping in a solution of either Amidoblack, or a benzoxanthene derivate (Hoechst 2495) dissolved in methanol/ acetic acid the protein is fixed and stained simultaneously. After washing out the excess dye ( 3 x 5 min.) quantitative evaluation can be performed in several ways: 1. By incubation in dioxane/n-butanol the sample is fixed and made transparent and evaluated densitometrically (Amidoblack 10B). 2. by in situ spot fluorometry (30) after staining with the fluorochrome Hoechst 2495. 3. The sample can be dissolved together with the acetate layer completely in dioxane, dimethylsulfoxide or n,ndimethylformamide and evaluated photometrically, or 4. fluorometrically. 5. The highest sensitivity is reached if the fluorochrome (Hoechst 2495) bound to the protein is eluted with 15% NH^OH and measured fluorometrically. There is a linear correlation with a correlation coefficient of 0.999 between the fluorescence and protein in the range of 10 ng - 20 |ig. In addition to its simplicity, the method has the further advantage of being either independent of various external influences, e.g., SDS, mercaptoethanol, Triton X-100, etc., or

217 being affected in a well defined manner. The staining of a protein with Amidoblack is influenced stSchiometrically by SDS (not by mercaptoethanol) whilst the staining with Hoechst 2495 is not affected at all. Since there is a linear correlation between the area of a spot on an acetate layer and the volume applied in the range between 0.5 and 5 p.1, which will only be influenced stSchiometrically by the protein concentration in that volume which in turn is measured by staining with Amidoblack, it is easily possible using a simple iterative calculation based on suitable calibration curves to determine a protein concentration in mg/ml even in an unknown volume between 0.5 and 5 jil. I like to thank Dr. C. Linington for carefully reading the manuscript.

References 1. Neuhoff, V.: Micromethods in Molecular Biology, in: Molecular Biology, Biochemistry and Biophysics, Vol. 14, Eds. A. Kleinzeller, G.F. Springer, H.G. Wittmann, Springer-Verlag Berlin, Heidelberg, New York 1973. 2. Neuhoff, V.: Microelectrophoresis. In Modern Methods of Electrophoresis, Ed. B.J. Radola, Verlag Chemie, Weinheim, in preparation. 3. Quentin, C.-D., Neuhoff, V.: Int. J. Neurosci. _4, 17-24 (1972) . 4. Bispink, G., Neuhoff, V.: In: Electrofocusing and Isotachophoresis, Eds. B.J. Radola and D. Greasslin, 135-146, Walter de Gruyter, Berlin. 5. Cremer, Th., Dames, W., Neuhoff, V.: Hoppe-Seyler's Z. Physiol. Chem. 353, 1317-1329 (1972). 6. Gustke, H.-H., Neuhoff, V.: Hoppe-Seyler1s Z. Physiol. Chem. 259, 1481-1489 (1978). 7. Diez, A.A., Lumbrano, T.: Anal. Biochem. J20, 246-257 (1967).

218

8. Diez, A.A., Lumbrano, T., Rubinstein, H.M.: Clin. Chim. Acta 27, 225-232 (1970). 9. Endou, H., Neuhoff, V.: Hoppe-Seyler's Z. Physiol. Chem. 356, 1381-1396 (1975). 10. Nachlas, M.M., Margulies, S.I., Goldberg, J.D., Seligman, A.M.: Anal. Biochera. J_, 317-326 (1960). 11. Hyldgaard-Jensen, J., Valeta, M., Jensen, S.E., Moustgaard, J. Clin. Chim. Acta 22, 497-509 (1968). 12. Neuhoff, V.: Arzneim.-Forsch.

35-39

(1968).

13. Gustke, H.-H., Neuhoff, V. : Hoppe-Seyler's Z. Physiol. Chem. 36J), 605-608 (1979). 14. Smith, I., Lightstone, P.-J., Perry, J.D.: Clin. Chim. Acta 19, 499-505 (1968). 15. Hosenfeld, V. , Hosenfeld, D.: Med. Welt 22, 749-752

(1971).

16. Green, S., Cantor, F., Inglis, N.R., Fishman, W.H.: Am. J. Clin. Pathol. 52-65 (1972). 17. Fishman, L.: Biochem. Med. 9, 309-315 (1974). 18. Cha, Ch.J.M., Mastrofrancesco, B., Cha, S., Randall, H.T.: Clin. Chem. 2J_, 1067-1071 (1975). 19. Gustke, H.-H., Neuhoff, V. : G-I-T Fachz. f.d. Lab. JJJD, 841-846 (1977). 20. Gosh, N.K., Fishman, W.H.: Biochem. J. 108, 779-792

(1968).

21. Rüchel, R., Mesecke, S., Wolfrum, D.I., Neuhoff, V.: Hoppe-Seyler's Z. Physiol. Chem. 354, 1351-1368 (1973). 22. Zimmer, H.-G., Neuhoff, V.: G-I-T Fachz. Lab. J[9, 481-484 (1975) 23. Neuhoff, V., Mesecke, S.: Hoppe-Seyler's Z. Physiol. Chem. 358, 1623-1637 (1977) 24. Lane, J.D., Zimmer, H.-G., Neuhoff, V.: Hoppe-Seyler 1 s Z. Physiol. Chem., in press. 25. Pöhling, H.-M., Neuhoff, V.: in preparation. 26. Zimmer, H.-G., Neuhoff, V. : In: Informatik-Fachberichte 8. Ed. H.H. Nagel, 12-20, Springer-Verlag, Berlin 1977. 27. Zimmer, H.-G., Kronberg, H., Neuhoff, V. : Proc. 4th Int. Joint Conf. on Pattern Recognition, 834-836, Kyoto 1978. 28. Zimmer, H.-G.: J. Microsc. JM6, Pt. 3, 365-372

(1979).

29. Neuhoff, V. , Philipp, K., Zimmer, H.-G., Mesecke, S.: Hoppe-Seyler 1 s Z. Physiol. Chem. 360, 1657-70 (1979). 30. Zimmer, H.-G., Kiehl, Fr., Neuhoff, V. : Hoppe-Seyler's Z. Physiol. Chem. 360, 1671-1672 (1979).

A SIMPLE, EFFECTIVE AND INEXPENSIVE EQUIPMENT FOR POLYACRYLAMIDE GEL ELECTROPHORESIS IN COOLED MICROCAPILLARIES

R. Peter Institut für Allgemeine Biologie, Biochemie und Biophysik der Universität Salzburg A-5020 Salzburg, Austria

Introduction The different methods of polyacrylamide gel microelectrophoresis in glass capillaries developed by Neuhoff and coworkers (1, 2, 3, 4) have found wide application due to their sensitivity, high resolution and simplicity. No special cooling devices are used in the original procedures since heat dissipation is much more effective than with macromethods. To improve malate dehydrogenase zymograms and protein separations of extracts from the poikilothermal flatworm Crenobia alpina, a simple cooled microelectrophoresis cell was constructed from Plexiglass and ready-made laboratory plastic ware.

Materials and Methods The flatworm Crenobia alpina (Platyhelminthes: Turbellaria tricladida) was obtained from a small mountain stream in Salzburg (Gaisberg, Parsch). Protein extracts prepared and processed as described previously (5) were separated by microelectrophoresis in polyacrylamide gradient gels (2) and by isoelectric focusing in microgels (4). Acrylamide and N,N'methylene-bis-acrylamide were purchased from Serva and recrystallized twice from chloroform and acetone respectively (6). The other reagents were from Merck, Serva and Chroma

© 1980 Watter de Gruyter & Co., Berlin • New York Electrophoresis '79

220

Fig. 1. Microelectrophoresis Cell: a) Longitudinal section (partial view), b) Cross section, c) Exploded view of upper electrode compartment. Ten Plexiglass tubes (A, A' etc.) forming the lower electrode compartments are mounted on a Plexiglass base (B) housing the wire connections. Tube A' is prepared for electrophoresis and contains electrolyte (H) and petroleum ether (I). A cooling jacket (C) surrounds these tubes. A platinum wire (D) 0,3 mm in diameter is inserted into each tube through a small bore and is soldered to a separate wire connection for individual power supply and control of every microgel. The corresponding cable enters the base (B) through the gasket (E). A V2A steel wire 0,5 mm in diameter (F) is cemented onto the top of the cooling jacket parallel to the row of Plexiglass tubes in such a way that it runs through small notches in these tubes. The cement simultaneously insulates the wire outside the tubes. The wire (F) serves as a contact between the power supply and the upper electrodes. The Plexiglass rods (G) prevent the user from touching the tubes. The upper electrode compartment (Fig. l.c) is built from commercially available plastic stoppers and glass tubes. Each gel capillary (J) is held in a glass tube 35 mm long (K) by a polythene stopper (L) from a Beckman microfuge tube. An appropriate hole for the capillary is bored by a hypodermic needle cut even and sharpened. Each glass tube is held by a stopper (M) similar to L which, in turn, is fixed in a laminar

221 Fig. 1 (continued) polythene plug CN). A platinum wire 0,5 mm in diameter (0) dips into the glass tube through a hole in M, penetrates the laminar plug (N) through a lateral hole, runs along the outside of this stopper to get contact to the V2A steel wire (F) of the cell and is bent a few millimeters around the lower edge of the plug (N) for fixation. The whole device is mounted on a Plexiglass rod (P). (analytical grade). Ordinary commercial products were used for the construction of the electrophoresis cell and power supply. The electrical components need not meet any special requirements except being suited for a voltage up to 300 V.

Construction and Function of the Microelectrophoresis Cell Construction and function of the cell are evident from figure 1. As silicon rubber swells in petroleum ether, the original adapters supplied with the microcapillaries cannot be used. The petroleum ether (Merck analytical grade, b.p. 60-80 °C) serves as a heat dissipation and electrically insulating medium. It should not be replaced by electrolyte solution since considerable leakage current will result in this case.

Construction and Function of a Simple Power Supply Any suitable power supply may be used with the cell described. For work in biological laboratories lacking special electrophoresis equipment a small unit was designed that can be connected to any low voltage microscope lamp transformer. This power supply has ten outputs providing independent voltage and current measurement for every microgel. In addition, the full voltage adjusted by the transformer or half this value can be chosen for every output. This facilitates and accelerates electrophoresis runs when inserting the capillaries one by one. The output voltage ranges from zero to 300 V and can be

222

Fig. 2. Connection diagram of the power supply. A: input for 0-9 V~< or 0-300 V = alternatively; B: output (a total of 10 independent channels are provided); T: transformers (each 9 V/ 220 V, 250 mA for the primary winding); D: rectifiers (250 V each); C: capacitors (1 pF each); F: fuses (50 mA each).

223 Fig. 2 (continued) Switches: a: on-off-switch for AC and DC input; b: polarity selector; c: range selector for microamperemeter (lx, 2x, 5x, lOx, 50x); d:range selector for voltmeter (lx, 2x, 3x); e: selector for total or single current measurement (position shown: single); f,f': voltage selectors with central zero position for a total of 10 outputs; g, g': channel selectors for voltage and current measurement (a total of 10 push button switches with mutual release; g is in measuring position). The voltmeter should have a resistance of at least 500 kiloohms. The adjustable resistances connected to c and d are chosen according to the corresponding values of the meters. adjusted continuously by the external lamp transformer. Alternatively, one or two stabilized DC supplies with single outputs may be used instead of a transformer. Proper cables and plugs are wired for the two operation modes. For use with the lamp transformer two diagonal connections are made at the input according to the dotted lines in figure 2.

Results and Discussion Proper construction provided, a convenient microelectrophoresis equipment of small overall dimensions can be obtained according to the above instructions (Fig. 3). The most pronounced improvement in comparison with runs at ambient temperature was achieved with malate dehydrogenase zymograms in gradient gels (Fig. 4a, b). Samples that just gave faint bands when run at room temperature yielded intensely stained patterns after electrophoresis in cooled gels. The improvement was less pronounced with malate dehydrogenase zymograms after isoelectric focusing (Fig. 4c, d). A distinct improvement of patterns stained with Coomassie Blue was observed, too: the cooled procedure gave somewhat sharper bands on a reduced background (Fig. 4e-h). No improvement was found for separations of human serum; this agrees well with the omission of cooling in the original procedures. It seems that considerable advance can be achieved by the equipment described in this paper for micro-

22k

Fig. 3. Overall view of the microelectrophoresis cell and the power supply. The syringe in the left foreground is 105 mm long.

5 mm

v

i r

Fig. 4. Microgels from cooled runs compared with runs at ambient temperature. All samples were from Crenobia alpina extracts. Malate dehydrogenase zymograms : a) two gradient gels, not cooled; b) two gradient gels, cooled; c) isoelectric focusing, not cooled; d) isoelectric focusing, cooled.

1

.

®f T

li

a

fi

b

cd

Coomassie Brilliant Blue R 250 protein staining: e) gradient gel, cooled; f) gradient gel, not cooled; g) isoelectric focusing, cooled; h) isoelectric focusing, not cooled.

I.

ef g h

electrophoresis of heat sensitive aizymes and protein

extracts

from poikiloth ermal organisms. Petroleum ether as a heat dissipation medium for gel electrophoresis has already bean

225 described in somewhat other context by Wieme (7). Alternative inexpensive equipment for microelectrophoresis

in capillaries

without cooling has been developed by RUchel and Wolfrum (8).

Acknowledgements The author thanks the Funds for the Promotion of Scientific Research in Austria for financial support (project no. 1767) as well as Prof. V. Neuhoff and coworkers, the German Academic Exchange Service and the Austrian Ministry of Science and Research for introductory studies of micro isoelectric focusing at the Max Planck Institute of Experimental Medicine in Gottingen. The technical assistance of Mr. A. Schreilechner and Miss E. Chloupek is gratefully

acknowledged.

References 1. Neuhoff, V.: Micro-electrophoresis on Polyacrylamide gels. In Micromethods in Molecular Biology (ed. Neuhoff, V.), pp. 1-83. Springer, Berlin 1973. 2. RUchel, R. , Mesecke, S., Wolfrum, D.I., Neuhoff, V.: HoppeSeyler's Z. physiol. Chem. 354, 1351-1368 (1973). 3. RUchel, R., Mesecke, S., Wolfrum, D.I., Neuhoff, V.: HoppeSeyler's Z. physiol. Chem. _35_5, 997-1020 ( 1974). 4. Bispink, G., Neuhoff, V., in: Electrofocusing and Isotachophoresis (eds. Radola, B.J., Graesslin, D.), pp. 135-146. Walter de Gruyter & Co., Berlin, New York 1977. 5. Peter, R., Wolfrum, D.I., Neuhoff, V.: Comp. Biochem. Physiol. J55B, 583-589 ( 1976 ). 6. Loening, U.E.: Biochem. J. 102, 251 (1967). 7. Wieme, R.J.: Ann. New York Acad. Sci. 121,

366-372

(1964).

8. RUchel, R., Wolfrum, D.I.: G-I-T Fachz. Lab. _18, 1098-1100 ( 1974) .

LINEAR POLYACRYLAMIDE GRADIENT GELS FOR MOLECULAR WEIGHT ESTIMATION OF PROTEINS

Patrick Lambin Centre National de Transfusion Sanguine, 75739 P a r i s Cedex 15, France.

Summary Electrophoresis in l i n e a r polyacrylamide gradient gels can be used for molecular weight (MW) determination of proteins either in the presence or i n the absence of sodium dodecyl s u l f a t e (SDS). In the presence of SDS, there i s a l i n e a r r e l a t i o n s h i p between the log of the MWs and the log of the polyacrylamide concentrations reached by the proteins a f t e r e l e c t r o phoresis in these g e l s . Under the conditions described, MWs between 10" to 10 6 can be estimated i n 3 to 30% l i n e a r gradients with an excellent accuracy (r = 0.998). In the absence of SDS, an i n d i r e c t method must be used. I t i s shown that distance migrated by a protein increases

linearly

as the square root of time of electrophoresis (between 1 to 8 hours). The slopes of the r e g r e s s i o n l i n e s calculated by t h i s method can be used for MW determination of native p r o t e i n s . In f a c t , there i s a l i n e a r r e l a t i o n ship between the log of the slopes and the log of the MWs f o r proteins between 2 x 10" to 106 daltons (r = 0.986).

Introduction The evaluation of the molecular weight (MW) of a protein by electrophores i s i s u s u a l l y performed in polyacrylamide gels of constant concentration with or without treatment by sodium dodecyl s u l f a t e (SDS) ( 1 - 3 ) .

Gradient

g e l s instead of constant concentration gels were proposed since they provide a valuable mean

for separing proteins of widely d i f f e r i n g s i z e s

(4).

In t h i s paper i t i s shown that by using l i n e a r gradient p r e f e r e n t i a l l y to concave g e l s , MW estimation of proteins from 10" to 106 daltons can be

© 1980 Walter de Gruyter & Co., Berlin • New York Electrophoresis '79

228 achieved. This estimation performed in presence or in the absence of SDS, is based upon 3 new relationships ( 5 - 7 ) which will be discussed in this paper.

Material and Methods

Check proteins.

Prealbumin (MW : 13,745), lysozyme (MW : 14,314), ribonu-

clease B (MW : 14,700), hemoglobin (MW : 15,500), avidin (MW : 16,000), soybean trypsin inhibitor (MW : 20,095), papain (MW : 23,426), < chain of IgG (MW : 23,500), chymotrypsinogen (MW : 25,666), carbonic anhydrase B (MW : 28,739), carboxypeptidase A (MW : 34,409), pepsin (MW : 34,700), glyceraldehyde-3-phosphate dehydrogenase (MW : 35,700), lactate dehydrogenase (MW : 36,180), aldolase (MW : 38,994), alcohol dehydrogenase (MW : 39,805), en-acid glycoprotein (MW : 40,000), ovalbumin (MW : 43,000), fibrinogeny chain (MW : 47,000), glutamic oxaloacetic transaminase (MW : 50,000), heavy chain of IgG (MW : 50,000), fibrinogen p chain (MW : 56,000), catalase (MW : 57,500), fibrinogen a chain (MW : 63,500), albumin monomer (MW : 66,290), heavy chain of IgM (MW : 72,000), transferrin (MW : 76,000), plasminogen (MW : 81,000), phosphorylase b (MW : 96,800), ceruloplasmin (MW : 124,000), albumin dimer (MW : 132,580), immunoglobulin G (MW : 150,000), immunoglobulin A (MW : 160,000), reduced a 2 macroglobulin (MW : 190,000), albumin trimer (MW : 198,870), immunoglobulin A dimer (MW : 320,000), thyroglobulin (MW : 330,000), fibrinogen (MW : 340,000), a2-macroglobulin subunit (MW : 380,000), immunoglobulin A trimer (MW : 480,000), a2-macrog1obulin (MW : 760,000), immunoglobulin M (MW : 950,000), were obtained as already described (6). Methods. Linear polyacrylamide gradient gels (2 to 10%, 3 to 20%, and 3 to 30%) were prepared as described in previous papers ( 5 - 7 ) . In the case of 3 to 30% gels, 2 different bisacrylamide percentages C (8) were used : 3.8% and 8.4%. In the other gels, C was always 3.8%, this latter concentration providing completely transparent gels. The polymerized gels (8 x 7,1 x 0,3 cm) enable the simultaneous migration of 12 samples containing one or several proteins. For MW determination in the presence of SDS, proteins (0.5 mg/ml) were incubated in 1% SDS phosphate buffer (0.01 M, pH 7.2) for 3 minutes in a 100°C bath. Cleavage of disulfide bridges was

229 obtained by the addition of 1% 2-mercaptoethanol. Electrophoresis was performed in a continuous buffer system with the same phosphate buffer containing 0.1% SDS only, at 40 V for 16 hr. Distance covered by each protein was measured and related to the Polyacrylamide concentration (T) in the gel. For MW determination in the absence of SDS, two protein mixtures (one of 5 and the other of 4 check proteins) were submitted to electrophoresis at a constant voltage (80 volts) in a Tris-borate EDTA buffer pH 8.2 consisting of Tris : 10.75 g, boric acid : 5.04 g, EDTA-Na2 : 0.93 g, H20 to 1 liter. One sample of each mixture was dispensed every hour in order to obtain times of electrophoresis from 1 to 8 hours. The best linear fits were computed by the method of the least squares.

Results a/ Molecular Weight Determination in the Presence of SDS By plotting the log of the MW against the log of the polyacrylamide concentration (T), reached by the check proteins, a linear relationship log(MW) = a log(T) + b

[1]

is clearly demonstrated. The extent of this relationship is substanted by the results obtained with more than 40 proteins and polypeptide chains between lO 4 to 10 s daltons. TABLE 1 : Statistical data obtained with 3 different structures of gel and 2 different protein incubation conditions (6) Gel Structure

Protein Treatment

Nb of Proteins

Slope

T = 3-30% C = 8.4%

SDS

34

-3.43

8.69

0.998

5.9

T = 3-30% C = 8.4%

SDS + 2-Mer.

31

-3.14

8.30

0.997

4.9

T = 3-30% C = 3.8%

SDS

33

-2.38

7.38

0.995

7.3

T = 3-30% C = 3.8%

SDS + 2-Mer.

31

-2.24

7.22

0.991

7.8

T = 2-10% C = 3.8%

SDS

8

-2.49

7.30

0.996

5.5

Intercept

r

Mean deviation

230

MW

io® -»

10 -

10

20

30

T %

20

10

30

TI %h

Fig.l. (a) Plot obtained with 34 proteins incubated in SDS after electrophoresis in a 3 to 30% gradient gel (C = 8.4%).

(b) Plot obtained with 31

proteins incubated in SDS and 2-mercaptoethanol after electrophoresis in a 3 to 3% gradient gel (C = 8.4%) ; (

) best linear fit ; (

) 95%

confidence limits (6). TABLE 2 : Results obtained by 3 to 30% gradient gel

electrophoresis

(C = 8.4%) of proteins with or without carbohydrates (incubation in SDS) (6)

Intercept

r

Mean deviation (%)

Protein

N

Slope

With carbohydrates

15

-3.421

8.68

0.998

6.6

Without carbohydrates

19

-3.426

8.68

0.996

5.3

All proteins

34

-3.430

8.69

0.998

5.9

231 The best linearity is obtained in 3 to 30% linear gradients with C = 8.4% : correlation coefficient (r) and mean deviation are respectively 0.998 and ± 5.9% in the case of proteins incubated in SDS only, and 0.997 and ± 4.9% in the case of proteins incubated in SDS and reducing agents (see Fig. la and lb and Table 1). Acceptable results are also obtained with the same profile of gels but with C = 3.8% (see Table 1). In addition, 2 to 10% linear gradient gels (C = 3.8%) provide good results for proteins with MWs above 10 5 daltons (see Table 1). It is worthy to note that glycoproteins behave in the same way as do other proteins as far as their MW estimation by this method is concerned (see Table 2). b/ Molecular weight determination in the absence of SDS. By different transformations of the coordinates, we found that the best linear fit is obtained by plotting square root of time versus distance of migration according to the equation /t = aD + b

[2]

t being the time of electrophoresis, D the distance, a and b the slope and Time (V hours )

Distance ( mm }

Fig. 2. Square root of time of electrophoresis versus distance migrated by 9 proteins in the absence of SDS. a : immunoglobulin M, b : fibrinogen, c : a2 macroglobulin, d : transferrin, e : ceruloplasmin, f : albumin dimer, g : albumin monomer, h : al acid glycoprotein, i : soybean inhibitor (7).

232 TABLE 3 : Electrophoresis

in 3 to 20% linear gradient of 9 native

proteins.

Best linear fit and correlation coefficient according to equation

/t=aD+b

Observed MW equation

and deviation

between expected and observed MW

according

to

: log(MW) = 1.54 log(a) + 6.74. Best 'linear fit Slope Intercept

r

Expected MW

Observed MW

Dev.

%

0.0321

0.2841

0 .9996

20,095

27,524

+ 37

ai acid glycoprotein

0.0427

0.1174

0 .9990

40,000

42,740

+

Albumin monomer

0.0508

0.0481

0 .9993

66,290

55,749

-

Transferrin

0.0677

0.2645

0 .9999

76,000

86,796

+ 14

Ceruloplasmin

0.0707

-0.0166

0 .9994

124,000

92,779

-

25

Albumin dimer

0.0752

-0.2131

0 .9988

132,580

101,949

-

23

Fibrinogen

0.1888

-0.0181

0 .9994

340,000

420,528

+ 24

a2 macroglobulin

0.2558

-1.1736

0 .9974

760,000

671,210

Soybean inhibitor

Immunoglobulin M

0.3474

-0.3053

0 .9975

950,000

1,075,340

-

7 16

12

+ 13

MW io't

Fig. 3 : log of MWs versus log of slopes, plot obtained according to the data of Table 3. (a, b, c .. . see Fig. 2) ( (

.02

04

.06 08 1

T" Slope

• ) Best linear fit ) 95% confidence limits

233 the intercept of the regression lines. Fig. 2 shows the experimental data and the best linear fits calculated with these coordinates. Slope

(a), intercept (b) and correlation coefficient obtained with each

protein are given in Table 3. When log of MWs are plotted against log of slopes (a) previously computed, another linear relationship can be evidenced (see Fig. 3). The latter corresponds to the equation log(MW) = c log(a) + d

[3]

In the experiment reported here, c and d are respectively 1.87 and 6.74. Deviations between the expected and the observed MWs are given in Table 3. The correlation coefficient between these 2 parameters is 0.986 and the mean deviation ± 19%. It is worthy to note that modifications of voltage from 40 to 100 volts does not influence noticeably the validity of equations [2] and [3]. Moreover, 3 to 30% or 3 to 20% linear gradient can be used indifferently. Similar results are obtained in Veronal Tris buffer pH 9.8 (7). However when electrophoresis is performed in phosphate buffer pH 7.2, a poorer accuracy is observed and mean deviation increases up to ± 25%.

Discussion In opposition with concave gradients, linear gradients are easily reproducible, this facilitates the establishement of proteins migration laws in pore gradient electrophoresis (9). Equation [1] (log(MW) versus log(T)) can be utilized to determine directly the MW of a protein treated by SDS. In fact, it is supported by the experimental findings proving that the correct parameter to use is T instead of covered distance. Equation [2] (migration distance increasing as the square root of time of electrophoresi s) provides an excellent linearity. As depicted in Table 3, six correlation coefficients out of 9 are above 0.999. Similar results were obtained over an extended range of conditions (7). Equation [3] (relationship between log(MW) and log of slopes calculated by equation [2]) can be used for MW estimation of native proteins. It must be emphasized that this relationship is linear provided that the electrophoretic run is performed at

23h a pH well above the isoelectric point of the protein under investigation. With the check proteins used (isoelectric points between 2.7 and 6.6) buffers above pH 8 are recommanded. With proteins treated by SDS, conformational changes and charges modifications induced by this agent enable a direct estimation of the MW. In the contrary, an indirect method must be used in the absence of SDS. This is the reason why a better accuracy is obtained by the SDS method ; in fact the mean deviation between expected and observed MWs is ± 7% with SDS and ± 19% without SDS. In conclusion, linear Polyacrylamide gradient gels can be applied in combination with SDS alone or SDS plus reducing agents for MW determination of protein subunits and of the chains that form these subunits. It can be also used without SDS for MW estimation of a native protein. Acknowledgments : We are indebted to Dr. J.M. Fine in whose laboratory this work was carried out and to Miss S. Bru for her editorial assistance.

References 1. Shapiro, A.L., Viriuela, E., Maizel, J.V. : Biochem. Biophys. Res. Commun. 28, 815-820 (1967). 2. Weber, K., Osborn, M.: J. Biol. Chem. 244, 4406-4412 (1969). 3. Rodbard, D.: In Methods of Protein Separation (Catsimpoolas, N. ed.), 2, 145-218 (1976) Plenum, New York. 4. Margolis, J., Kenrick, K.G.: Anal. Biochem. 25, 347-362 (1968). 5. Lambin, P., Rochu, D., Fine, J.M.: Anal. Biochem. 74, 567-575 (1976). 6. Lambin, P.: Anal. Biochem. 85, 114-125 (1978). 7. Lambin, P., Fine, J.M..: Anal. Biochem. 98, 160-168 (1979). 8. Chrambach, A., Jovin, T.M., Svendsen, P.J., Rodbard, D.: In Methods of Protein Separation (Catsimpoolas. N., ed.) 2, 27-144 (1976) Plenum, New York. 9. Rodbard, D., Kapadia, G., Chrambach, A.: Anal. Biochem. 40, 135-157 (1971).

SOLUBLE POLYACRYLAMIDE GRADIENT GELS IN THE SEPARATION AND ESTIMATION OF PROTEINS

M.P. Brown, A.V. Braes School of Pharmacy, Sunderland Polytechnic, Chester Road, Sunderland SRI 3SD, U.K. and A.L. Latner Department of Clinical Biochemistry, Royal Victoria Infirmary, Newcastle upon Tyne NE1 4LP, U.K.

Introduction Two-dimensional techniques, in which polyacrylamide gel isoelectric focusing is followed by electrophoresis through a polyacrylamide gel concentration gradient, have been widely used to separate the protein components of serum or plasma (1-3).

We have used one such technique (4)

to give repeatable, high-resolution separations (Fig. 1A) which allow easy visual identification of abnormalities.

Such a qualitative assessment is

often very useful and informative and may be all that is required. However, in some cases more accurate quantitative data are needed, involving proteins or protein fractions for which specific assay methods, such as may be provided by immunodiffusion against specific antisera, are not readily available.

Therefore we have investigated two procedures both of which

enable protein to be recovered in solution from polyacrylamide gels and then estimated colorimetrically.

Methods and Results A.

Electrophoretic elution

An apparatus was constructed, similar to one described by KSrsnas and Roos (5) for elution into a collodion bag.

© 1980 Walter de Gruyter & Co., Berlin • New York Electrophoresis '79

The apparatus incorporated a

236 cellulose acetate thimble attached to the lower end of a glass tube with a suitable watertight connection; both the tube and thimble were components of an ultrafiltration apparatus (Selectron apparatus;Schleicher & Schull, Dassel, F.R.G.) and, in position, provided electrical continuity between two buffer reservoirs.

As with the earlier apparatus (5) elution and any

subsequent concentration and dialysis was achieved in the same thimble. Snail volumes (two m l ) of polyacrylamide gel (7% T at k'L C) were cast using N,N' - methylenebisacrylamide (BIS) as cross-linking agent and the acrylamide and diluent solutions given by Emes et al. (4). Bovine serum albumin, with bromophenol blue as marker, was incorporated into the gel mixture.

After polymerization the gel block was broken into fragments and

sewn into a small nylon bag.

T he elution apparatus was filled with either

0.15M-glycine, NaOH buffer, pH 8.8 (5) or Xris, borate, EDTA buffer, pH 8.3 (6) and the nylon bag containing the gel was then suspended inside the glass tube above the ultrafiltration thimble.

Elution was carried out at

200V for 4.5h, a current of about 5mA being recorded.

After elution the

albumin was concentrated by ultrafiltration using the elution buffer and assayed (7) in the final concentrate. Both buffer systems gave similar recoveries of protein after elution (Table I). The recovery remained high even when the albumin concentration in the gel was reduced.

However a wide range of recoveries was obtained in

duplicate experiments and some adsorption of albumin onto the ultrathimble also occurred. Albumin (jig) 250 100 30 Table I.

Recovery (%)* Glycine, NaOH Tris, borate,EDTA buffer, pH 8.8 buffer, pH 8.3 74; 72-76 (3) -

79; 71-88 (4) 79; 53-100(3) 84; 73-94 (2)

Electrophoretic elution of albumin from polyacrylamide gel.

Bovine serum albumin cast in two ml of polyacrylamide gel, eluted at 200V for 4.5h and concentrated by ultrafiltration. *Data recorded as: mean recovery; range of recoveries obtained from individual experiments (no. of experiments).

237 Attempts to remove this albumin by reversing the polarity of the applied voltage for a few minutes, after the elution period, were unsuccessful and failed to give any noticeable increase in the yield of protein. B.

Polyacrylamide gels cross-linked with ethylene diacrylate

Because of the difficulty in obtaining reproducible results by electrophoretic elution, the use of soluble polyacrylamide gels was investigated.

Ethylene diacrylate was used as the cross-linking agent in

place of BIS since the resultant gels were soluble in lM-NaOH; initially it was thought that these solutions would be suitable for the direct estimation of protein and also radioactivity (8). Small volumes of polyacrylamide gel were cast using the acrylamide and diluent solutions described previously (4) with BIS replaced by ethylene diacrylate of the same molarity. incorporated into the gel mixture.

Bovine serum albumin was again After polymerization, samples (0.5g)

of the gel were dissolved in 9.5ml lM-NaOH overnight at 37° and dialysed against 2 xl 1 volumes of Tris, borate, EDTA buffer, pH 8.3 (6).

Protein

was estimated (7) in the dialysis residue, taking into account any interference by the buffer. Table II shows the results obtained using soluble gels of several acrylamide concentrations.

At high acrylamide concentrations recoveries

of protein were the same as, or better than, those obtained by electrophoretic elution.

At lower concentrations recoveries were

possibly a little lower than those seen in Table I but there was generally much better agreement between duplicate experiments. Polyacrylamide gel concentration gradients were constructed using ethylene diacrylate as cross-linking agent.

Hie gels were intended to

reproduce the separation conditions given by gradient gels cross-linked with BIS.

Concave concentration gradients were obtained using the gradient

mixer (9), casting tower (6) and associated equipment available from Universal Scientific Ltd., London E13, U.K.

The acrylamide and diluent

solutions (Table III) were mixed in the proportions required to give a 5-30% acrylamide concentration gradient. tetramethylethylenediamine

T he concentration of N,N,N'N' -

(TEMED) was arranged so that, as is usual,

polymerisation started at the top of the gels and progressed downwards.

238 Polyacrylamide gel concn. (%) 4 8 15 24 Table II. gel.

Recovery of albumin (7.)* 71; 68; 92; 80;

67-74 66-69 82-98 76-83

(2) (2) (3) (3)

Recovery of albumin after dissolution of soluble polyacrylamide

Bovine serum albumin (0.5 - l.lmg) in polyacrylamide gel cross-linked with ethylene diacrylate. T he gel was dissolved in lM-NaOH, the solution dialysed and protein estimated in the dialysis residue. *Data recorded as: mean recovery; range of recoveries obtained from individual experiments (no. of experiments). When used as part of a two-dimensional procedure with gel isoelectric focusing (10) providing the separation in the first dimension these gradient gels gave a pattern of separated proteins (Fig. IB) which was virtually identical to that seen with a similar procedure using a gradient gel cross-linked with BIS (Fig. 1A).

Acrylamide soin. Acrylamide 71.8g Ethylene diacrylate 4.2g Tris, borate, EDTA buffer, pH 8.3 to 195ml Ammonium persulphate, 10% (w/v) 2.5ml TEMED, 0.3% (v/v) 1.5ml

Diluent soin.

195ml 2.5ml 4.0ml

Table III. Solution used to construct polyacrylamide gradient gels cross-linked with ethylene diacrylate. Anmonium persulphate and TEMED were added after dissolved air had been removed under reduced pressure. The solutionswere mixed in the proportions required to give gels containing a 5-307„ concentration gradient.

239

A

B

Fig. 1. Gel isoelectric focusing followed by gel gradient electrophoresis of 5 ]il of normal serum. Proteins stained with Coomassie Brilliant Blue G (13). As Gradient gel cross-linked with BIS (4). 1, IgA; 2, IgG; 3, IgM;

linked with ethylene diacrylate.

Discussion The mean yields of protein obtained with electrophoretic elution are in close agreement with data obtained elsewhere (5), but we were not able to obtain reproducible results from duplicate experiments (Table I).

There

was much better agreement between the results obtained from duplicate soluble gels.

We found that gels of about 15% acrylamide concentration

showed good physical properties, as did those of Alpers and Glickman (8), and these gels were easy to handle, this being reflected in the high protein recoveries shown in Table IX.

Gels of low acrylamide concentration

were sufficiently rigid for samples to be taken for dissolution but they tended to be sticky and therefore handling difficulties reduced protein yields.

High concentration gels had good handling properties themselves

but dissolved to give very viscous solutionsjthis viscosity probably led to the somewhat lower protein recoveries seen with 24% gels. In the formation of soluble gradient gels the overall acrylamide concentration was increased to give a 5-30% acrylamide gradient compared with 4-24% in gradient gels cross-linked with BIS (4) and the cross-linker: acrylamide ratio was slightly increased, as suggested by Peacock and

2k0 Dingman (11).

These changes resulted in gels with properties almost

identical to those of gradient gels cross-linked with BIS.

By comparing

Figs. 1A and IB it will be seen that the configuration of the concave concentration gradient is not absolutely identical in the two gels shown and that some lateral swelling of the soluble gel has occurred during staining and intensification.

This swelling has increased the resolution

of certain proteins in the soluble gel. T he high acrylamide concentration required at the dense, lower end of the gradient would limit the use of these soluble gels to proteins of molecular weight greater than about 60,000.

This is in agreement with

O'Connell and Brady (12) but is unlikely to prove a serious limitation where, for example serum proteins are under investigation.

In fact the

soluble gels seem to perform best in the 'middle range' of acrylamide concentrations: a large number of serum proteins lie within this range in the gel after separation. References 1.

Latner, A.L. : Biological and Biomedical Applications of Isoelectric Focusing. (Catsimpoolas, N., Drysdale, J., eds.) pp. 303-345, Plenum, New York, London 1977.

2.

Anderson, L., Anderson, N.G. 74, 5421-5425 (1977 ).

3.

Ruchel, R.

4.

Bnes, A.V., Latner, A.L., Martin, J.A. 64, 69-78 (1975).

s

: Proc. Natl. Acad. Sci. U.S.

J. Ghromatogr.

132,

451-468

(1977).

: Clin. Chim. Acta

5.

KSrsnas, P., Roos, P.

6.

Margolis, J., Kenrick, K.G.

7.

Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. J. Biol. Chem. 193, 265-275 (1951).

8.

Alpers, D.H., Glickman, R.

9.

Margolis, J.

10.

: Anal. Biochem. 77_,

168-175

: Anal. Biochem.

s Anal. Biochem.

: Anal. Biochem.

(1977).

25, 347-362 (1968).

35 ,

:

314-3 20

(1970).

27_, 319-322 (1969).

Doerr, P., Chrambach, A. : Anal. Biochem.

(1971).

11.

Peacock, A.C., Dingman, C.W.

12.

O'Connell, P.B.H., Brady, C.J. : Anal. Biochem. 7£, 63-73 (1976). ?i Diezel, W., Kopperschlager, G., Hofmann, E. : Anal. Biochem. 48, 617-620 (1972).

13.

: Biochemistry

96-107

6, 1818-1827

(1967).

QUANTITATION OF PROTEINS SEPARATED BY TWO-DIMENSIONAL PAGE: A NOVEL DYE ELUTION TECHNIQUE COMPARED WITH A DENS ITOMETRIC

O.H.W. M a r t i n i ,

PROCEDURE

J . K r u p p a , R. Temkin

I n s t i t u t f ü r V i r o l o g i e und I m m u n b i o l o g i e der U n i v e r s i t ä t Würzburg D - 8 7 0 0 W ü r z b u r g , Germany und P h y s i o l o g i s c h - C h e m i s c h e s I n s t i t u t der U n i v e r s i t ä t Hamburg D - 2 0 0 0 Hamburg, Germany

Introduction

F o r the q u a n t i t a t i v e e v a l u a t i o n o f s t a i n e d p r o t e i n p a t t e r n s o b t a i n e d two-dimensional

(2-D)

matic technique

( 1 ) , w h i c h employed an O p t r o n i c s A u t o d e n s i t o m e t e r ,

o n l y of

polyacrylamide gel

limited a p p l i c a b i l i t y

g e l s and the p h o t o g r a p h i c be f a i t h f u l ; streaks,

b e c a u s e 1)

the d e n s i t o m e t e r

(PAGE) an a u t o was

scans films

of

r e p r e s e n t a t i o n o f d e n s i t y d i f f e r e n c e s may not

2) computer programmes may m i s j u d g e s p o t b o r d e r s due t o

p r o t e i n s p o t asymmetry,

and b a c k g r o u n d v a r i a t i o n ; tion curves

electrophoresis

by

incomplete

r e s o l u t i o n o f the

proteins,

3) c e r t a i n e l e m e n t a r y knowledge a b o u t

r e l a t i n g dye amounts t o p r o t e i n amounts

calibra-

i s t h e r e f o r e hard

to

o b t a i n. Here we d e s c r i b e an a l t e r n a t i v e q u a n t i t a t i o n

t e c h n i q u e based on dye

e l u t i o n , w h i c h we have a p p l i e d t o the s t u d y o f e u k a r y o t i c

ribosomal

t e i n s . We compare r e s u l t s o b t a i n e d by dye e l u t i o n w i t h e a r l i e r metric

results

pro-

densito-

(1).

Results

I n f o r m a t i o n a b o u t the dye c o n t e n t o f e l e c t r o p h o r e t i c a l l y s t a i n e d p r o t e i n s p o t s may be d e s i r e d f o r parison:

two d i f f e r e n t

separated

types of

com-

1) e s t i m a t i o n o f t h e amount o f a c e r t a i n p r o t e i n s p e c i e s

particular

sample r e l a t i v e

to the amount o f the same s p e c i e s

© 1980 Walter de Gruyter & Co., Berlin • New York Electrophoresis '79

in a

in a

2k2 different sample; 2) estimation of relative amounts of different protein species present in the same sample.

In the first case calibration curves,

for the protein to be studied, are needed correlating protein amounts electrophoresed with dye contents of their spots; for the second type of comparison additional

knowledge

is required concerning the dye affinities

of the proteins to be compared. W e first established calibration curves for each of the 'tOS subunit proteins of rabbit reticulocyte

ribosomes,

assessing at the same time the reliability of our dye elution procedure, and then studied the variation of the dye affinities within the set of some 30 basic proteins. Preparation of proteins and 2-D electrophoresis. We prepared the proteins either from salt-washed

(300 mM KC1, 10 mM M g C l 2 , 40 mM Tris-HCl, pH 7.6)

rabbit reticulocyte polysomes or from 40S subunits derived from polysomes by dissociation with puromycin at high ionic strength (2). Protein was extracted from ribosome suspensions with two volumes of acetic acid in the presence of 66 mM Mg

(3), dialysed against lower acetic acid con-

centrations, and lyophilized. The protein was dissolved

in the sample

buffer for 2-D electrophoresis

(4) and its concentration determined

For 2-D PAGE w e chose a method

CO

acrylamide gel cylinder

where separation at pH 4.5 in a 5 %

is followed by separation in a 10 X gel

ing SDS. The cylindrical gel

(5).

contain-

is adapted by dialysis to second dimension

buffer conditions. This system is well suited for quantitation studies because 1) at pH h.5 protein losses at the origin are minimized; 2) no residual protein remains stuck in the first dimension gel; 3) visible streaking which may occur in the second dimension is usually to a few proteins not causing serious problems protein patterns the resolution be estimated

restricted

(Fig. 1a); 4) on subunit

is high; 5) molecular weights can easily

(6).

Dye elution. After staining with Coomassie Brilliant Blue R-250 and destaining

in acetic acid/methanol/water

(10:10:80 by volume) under stan-

dardized conditions the protein spots were punched out with dentists' copper rings. For spots which overlapped w e used a scalpel blade to cut the gel between them at minimal colour density. The gel pieces were weighed, transferred to conical centrifuge tubes, and 3 volumes

(volume

243 always refers to gel weight) of 1.5 M NaOH was added to overcompensate the acetic acid of the gel pieces by approximately 1 M. The gel pieces were carefully disrupted using fitting perspex pestles. The tubes were incubated in a water bath at 75°C for 1 h. After cooling a twofold volume of isoamyl alcohol was added and the tube contents were mixed thoroughly on a vortex, moving the pestles up and down. The contents were reacidified with 1 volume of 30 % acetic acid, mixed briefly and centrifuged at 1000 rpm and it°C. The absorbance at 560 nm of the alcohol phase was measured in microcuvettes of 1 cm path length. Dye was also eluted from protein-free gel pieces in order to determine the background. The relationship

/.S «B x 560 ~ 560

,,S x

„ =

was used to calculate the dye content.

. x

y e

^ c o n t e n t

In the formula S means spot, B

means background, and K is the product of the absorption coefficient of the dye and the path length of the cuvette. For simplicity we ignored K and expressed the dye content in arbitrary units. In the first round of extraction nearly 90 % of the alcohol were recovered containing

>80% of the dye. With a second round the dye reco-

very could be increased to 95 %• Correlation between protein and dye contents of spots. Six different amounts (in fig: 1*3, 80, 131, 216, 35*», 582; at higher protein loads resolution becomes unsatisfactory) of the same preparation of ^0S subunit protein were electrophoresed. A typical separation is shown in Fig. 1a. By plotting the dye amounts extracted from corresponding spots of the six separations against the protein amounts used, calibration curves (Fig. 1c) were obtained for the complete set of 33 proteins. The curves for the larger proteins (>16 000) were linear between 130 and 580 ^g total protein load. Since, with few exceptions, the ribosomal proteins may be assumed to be present in equimolar amounts (8), we could calculate from the individual molecular weights and their sum, that the linear range corresponds to approximately 5 to 30 ^ig of protein per spot. In this range the results of single measurements rarely deviate more than 10 % from the value indicated by the calibration curve. At low protein loads and for the low molecular weight proteins the percentage error be-

2kk

O 2QO

2b?r

©-

c 33

1.D.

°o0 0

4Q

• I ? O5

©

12 b fcO ° 0 8 12e 13a I 8 3 9) C < ^ J 1 313b .0^013c 150

B

applied as an 8 cm band, are separated by preparative TLG-IF in 2 mm Sephadex G-75, 1 $ Ampholine pH 3.5-11, 6 M urea. pH

Fig. 3. Analytical TLG-IF separation of membrane extract of Ehrlich ascites tumor cells. Sephadex G-100 superfine, Ampholine pH 4-6 and 3.5-10 (1:1), Triton X-100. Detection: phenanthrenequinone and Coomassie Blue G-2 50. START

TLG

CNBr fragments. In protein sequence work purification of large fragments, that are generally aggregated and poorly soluble, has become a bottle neck. Fig. h shows analytical separations of CNBr fragments which, however, have an unfavourable charge heterogeneity at their COOH-terminus, i.e. homoserine or homoserinelactone. Turnip peroxidase TP 7 (prepared by Dr. G.Mazza) yields three large fragments, 1 (l83 residues), 2 (70 residues), and 3 (^0 residues). 3 contains no lysine and does not bind Coomassie G-250, but stains with both the Pauly and the arginine-specific reagents. The bacterial K88 antigen (prepared by Dr. P.Klemm) never had the two similar and highly aggregated CNBr fragments separated previously.

253 1

1 1

PH 2

°

RN TLG

B D

BPB

" • 5 3

1

A

IF

- 6

j

pH

TLG

1 2 |

M 1

PAULY A RGIN1

- 5 6 -

1 II 1

NE

59 3

3

- 7 0 • 7 9 - e - 9

8 2

9.3

j S i ^

8

START

START

Fig. 4. Analytical TLG-IF separation of CNBr fragments (l mg samples) in Sephadex G-75. (A) Three CNBr fragments of TP 7 (pi 11.6) are located after IF in 1$ Ampholine pH 3.5-11, 6 M urea, (B) TWO strongly aggregated CNBr fragments of K88 (pi 5) are separated in 1% Ampholine pH 3-5» 6 M urea, 1% Triton X-100

Conclusion Two-dimensional TLG-IF separation permits a fast determination of apparent MW and of pi on minute amounts of soluble sample and allows an upscaling for preparative purposes.

References 1.

Radola, B.J.: Biochim.Biophys.Acta 295, 412-428

2.

TLG. Manual from Pharmacia Fine Chemicals AB.

3.

Reisner, A.H., Nemes, P. & Bucholtz, C.: Anal.Biochem. 64, 509-516 (1975). Vesterberg, 0., Hansen, L. & Sjosten, A.: Biochim.Biophys. Acta 4¿I, 160-166 (1977)-

4.

(1973).

1

ENHANCEMENT OF LOAD CAPACITY IN ISOTACHOPHORESIS

F.M. Everaerts, F.E.P. Mikkers and Th.P.E.M. Verheggen Department of Instrumental Analysis, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands

Introduction Often minute amounts of substances in complex mixtures need to be analysed and sample pre-treatment procedures, such as extraction, column techniques or salting-out procedures have to be used. Using the column-coupling system (1) a large amount of sample can be introduced, often without any sample pre-treatment. Moreover the equipment described has a greater potential than simply increasing the maximal load capacity (2,3). Combination with other separation techniques, such as liquid chromatography or zone electrophoresis, can be considered.

Instrumentation The column-coupling device consists essentially of three sections: - The pre-separation compartment; - The bifurcation block with a linearised conductivity detector (so-called "tell-tale" detector); - The final separation compartment. Besides the bifurcation block, all other compartments and the way of mounting the detectors and the capillaries are extensively discussed in the reference 4. The bifurcation block consists of three different channels: 1. A circular wide boring of e.g. 0.8 mm, in which the "tell-

1980 Walter de Gruyter & Co., Berlin • New York Electrophoresis '79

256 tale" detector is mounted. This boring is connected with a comparable wide bore PTFE pre-separation tube. 2. A circular narrow boring of e.g. 0.2 mm, in line with the wide boring. Onto this boring the narrow bore PTFE separation tube is mounted. 3. A rectangular channel (1 mm width and 0.05 mm height) perpendicular on the borings of 0.8 mm and 0.2 mm. This channel forms the connection with the counter-electrode compartment, applied during the pre-separation. The dimensions of the rectangular channel are chosen in such a way that diffusional effects are minimized and an optimal trapping is allowed. Since the separation compartments are in line with each other, there is only a minimal mixing of already separated zones (pre-separation) during trapping. It should be emphasized that the distance between the pre-separation and separation compartment in the bifurcation block is only 0.05 mm. Because the heat-transfer in this rectangular channel is excellent, high pre-separation currents are still permitted. The "tell-tale" detector identifies the pre-separation. Because the zones, under carefully chosen operational conditions, migrate all with equal velocity, the time needed for a zone-boundary to cover the distance between the "telltale" detector and the bifurcation is constant. The delay time, t^, is readily determined by measuring the dV-(dt)

1

of the

current stabilized power supply in a leading electrolyte/ terminating electrolyte experiment or measuring the migration time of a dye. Once the zone(s) have been identified by the "tell-tale" detector, the moment at which they reach the branching-off is well known. Hence the zone(s) of interest can be easily selected, even if they are not migrating consecutively. These zone(s) are further analysed and detected in the analytical column via both the UV and a measured conductivity detector. The maximal load capacity (2,3) can be increased with an order of magnitude, without increasing the total analysis time. The automation of the column-coupling

257 system is discussed in ref. 5.

Experimental The equipment with the coupled-columns was tested and compared with the conventional equipment (1,6,7). Straight calibration curves, injected amount versus zonelength, were obtained and no loss of material due to the construction of the bifurcation block could be measured. Especially the equipment proved to be very usefull, if ionic material at urin and serum level need to be analysed. Reproducibilities, even in the day to day variation, better than 2% were obtained. Analytical results obtained via isotachophoretic analyses were compared with established clinical determinations.

Uric acid determination in serum in aid of the study of primary gout. Isotachophoretic uric acid determination (injected volumes 1-3 ul serum) were compared with enzymatic assays, performed at the laboratory of the department of Neurology (Faculty of Medicine, Nijmegen, The Netherlands) with an ABA 100 bichromatic analyser (ABBOTT). The determination of uric acid is based on the successive action of three purified enzymes which are added to the reaction mixture: uricase, katalase and aldehyde dehydrogenase (7) . The formation of NADPH from NADP + in the latter reaction (measured both at 380 and 340 nm) is used for the quantification of uric acid. As standards sera with known concentrations of uric acid were used. To serum, extensively dialysed against 0.9% NaCl, a standard amount of uric acid was added, giving a final concentration of 474 iiM. The isotachophoretic analysis performed under

258 Table 1. System, pH 5.0, for the isotachophoretic determination of uric acid.

Anion Concentration Counter ion pH Additive

leading

terminating

electrolyte

electrolyte

CI" 0.005 M

MES~

EACA +

0.005 M •+ m Tris

5.0

6.5

0.25% HEC

-

conditions listed in Table 1 yielded recoveries of 99.0-100.5%. To estimate the amount of uric bound to serum proteins under our experimental conditions the recovery from ultrafiltered and nonfiltered samples was compared. When undialysed serum was passed through an Amicon CF 25 filter (M.W. cut off: 25.000) 85.1% of the total serum uric acid was recovered in the ultrafiltrate, indicating that approximately 15% was bound

Fig. 1 Isotachophoretic analysis of the determination of urate in serum: a = non-ultrafiltered; b = ultrafiltered (M.W. cut off: 25.000).

259

to protein with a M.W. exceeding 25.000 (fig. 1). The lower amounts of uric acid in the ultrafiltrate as compared to nonfiltered samples was not due to the CF 25 filter: when a standard solution of uric acid (474 uM in water) was passed through it, the recovery was 99.4%. In addition, the effect of high pH on the binding of urate to serum protein was studied. The pH of normal serum samples (pH 7.2-7.4) was adjusted to pH 10.0 and after ultrafiltration still approximately 7% (instead of 15%-20%) of total urate was bound. A small part of the sera showed some turbidity, as judged from visual inspection. Those samples were rapidly passed through a Millipore filter (Millex: 0.22 y). This did not affect the recovery of uric acid. The identity of uric acid was confirmed in various manners. After pre-incubation of the sample with uricase, it was demonstrated that the uric acid zone was abolished in the traces of the conductivity detector and the UV-absorption detector. Injection of an extra small amount of uric acid, gave an increased length of the uric acid zone. Under the operational conditions, listed in the Table 1, the molecular extinctioncoefficient of urate was identical at 254 and 280 nm.

Comparison between Enzymatic and Isotachophoretic Results Table 2 shows that there exists a good correlation between the data obtained with sera (not filtered and ultrafiltered over CF 25) from 4 controls analysed with the enzymatic and the isotachophoretic method. The day to day variance was about 2% with the isotachophoretic and approximately 10% with the enzymatic method. It is well known that several metabolites and drugs can interfere with the enzymatic determination. When homogentisic acid was added to serum samples (0.5-5.0 g /l) higher values for uric acid were obtained with the enzymatic method, whereas no effect was seen with isotachophoresis.

260 Table 2. Results of Enzymatic and Isotachophoretic Uric Acid Determinations in Serum from 4 Healthy Control Individuals. Sample no.

enzymatic method NF UF % bound

NF

isotachophoresis UF % bound

1

383

283

26%

392

282

2

292

233

20%

294

224

28% 24%

3

483

400 317

483 361

14%

375

17% 15%

415

4

298

17%

Uric acid concentrations in pM. NF: not filtered ; UF: ultrafiltered (CF 25 filter). The present isotachophoretic method for the determination of uric acid levels in serum is quantitative, reliable and reproducible. In contrast to some HPLC procedures (8) there is no need for deproteinisation: the samples can be applied directly, but also a HPLC system without deproteinisation has been described (9). However, the advantage of isotachophoresis over HPLC is the flexibility of the system. Once an electrolyte system has been chosen the analyses can be done with very low coefficients of variation (2%). In the present stage the isotachophoretic serum uric acid determination is more accurate than the enzymatic method, although the latter is faster when automatised. A decisive advantage of isotachophoresis (and also of HPLC) is the possibility to run whole metabolite profiles. In contrast to HPLC, there is no need to equilibrate columns enabling a fast switching from one electrolyte system to another when different conditions have to be tested. Both for experimental and clinical purposes alternative possibilities are opened up, such as for the pharmacokinetic analyses of drug metabolism, or for studies on the binding of metabolites te serum proteins.

261

Valproic acid determination in serum in aid of epileptic patient control. Sodium valproate is known as an useful anticonvulsant drug in primary generalized epilepsy. The determination of the anticonvulsant in serum is of importance for the correct treatment of epileptic patients, especially in establishing the pharmacotherapy. Optimal therapeutic serum levels are known to be ca. 60 pg/ml. Several gaschromatographic methods have been described (10-16), each with its own limitations and advantages. A disadvantage common to all chromatographic procedures is the sample treatment prior to chromatography. Depending on the method solvent extraction, derivatisation and evaporation have to be used. Isotachophoretic valproic acid determinations were compared with gaschromatographic determinations, performed in the clinical laboratory of the Kempenhaeghe Epileptic Centre in Heeze (The Netherlands). For the gaschromatographic determination a Packard Becker 421 gaschromatograph was used with a glass column, packed with 5% FFAP on Chromosorb WHP. The injection temperature was 160°C, whereas the oventemperature was kept isothermal at 150°C. Nitrogen was the carrier gas and FID detection at 175°C was used. Cyclohexanecarbonic acid was applied as the internal standard. The isotachophoretic analysis was performed in the device with coupled-columns and operational conditions were used as listed in Table 1. The calibration line (injected amount versus zone-length) was made from calibration points, determined from standard solutions in water and in serum. Additionally several test-sera, containing various other drugs, were analyzed. A good linear relation was found with a correlation-coefficient of 0.99914. The response was found to be 6.12 ng/sec and the mean error per point was 3.8 ng (n=26). Using these calibration data,valproate levels in twenty

262 patients were determined. Isotachophoretic results were compared with the routine gaschromatographic results. The experimental slope deviates only 1% from the ideal value of unity (17). A positive intercept of 4.9 ng however is present, although the determination of 4 test-sera in calfserum was in agreement with the expected values.

References 1. Everaerts, F.M., Verheggen, Th.P.E.M. and Mikkers, F.E.P., J. Chromatog., 169, 21 (1979). 2. Mikkers, F.E.P., Everaerts, F.M. and Peek, J.A.F., J. Chromatog., 168, 293 (1979). 3. Mikkers, F.E.P., Everaerts, F.M. and Peek, J.A.F., J. Chromatog., 168, 317 (1979). 4. Everaerts, F.M., Beckers, J.L. and Verheggen, Th.P.E.M., Isotachophoresis, Elsevier, Amsterdam, Oxford and New York, 1976. 5. Verheggen, Th.P.E.M., Mikkers, F.E.P., Kroonenberg, D.J. and Everaerts, F.M., First International Symposium of Isotachophoresis, Elsevier Symposium Series on Chromatography and other Analytical Techniques, in press, 1979. 6. Oerlemans, F., Verheggen Th.P.E.M., Mikkers, F.E.P., Everaerts, F.M. and Bruyn, C. de, Adv. Exp. Med. Biol., in press, 1979. 7. Verheggen, Th.P.E.M., Mikkers, F.E.P. and Everaerts, F.M., Prot. Biol. Fluids, 27th Coll., in press, 1979. 8. Milner, J.A. and Perkins, E.G., Anal. Biochem. 8J3, 560 (1978). 9. Slaunwhite, W.D., Pachla, L.A., Wenke, D.C. and Kissinger, P.T., Clin. Chem., 21, 1427 (1975). 10. Jacobs, C., Bojasch, M. and Hanefeld, F., J. Chromatog., 146, 494 (1978). 11. Willox, S. and Foote, S., J. Chromatog., 151, 67 (1978). 12. Berry, D. and Clarke, L. , J. Chromatog., 156, 301 (1978). 13. Libeer, J., Sharpe, S., Schepens, P. and Verkerk, R., J. Chromatog., 160, 285 (1978). 14. Gyllenhaul, 0. and Albinsson, A., J. Chromatog., 161, 343 (1978). 15. Chabert, G., Bondon, P., Revol, M. and Plasse, J., in Recent Developments in Chromatography and Electrophoresis,

263 Chromatography Symposia Series, Vol. 1, A. Frigerio and L. Renoz (Editors), Elsevier, Amsterdam, Oxford and New York, 123 (1979). 16. Libeer, J., Schepens, P. and Lakiere, F., ibidem 16 3. 17. Mikkers, F.E.P., Verheggen, Th.P.E.M., Everaerts, F.M., Hulsman, J. and Meyer, J., J. Chromatog., submitted, (1979).

A MODEL FOR THE USE OF MORE THAN ONE SINGLE BUFFERING COUNTERION

IN

COMMON

ISOTACHOPHORESIS

P. J U S T S V E N D S E N

AND

C.

SCHAFER-NIELSEN

The Protein Laboratory, University of Copenhagen, 34 Sigurdsgade, 2200 Copenhagen N, Denmark.

Introduction Isotachophoresis is generally described to comprise three ions in a steady state: The leading ion, the terminating ion and the common counterion (1,2,3,4). The buffering action of the counterion, i.e. its pK-value, has a great influence on the stability and steady state conditions of the system, i.e. the pH in the terminating ion zone and thereby the net mobility of the terminating ion. Each counterion therefore has a limited pH-range within which it is useful, and scanning for optimal separation conditions will thus involve the use of different counterions in separate experiments. A counterion with more than one charge and pK-value could be considered to expand the buffering-range, but the user would still be restricted by the availability of such compounds, even though multivalent ions are easily included in the calculations (2,4,5). Below we describe a model for using more than one counterion at the same time. This will enable the user to compose any combination of buffering counterions, monovalent and/or multivalent, so that buffering counterions will allways be present in both the leading ion electrolyte and the terminating ion electrolyte. The model also allows for the presense of an interfering ion, an ion migrating in the same direction as the system itself

© 1980 Walter de Gruyter & Co., Berlin • New York Electrophoresis '79

266 and present throughout the system. The net mobility of the interfering ion must exceed that of the leading ion or be less that of the terminating ion. In the cases where the interfering ion is equal to the leading ion or the terminating ion the reader is referred to reference (6). The fully extended model for isotachophoresis and steady state electrophoresis, covering monovalent and multivalent systems, will be published elsewhere (5) as well as a computer program (FORTRAN IV) (7), based on this extended model.

Calculations and Discussion Symbols: C L , C T

Concentration of counterion in leading -and terminating ion zone

L, T

Concentration of leading ion and terminating ion Concentration of interfering ion in leading and terminating ion zone

IL, IT Al, At

Concentration of alien counterion in leading -and terminating ion zone

NZ

Molar net charge

H^, 0H l

Hydrogen -and hydroxy1 ion concentration in leading ion zone

H t , 0H t

Hydrogen -and hydroxy1 ion concentration in terminating ion zone

R_

Relative concentration of counterion

Q

Molar conductivity

The user sets the pH, the concentration of the leading ion, the concentration of the interfering ion and alien counterion, and the concentration-ratio of the counterions relative to counterion 1. When the pK/mobility sets of each ion is known, C T . can be calculated from Li C

after C ^

Li =

is found from

C

Lr

R

CLi

267 n m NZ l .L + OH l -H l + x NZ i l ..I L i - Z NZ a l ..A l . CL1

\

=

i

(2)

J^CLi^CLi where k, m, and n are the number of counterions, alien counterions and interfering ions. The corresponding concentrations of each counterion in the terminating electrolyte can be found by

C

Ti

_

nm

T -(nmL + nmCLi) nm L •(nmT + n m C T i ) ' c L i

(3)

where the nm values refer to the net mobility of the ions indicated by the indices. The concentrations of the alien counterions in the terminating electrolyte is found by using the same equation, but these ions are treated separately, since they are present in constant concentration, set by the user, in the leading electrolyte. The steady state concentration of the interfering ion in the terminating electrolyte is found by nm T . (nmL - nm I L i > I T i - nm L . (nmT - n m I T i

'^i

(4)

The steady state concentration of the terminating ion is obtained from k m n nm T .(L.Q L + z C L i - Q C L i + . 2 A L . - Q A L i + 2 I L . . Q I L i + H L . M H + O H L . M O H ) 1=1 i=1 i=1 T=nmT • Q m L T . 2 C Ti - Q CTi + .

1=1

1=1

'°ATi+ . V T i 1=1

QT

-QlTi+W^T^OH (5)

268 where M., and M... are the mobilities of the hydrogenion and H Un hydroxylion. The ion-fraction, X n , for a constituent with z charges isfound from (in case of a weak acid)

X

=

n

10

n n-pH - 2 pK i =1

(6)

k z k-pH - E pK 1 + E 10 m=1 m k=1

which is also valid for a weak base by multiplying the exponents with -1. It is evident that the pH in the terminating ion zone must be known to solve the equations 3-6, but this cannot be calculated and must be found by iteration. The user (i.e. the program when executed) suggests an initial pH in the terminating elec• trolyte, pH T , according to which the calculations are performed. The program will then calculate a new pH in the termina*

ting electrolyte, pH T , by matching both sides of the electroneutrality equation and test for unity m

k *

•

PH T = pH T - log

, Z 1 C Ti- N Z CTi + .1 j=:1i A Ti- N Z ATi + H T i=1 n T

-

NZ

+

I

T J1 Ti'

NZ

ITi

+0H

(7)

T

*

pH T is then used for the next iteration until the change between the iterations of one of the parameters T, C T and pH^, is sufficiently small. We discontinue the iteration procedure when T changes less than 10 ^ (molarity) between two subsequent iterations. The program is designed so, that also the conditions in a leading ion electrolyte can be calculated to match the desired properties of a terminating ion electrolyte.

269 In the following computer-simulated experiments the leading ion is acetate 0.05 molar. The pK/mobility data-sets are from the littérature (8,9).

p H L Acttat*, 4.71

Figure 1. Net mobility function of glycine versus the pH in the leading ion zone. The net mobility function of glycine versus the pH, pH T , in the leading ion zone is shown in fig. 1, using three different single counterions, Tris (A), pyridine

and ammediol ($).

The pH, pH^/ in the terminating ion zone is shown on the right ordinate. The curves A, B, C are the net mobility functions of glycine when 6 counterions are used at the same time, and it is clearly seen that the function is strongly linearized when the counterions are present in equimolar concentrations in the leading ion zone, B (Pyridine, 4-picoline, imidazole, Tris, morpholine and ammediol). By varying the ratio, the slope can be manipulated, A=1:2:3:4:5:6 and C= 6:5:4:3:2:1, and adjusted furhter. By employing such a system over a wide pH-range for optimizing a separation problem, the environment is kept more constant since the same counterions are constantly present. It is even possible to calculate systems with constant concentration-ratio in the terminating ion zone. The net mobility function of 6 terminating ions, Mes, Glygly,

270 asparagine, glycine, g-alanine and e-amino-caproic acid (EACA) is shown in fig. 2, and the same effect can be observed (ratio for the 6 counterions = 1:1:1:1:1:1). The dotted lines indicate the net mobility function when only Tris is used. NM, GLYGLY, 1.21 ASP t(?

20 18 _ 16 14

G

12 _

//

10

/

8 6

//

4

2

4

r

_

i—i—i—i—i—i—r 5

6

7

8

P^t Acute, 4 Tl Figure 2. Net mobility function of 6 terminating ions versus pH . By comparing the single-counterion systems with the multiple counterion systems it appears that the pH in the leading ion zone becomes less critical since the actual increase in net mobility using single counterions occurs over a considerably narrower pHT-range. The massbalance for each individual counterion (over an acetate/asparagine boundary) is demonstrated in fig. 3. The concentration-ratio for the 6 counterions is 1:1:1:1:1:1

(•) in the

leading electrolyte. The steady state concentration for each counterion is drawn versus

pH in the leading electrolyte,

pyridine (A) , 4-picoline (•) , imidazole (•) , Tris (A) , morpholine (O) and ammediol ($). Included in the figure is also the constant concentration of Temed in the leading ion zone and

271 C T , Aiparagina Zone

Figure 3. Massbalance for multiple counterions and Temed over an Ac/Asp-boundary. the corresponding concentration in the terminating electrolyte. The presence of Temed, which is necessary for polymerizing acrylamide, in the leading electrolyte is easily included in the calculations as a divalent alien counterion and thereby obtaining most correct values for T, pH T and nm T , and for preparing the buffers as precisely as possible. The correction for Temed is shown in fig. 4. The lower dotted line is the net mobility function of glycine with Tris as the counterion and without correction for Temed. The upper dotted line indicates the corresponding buffering capacity of the leading ion electrolyte. The net mobility curve when corrected for Temed ($) shows some deviation, which is most pronounced

272

PH,Acatate, 4.72 Figure 4. Buffering capacities and correction for presense of Temed. at low pH , since the required constant concentration of this Li compound will be more and more dominating with decreasing pHLThe buffering capacity is also affected, curve (•). The net mobility function using the 6 counterions mentioned above, curve (•) deviates from the corresponding uncorrected curve, compare with curve B in fig. 1, and also note that the concentration of Temed is the same as for the counterions at pH^. — 4.6 see fig. 3, and we recommend to correct for the presense of Temed at pH — 0) -C • — C Ol :0 S

C Q. in""2

i F k : ( x ' y ) d x d y •

Spots j and k are considered to be connected if their overlap greater than some threshold t.

is

The net building algorithm proceeds in

the following manner: Any spot j, which is not already a member of another net, is selected to be a seed, and is placed at the head of a list which will eventually enumerate all PGP's which form this

33^ complex. list.

All spots k with

> t are then added to this

The list is then examined, one element at a time, to

find additional spots with an overlap greater than Twith at least one member of the list.

These additional spots are

appended to the list, and the procedure continues, until no more PGP's can be added. The sorting of the parameter file by spot position reduces the search time (and main memory requirements) considerably.

The algorithm is

modified to induce a premature termination of the complex growing if too many spots are being added.

Only the first p (usually 15) spots in

the membership list are considered when searching for additional members.

The spots in the list which are added after the first p

elements are processed in the usual way during the fitting procedures, but their parameters will not be updated. membership in other complexes.

They remain eligible for

Spots from the first complex that are

overlapped with these special spots can be included in other complexes in a similar manner.

In this way very large groups of spots can be

considered as smaller complexes, with boundary zones to minimize the effect of not fitting all the spots in the group simultaneously.

The

procedure is written in both PL/I and FORTRAN, and can process a file of 1000 spots in about 15 seconds on the PDP-11/60 computer. The total image is processed from top to bottom, one line at a time. As each complex is encountered, a region of main memory is dynamically allocated to contain its data structures, and the complex is marked as active. read in.

Processing of this complex continues as new image lines are Details of the actual processing will be discussed below.

There may be several complexes active at any one time.

After the

entire region of a complex has been processed, the parameters are updated, convergence is checked, and the memory is deallocated.

The

cycling of the grouping program and the processing program continues until all complexes are converged.

Complexes are considered converged

if all of their member spots are converged.

Conversely, if a complex

is not converged, then all of its spots are marked not converged, regardless of the changes of their parameters on the previous cycle.

335 The F i t t i n g

Kernel

The parameters are optimized in a l e a s t squares sense using a l i n e a r d i f f e r e n t i a l - c o r r e c t i o n technique ( 9 ) . image I ( x , y )

The method assumes that the

can be most c l o s e l y approximated by the f u n c t i o n n

F(x,y,c^,

...

The c

!s

r e p r e s e n t the spot parameters

k=1 f o r a l l the spots in the complex, and are t o be determined by the o p t i m i z a t i o n process.

The estimates f o r the c ' s at any g i v e n stage in

the f i t t i n g process are denoted by c ' , c ' ,

. . . , cm '. I t i s assumed that the estimated parameters are c l o s e t o the a c t u a l , or converged, parameters and that ar ^JL

m F ( x

-y-C1

c m>

~

^

+

The p a r t i a l d e r i v a t i v e i s evaluated at c. =c.' . 1 1 r(x,y)=F(x,y,c,, 1 the current

...

,c ) - I ( x , y ) m

.

The r e s i d u a l

can then be approximated in terms o f

parameterization: m

r(x,y)

(C.-C!)

~

[F(x,y,c]

. . , c ^ - I (x , y ) ]

+

I

3F

Sci

,

where 6c. i s d e f i n e d as c . - c . ' . These are the values that need to be l 1 1 approximated to determine the new parameters. The c a l c u l a t e d r e s i d u a l R(x,y)

(based on the current p a r a m e t e r i z a t i o n ) R(x,y)

= F(x,y,c!_, . . .

i s d e f i n e d to be

-

I(x,y)

.

The square o f the r e s i d u a l i s i n t e g r a t e d over the region o f the complex (denoted by fi ) to form the f u n c t i o n Q, which i s t o be minimized: Q = //

ii

r2(x,y)dxdy

~

// R ( x , y )

a

+

2 r - ^6cc .. I T d x d y . . 3c. 1 1=1 l J

Q i s considered to be a function o f the ^ c ^ ' s .

r1

The equations

336 3Q — — = 0 hold at the minimum of Q for k from 1 to m. 36c Evaluating the k-th p a r t i a l d e r i v a t i v e , we get 3Q 3 6 c,

2//

R(x,y)

+

I i=1

3 c

i

\

3F 3c,

dxdy

The above formula represents a s e t o f simultaneous equations which can be put i n t o the matrix form

B A = D, where B i s an m by m matrix with B. . = f i ^ a

The column vector D

IL-

^

dxdy

A has elements 6c ^ , and the vector D has elements = //[I(x,y)-F(x,y)] H

The matrix equation i s e a s i l y solved for the parameters.

1

dxdy

A , which i s used to update

Note that in the matrix equation above, only the

vector D i s a function of the image d a t a .

This f a c t eases the memory

a l l o c a t i o n requirements s i n c e the m X m matrix does not have to be c a l c u l a t e d u n t i l a f t e r the l i n e processing of image data i s

finished.

The region fi over which the i n t e g r a t i o n s are performed i s s e t to be t h a t area for which F(x,y,c!|

c^) > e .

The value of e i s small

and i s chosen to be c o n s i s t e n t with the overlap threshold grouping.

In p r a c t i c e , the region i s usually taken to be

T

used in the to

for

both x and y for the B matrix, as these i n t e g r a l s are close to those over the f i n i t e region and are much e a s i e r to compute.

However, t h e r e

often a r i s e two conditions which require that c e r t a i n subregions be omitted from the i n t e g r a t i o n s .

The f i r s t condition i s the problem of a

337 spot occurring on the edge of the scanned image, and the second condition is the existence of an area where the pixel values are overrange.

Both conditions result in incomplete data for a spot from

the integration.

In the case of the B matrix, the integrations are

performed over the omitted regions and subtracted from the integrals from — oo to oo .

Test Results The fitting system has been tested on several images with up to 800 spots being processed per pass. 8 cycles.

Spots are generally converged in 7 or

The left frame of Figure 1 shows a detailed view of one of

the more difficult spot complexes to fit.

There are three strongly

overlapping spots in the foreground, and the center one is overrange. The parameterization of this region is shown in the right frame.

Note

that the overrange region of the image is predicted by the model.

Future Enhancements The initial tests have been very encouraging. concentrated in three areas.

Future work will be

The first area is the increased

utilization of the array processor, as the current implementation uses the array processor only for the equation solving.

The second major

area is the calculation of error estimates for each of the parameters. The third area is the inclusion of different distributions to handle highly loaded gels and patterns with prominent streaks.

The inclusion

of these features should allow the routine and rapid processing of gel patterns on a production basis.

338

F i g u r e 1.

A complex of o v e r l a p p i n g and o v e r r a n g e PGP's

shown on t h e l e f t and i t s model on t h e r i g h t .

Acknowledgment The a u t h o r s would l i k e t o e x p r e s s a p p r e c i a t i o n t o J . I . G a r r e l s of t h e Cold Spring Harbor L a b o r a t o r y f o r t h e use of h i s p r o f i l e p l o t t i n g program used t o produce F i g u r e 1.

References 1. O ' F a r r e l l , P. H.: J . B i o l . Chem. 250, 4007-4021

(1975).

2. Anderson, N. G. Anderson, N. L . : A n a l . Biochem. 85, 331-340 (1978).

339 3. Anderson, N. L. Anderson, N. G.: Anal. Biochem. 85,

3^1-354

(1978). 4. Anderson, N. G., Anderson, N. L . : Behring I n s t . M i t t .

63,

169-210 ( 1 9 7 9 ) . 5. Anderson, N. G., Anderson, N. L . , T o l l a k s e n , S. L . :

Clinical

Chemistry 25, 1199-1210 ( 1 9 7 9 ) . 6. B o s s i n g e r , J . , M i l l e r , M. J . , Vo, K . - P . , Geiduschek, E. P . , Xuong, N . - H . : J . B i o l . Chem. 254, 7986-7988

(1979).

7. G a r r e l s , J . I . : J . B i o l . Chem. 254, 7961-7977 8. Diamond, R . : Acta C r y s t . A27,436-452

(1979).

(1971).

9. McCalla, T. R . : I n t r o d u c t i o n t o Numerical Methods and FORTRAN Programming, John Wiley & Sons. New York,

1967.

INTERNAL CHARGE STANDARDIZATION FOR TWO-DIMENSIONAL ELECTROPHORESIS

B. J . Hickman, N. L. Anderson, K. E. W i l l a r d ,

N. G. Anderson

M o l e c u l a r Anatomy Program, D i v i s i o n o f B i o l o g i c a l Argonne N a t i o n a l L a b o r a t o r y Argonne, I l l i n o i s 60439, USA

and Medical

Research,

Introduction Two-dimensional the f i r s t

electrophoresis

employing i s o e l e c t r i c

dimension and e l e c t r o p h o r e s i s

f o c u s i n g i n urea i n

i n the presence o f sodium dodecyl

s u l f a t e i n the second (1-4) g i v e s a high degree r e s o l u t i o n o f the p r o t e i n s i n a sample.

However, i t i s necessary to be a b l e to s p e c i f y the

o f each spot w i t h p r e c i s i o n .

Ultimately,

spots should be l o c a t e d to the

l i m i t s o f r e s o l u t i o n o f the system to e s t a b l i s h p o s i t i o n a l spots on two d i f f e r e n t g e l s .

position

identity

of

I f spot l o c a t i o n s were to be compared by

s i m p l e measurement, spot p o s i t i o n s would have to be r e p r o d u c i b l e on consecutive gels within a f r a c t i o n of a m i l l i m e t e r . f e a s i b l e w i t h c u r r e n t systems and equipment. ments may be made a l o n g t h e i s o e l e c t r i c

This i s hot a t

Alternatively,

present

pH measure-

f o c u s i n g gel and m o l e c u l a r mass

standards may be run a l o n g the edge o f the second dimension SDS g e l , and pH and m o l e c u l a r mass p l o t t e d a l o n g the edges o f the p a t t e r n .

This

procedure i s f r e q u e n t l y used but p r o v i d e s o n l y an a p p r o x i m a t i o n o f the c o r r e c t v a l u e s and the methods a r e t e d i o u s and i n c o n v e n i e n t f o r our purposes.

Measurements o f pH i n 8 M urea a r e not f u l l y u n d e r s t o o d , and

cannot be made a t a l a r g e number o f p o i n t s and r e l a t e d w i t h p r e c i s i o n to the f i n a l

p o s i t i o n o f each s p o t , and hence do not show the small and

s h o r t - r a n g e n o n l i n e a r i t y o f ampholyte-generated pH g r a d i e n t s . mass i n d i c a t i o n s

Molecular

based on standards run a l o n g the edge do not take

into

account any c u r v a t u r e o f the p a t t e r n , and a l m o s t always employ a v e r y l i m i t e d number o f p r o t e i n s .

We have s o l v e d the problem o f m o l e c u l a r mass

s t a n d a r d i z a t i o n by u s i n g h e a r t muscle p r o t e i n s added to t h e agarose used to seal

the f i r s t dimension gel

© 1980 Walter de Gruyter & Co., Berlin • New York Electrophoresis '79

in place.

The r e s u l t i s a l a r g e number

3^2 of horizontal

lines

(over 70) r a n g i n g o v e r the e n t i r e pattern which have

been c a l i b r a t e d f o r general (or i s o e l e c t r i c

use (5, 6 ) .

The problem o f i n t e r n a l

charge

p o i n t ) standards i s somewhat more d i f f i c u l t to s o l v e , and

r e q u i r e s the p r e p a r a t i o n o f mixtures c o n t a i n i n g l a r g e numbers o f d i f f e r i n g from each other by s i n g l e c h a r g e s . requirement i s that the p r o t e i n s a l l

For s t a i n e d g e l s , the o n l y

be v i s i b l e by s t a i n i n g , w h i l e for

a u t o r a d i o g r a p h y or f l u o r o g r a p h y , r a d i o l a b e l i n g i s r e q u i r e d . d e s i r a b l e to have f l u o r e s c e n t l y l a b e l e d charged s t a n d a r d s tively,

It is

(or,

b r i l l i a n t l y dyed s t a n d a r d s ) , so t h a t the i s o e l e c t r i c

g r a d i e n t may be monitored d u r i n g a l l

proteins,

stages.

also

alterna-

f o c u s i n g pH

T h i s i s important

anomalies a s s o c i a t e d w i t h the s t r e a k i n g at the a l k a l i n e end o f

if

isoelectric

f o c u s i n g g e l s are to be understood and minimized. We have p r e v i o u s l y d e s c r i b e d the use o f p r o g r e s s i v e l y carbamylated d e r i v a t i v e s as i n t e r n a l

standards for i s o e l e c t r i c

focusing

(7).

protein

Depending

on the p r o t e i n used, as many as 35 s p o t s extending a c r o s s a g i v e n pH range may be prepared by c a r b a m y l a t i o n o f one p r o t e i n .

Since each o f the

s p o t s i n the carbamylation " t r a i n " thus produced can be unambiguously i d e n t i f i e d by c o u n t i n g from the n a t i v e s p o t , the t r a i n forms a natural internal

c o o r d i n a t e system f o r i s o e l e c t r i c

dimensional

p o i n t measurement i n two-

systems.

Here we d e s c r i b e f u r t h e r i n v e s t i g a t i o n s charge s t a n d a r d i z a t i o n .

i n t o the u s e f u l n e s s o f

internal

F i r s t , we present approximate measurements of the

i s o e l e c t r i c p o i n t ( i n 8 M urea) of s e l e c t e d elements in the c r e a t i n e phosphokinase

(CK) charge t r a i n .

Next we d e s c r i b e t e s t s of the u s e f u l -

ness o f such standards i n n o n - e q u i l i b r i u m dimensional protein will

systems.

[NEPHGE ( 8 ) or BASO ( 9 ) ] two-

The q u e s t i o n to be answered i s whether a g i v e n

appear at the same charge s t a n d a r d c o o r d i n a t e i n the e q u i l i -

brium systems (which are u s u a l l y s t a r t e d at the b a s i c end of the g r a d i e n t ) as i n n o n - e q u i l i b r i u m systems

( u s u a l l y s t a r t e d at the a c i d end) under a

range o f running c o n d i t i o n s .

L a s t l y , we i l l u s t r a t e some p o t e n t i a l

f l u o r e s c e n t l y l a b e l e d charge

standards.

use o f

343 M a t e r i a l s and Methods Carbamylation was performed u s i n g p u r i f i e d r a b b i t muscle c r e a t i n e k i n a s e obtained from Sigma. mercaptoethanol

phospho-

The p r o t e i n was s o l u b i l i z e d i n 8 M urea, 1%

at a c o n c e n t r a t i o n o f 5 mg/ml .

A l i q u o t s were put i n g l a s s

tubes each c o n t a i n i n g a small magnetic s t i r r i n g bar, and were then heated i n a b o i l i n g water bath f o r v a r i o u s t i m e s , 0, 2, 4, 5, 6, 8 , 9, 10, and 12 minutes.

A f t e r h e a t i n g , the tubes were put d i r e c t l y on i c e to

f u r t h e r m o d i f i c a t i o n o f the p r o t e i n s .

inhibit

A f t e r running two-dimensional

gels

o f each time p o i n t to determine p r o p o r t i o n s needed to make homologous " t r a i n " of s p o t s , a mixture o f the intermediate phases o f the p r o t e i n was made. use.

The standard mixture i s then f r o z e n down i n 50 A a l i q u o t s f o r

later

For i n t e r - l a b o r a t o r y comparisons, the samples ccul d be l y o p h i l i z e d

and rehydrated when needed. The c e l l

l i n e s used f o r determining measurements on CK charge t r a i n were

e i t h e r normal human peripheral line

lymphocytes o r a human l y m p h o b l a s t o i d c e l l 35 (GM 607) r a d i o a c t i v e l y l a b e l e d with S - m e t h i o n i n e . The p r e p a r a t i o n

o f lymphocytes has been d e s c r i b e d elsewhere ( 1 0 ) .

For comparison o f the

IS0-DALT system ( 3 - 4 ) and the BAS0-DALT (9) system, the same sample was used f o r both, l o a d i n g 20 A c e l l dimension g e l s .

sample plus 2 A CK s t a n d a r d s on the

For the I S O , we ran the sample f o r 13,000

first

volt-hours,

shown by previous experiments to be an a p p r o p r i a t e amount o f time f o r p r o t e i n s to completely f o c u s .

However, small v a r i a t i o n s

i n the i n t e g r a t e d

v o l t - h o u r s do not appear to move the p r o t e i n s a p p r e c i a b l y once they have found t h e i r i s o e l e c t r i c f o c u s i n g p o i n t . for v a r i o u s time p o i n t s :

In comparison, the BAS0 run was

2000, 3000, 4000, 5000 v o l t - h o u r s ; each time

p o i n t s h i f t e d the more b a s i c p r o t e i n s toward the a l k a l i n e end, so that the s e p a r a t i o n never appeared to reach e q u i l i b r i u m .

After

second-dimension

e l e c t r o p h o r e s i s , the g e l s were s t a i n e d i n 0.4% Coomassie Blue and d e s t a i n e d i n ethanol/5% a c e t i c a c i d for examination o f the " c o l d " or proteins.

non-radioactive

A f t e r photographing the s t a i n e d g e l s , those l a b e l e d with 35 S -

Methionine were soaked i n 2% g l y c e r o l were soaked i n 2% g l y c e r o l

before d r y i n g on b l o t t i n g

paper

before d r y i n g on b l o t t i n g paper, and were then

pi aced a g a i n s t Kodak X-R 2 X-Omat f i l m and exposed f o r two weeks f o r

344 autoradiography. For fluorescent l a b e l i n g of p r o t e i n s , f l u o r e s c e i n isothiocyanate was purchased from Sigma.

(FITC)

FITC i s added to p u r i f i e d proteins a f t e r they

have been o r i g i n a l l y stripped of possible bound low molecular weight components on P-6 Biogel 100-200 mesh (Bio-Rad Lab.)

The f l u o r e s c e i n i s

allowed to react with the protein for approximately one minute in the absence of urea.

After the reaction has taken place, a 0.1 volume of

1 M ethylenediamene i s added to react with any unbound f l u o r e s c e i n , and to prevent excess f l u o r e s c e i n from attaching to unknown foreign substances. The p r o t e i n - f l u o r e s c e i n complex i s passed over another P-6 Biogel

100-200

mesh (Bio-Rad) column, to s t r i p the protein-bound f l u o r e s c e i n from the f l u o r e s c e i n now complexed with ethylenediamene:

The protein s o l u t i o n

collected was concentrated f i v e f o l d on a minicon (Amicon).

After i s o -

e l e c t r i c focusing of hemoglobin-bound f l u o r e s c e i n , sharp fluorescent bands were v i s i b l e for the a-hemogl obin and B-hemoglobin.

For pH measurement,

a selected ruled 1/4 inch area, with each band in the center, was out and soaked in degassed 8 M urea for f i v e hours.

cut

The s o l u t i o n was

degassed again before d i r e c t pH measurements were made.

Results and Discussion Our objective i s to describe spot positions in two-dimensional gels in a reproducible manner, but we are here concerned only with the f i r s t , or i s o e l e c t r i c focusing dimension. The p o s i t i o n of s p e c i f i c proteins in human lymphocyte patterns may be located in reference to creatine phosphokinase charge standards as shown in Figure 1.

The difference between charge standard spots -1 and -4 i s

approximately 0.2 pH u n i t s .

The small s i z e of the charge standard spots

allows l o c a l i z a t i o n of nearby pattern spots to approximately 1/5 the distance between spots centers, allowing l o c a l i z a t i o n , in the best instances, to between 0.01 and 0.02 pH u n i t s .

F i g . 1. An autoradiograph of a two-dimensional pattern of human peripheral lymphocytes labeled with 3 5 S-Methionine with i n s e t s of the same; L e f t : the autoradiograph, Right: the Coomassie Blue stained gel co-electrophoresed with CK carbamylation t r a i n standards and rat heart molecular weight standards. P a r t i a l s of the two i n s e t s were used to obtain the total picture. Gels are oriented with the acid end to the l e f t , the basic end to the right. 1(pi = - 2 4 . 0 ) ; 2(pi = - 2 2 . 0 ) ; 3(pl = - 1 8 . 0 ) ; 4, actin (pi = - 1 7 . 5 ) ; 5(pi = - 1 7 . 0 ) ; 6(pi = - 1 2 . 5 ) ; 7(pl = - 4 . 0 ) ; 8(pl = - 2 . 0 ) .

346 Different proteins give charge standard trains having different pH ranges, and different interspot distances, depending on the amino acid composition. Proteins having a high charge density give much smaller interspot distances when carbamylated.

Under so-called NEPHGE (8) or BASO (9) conditions, patterns are quite different since the samples are purposefully not run to equilibrium. Here charge standards are useful for identification and localization purposes, as shown in Figure 2, especially at the molecular mass level of the charge standard in the gel.

However, they do not appear to indi-

cate the position of a certain pH in the alkaline end of the isoelectric focusing gel.

Surprisingly, in the more acid regions of BASO gels,

consistent positional

data are obtained and actin is found at -17.5 CPK

charge units in both ISO and BASO gels.

To investigate fluorescent charge standards we have labeled human hemoglobin by very brief labeling with fluorescein isothiocyanate.

The

results as seen in fluorescent light are shown in Figures 3 and 4.

Since

the standard can be seen during focusing we now are able to monitor the rate of approach to final focusing position.

Note that the fluorescein

labeling is very brief, so that at m o s t only one molecule of dye attached to one hemoglobin subunit, and many are left unmodified.

It is

then possible to locate both the modified and unmodified subunits with reference to CK charge standards.

Unless high pH ampholines are used,

the unmodified alpha chain of hemoglobin is rarely seen.

However, as

shown in Figure 5, one fluorescein molecule moves the beta chain from a position of greater than 0 CK units to a position of -3.5 CK units. Similarly, the alpha chain of hemoglobin is moved approximately the same distance by one fluorescein molecule added, but the proteins are too acid to measure on the CK train.

The modified beta hemoglobin is only

about 1/20 of the beta hemoglobin present, hence doubly modified beta chains would probably not be visible.

347

F i g , 2. Two-dimensional electrophoretic patterns of human lymphoblastoid c e l l s (GM 607) showing charge comparisons of the ISO-DALT and BASO-DALT systems. Gels are oriented as i n F i g . 1. l ( p l = - 1 7 . 0 ) ; 2, actin (pi = - 1 7 . 5 ) ; 3(pl = - 1 3 . 0 ) ; 4 and 5 present only on BASO gels and varying with volt-hours (V-H). 4[2,000 V-H, pi = - 4 . 0 ; 3,000 V-H, pi = - 2 . 5 ; 4,000 V-H, pi = - 2 . 5 ; 5,000 V-H, pi = - 1 ] 5 i s too basic to measure on CK charge t r a i n , but v a r i e s with V-H.

348

Fig. 3. I s o e l e c t r i c focusing gels of red blood cell lysate labeled with f l u o r e s c e i n . Photographed with an u l t r a - v i o l e t l i g h t source and a green fluorescence f i l t e r . Gels are oriented as in Fig. 1. 1, modified alpha - hemoglobin; 2, modified beta - hemoglobin

Fig. 4. Two-dimensional electrophoretic pattern of the same gel as in F i g . 3, f l u o r e s c e i n labeled hemoglobin chains. Gels are oriented as in F i g . 1 . 1, modified alpha - hemoglobin; 2, modified beta - hemoglobin.

349

- 2 5 -20 i

I

-15

-10

l i l i

I 4

Fig. 5. Two-dimensional pattern of red blood cell lysate stained by Coomassie Blue after fluorescein labeling and using the CK carbamylation train standards. Other RBC lysate proteins are evident which have been identified elsewhere (11). Gels are oriented as in Fig. 1. 1, Native beta - hemoglobin (pi = > 0) 2 , Modified beta - hemoglobin (pi = -3.5) 3, Native alpha - hemoglobin (too basic to measure) 4, Modified alpha - hemoglobin (too basic to measure)

The use of CK carbamylation train as standards is useful when the pH range is 5-7.

These charge shift markers will reveal the variability between

ampholine batches which we have found frequently occur.

350 Conclusions

By u s i n g c u r r e n t methods o f 2 - D gel sel ves v i r t u a l l y

without standardization.

and f l u o r e s c e i n l a b e l e d p r o t e i n s standards

for

s p o t i n t e r c o m p a r i s o n s , we f i n d

isoelectric

However, both

carbamylated

have been shown t o be u s e f u l

focusing

in polyacrylamide

our-

internal

gels.

Acknowl edgment

We w i s h to thank Monica S h a n d e r from W i s t a r Pennsylvania, and

Dr.

f o r her t h o u g h t f u l

Institute,

g i f t o f human l y m p h o b l a s t o i d

P e t e r S p r a g g , Birmingham U n i v e r s i t y ,

his discussions

Philadelphia,

l e a d i n g to t h e u s e o f f l u o r e s c e i n - l a b e l e d

T h i s work was s u p p o r t e d by t h e U. S.

cells,

Birmingham, England,

for

proteins.

Department o f E n e r g y u n d e r

contract

No. W-31 - 1 0 9 - E N G - 3 8 .

References

1.

Stegeman, H. :

Angew. Chem. 82,

P. H. :

J. B i o l .

640

(1979).

2.

O'Farrell,

3.

Anderson,

N. G. , A n d e r s o n ,

N. L. :

Chem. 2 5 0 , 4 0 0 7 - 4 0 2 1 Anal.

Biochem. 85_, 3 3 1 - 3 4 0

(1978).

4.

Anderson,

N. L . , A n d e r s o n ,

N. G. :

Anal.

Biochem. 8 5 , 3 4 1 - 3 5 4

(1978).

5.

Anderson, (1 9 7 9 ) .

N. G . , A n d e r s o n , N. L. :

6.

Giometti, Anderson,

C. S . , A n d e r s o n , N. G. , T o l l a k s e n , N. L. : A n a l . Biochem. i n p r e s s .

Behring

Anal.

(1975).

Inst. S.

Mitt.

63,

L. , E d w a r d s , J.

7.

A n d e r s o n , N. L . , Hickman, B. J . :

8.

O'Farrell,

Biochem. 93_, 3 1 2 - 3 2 0

9.

W i l l a r d , K. E. , S m i t h , C. F . , A n d e r s o n , N. L. , O ' C o n n o r , T. A n d e r s o n , N. G. : A n a l . Biochem. i n p r e s s ( 1 9 7 9 ) .

P. Z . , Goodman, H. M . , O ' F a r r e l l ,

K. E . , A n d e r s o n ,

N. L. :

169-210

10.

Willard,

11.

E d w a r d s , J. J . , A n d e r s o n , N. G. , N a n c e , S. B l o o d 53, 1 1 2 1 - 1 132 (1 9 7 9 ) .

P. H. :

Electrophoresis

J.,

(1979).

Cell E.,

1 979.

L . , A n d e r s o n , N. L. :

A METHOD FOR STUDYING PROTEINS IN 2-D GELS USING THERMAL DENATURATION ANALYSIS

S. L. Nance, B. J . Hickman, N. L. Anderson Molecular Anatomy Program, Division of B i o l o g i c a l and Medical Research, Argonne National Laboratory Argonne, I l l i n o i s 60439, USA

Individual proteins are c h a r a c t e r i s t i c a l l y unfolded and denatured over a s p e c i f i c temperature range which i s generally quite small.

The thermal

denaturation curve may be s h i f t e d by a l t e r i n g the environment of the prot e i n , e.g., by adding a substrate or cofactor, changing the pH or s a l t concentration, e t c .

By determining the behavior of a protein at a number

of d i f f e r e n t temperatures and in d i f f e r e n t environments, i t i s possible to obtain a spectrum of results that i s s u f f i c i e n t l y

frequently distinct

from that of other proteins under the same conditions as to be useful as an i d e n t i f y i n g

characteristic.

We have employed a stable thermal gradient device to treat multicomponent samples, which were subsequently analyzed for enzymatic a c t i v i t i e s and by two-dimensional acrylamide gel electrophoresis under denaturing conditions The results i l l u s t r a t e that this technique can indeed be used to i d e n t i f y proteins in a 2-D gel pattern and to provide information on cofactor or substrate binding.

Materials and Methods Rabbit psoas muscle was homogenized in a Waring blender with three volumes of phosphate buffered saline (PBS).

The homogenate was centrifuged for

one hour at 15,000 rpm, 5°C, and the p e l l e t was discarded.

After

further

centrifugation at 100,000 x £ for one hour, the supernatant was applied to the top of a P-4 Biogel column equilibrated with PBS, and the proteins in the excluded peak, stripped of low molecular weight compounds

© 1980 Walter de Gruyter & Co., Berlin • New York Electrophoresis '79

(i.e.,

352 with the compounds of less than 4000 daltons removed), were collected as the sample for analysis.

Mercaptoethanol

was then added to a concentra-

tion of 0.1%. Thermal denaturation was carried out as described previously (1) using the stable thermal gradient device shown in Fig. 1.

Aliquots of 200 yl

of the stripped soluble rabbit muscle were positioned at 20 temperature points over the range 36.5 to 75.65°C.

In addition, rows of aliquots of

the muscle sample supplemented with 5 mM NADH or 5 mM MgADP were similarly positioned on the gradient.

All 60 samples were treated

simultaneously

for 5 minutes, then cooled on ice for 2 minutes and centrifuged in a Beckman Microfuge B for 2 minutes to sediment denatured

protein.

From each supernatant 50 yl was used for enzyme activity analyses in an ElectroNucleonics (Fairfield, N. J.) GEMSAEC centrifugal analyzer

(2,3).

With this instrument it was possible to assay the samples from an entire row rapidly and in parallel, thus eliminating errors that would result from sequential measurements of an unstable enzyme that continued to denature with time even when chilled. To another portion of each supernatant was added an equal volume of 9 M urea, 4% NP-40, 2% 2-11 Brinkman ampholines, 0.5% mercaptoethanol.

All

60 samples were then analyzed by 2-D electrophoresis, first on isoelectric focusing gels (2-11 Brinkman ampholines) using the method

previously

described for very basic proteins (4,5) and then on 10-20% gradient acrylamide SDS slab gels (6). Activities of the following enzymes were measured using modifications of the methods referenced: EC 4.1.2.7

adenylate kinase (ADK), EC 2.7.4.3 (7); aldolase,

(8); aspartate aminotransferase (GOT), EC 2.6.1.1

phosphokinase

(CPK), EC 2.7.3.2 (10); enolase, EC 4.2.1.11

(9); creatine

(11); glyceral-

dehyde-3-phosphate dehydrogenase (G3PDH), EC 1.2.1.12 (12); lactate dehydrogenase (LDH), EC 1.1.1.27 (13); malate dehydrogenase (MDH), EC 1.1.1.37 (14); pyruvate kinase (PK), EC 2.7.1.40 (15); phosphohexose

isomerase

(PHI), EC 5.3.1.9 (16); triosephosphate isomerase (TPI), EC 5.3.1.1

(17).

353

Fig. 1. Stable thermal gradient device. The aluminum conductor strip (A), along which the temperature gradient is formed, has 5 rows of 20 wells and is supported in a horizontal position by two piers. The left pier (B) is wrapped with an electric heating element (D), and the right pier (C) has an internal serpentine cooling chamber, provided at the back with inlet and outlet hose connections. Mercury thermoregulators (F) are mounted in vertical holes in the left and right piers. These are connected to a controller (not shown) which switches (1) the heater on and off to maintain the left pier at 90"C and (2) the flow of tap water on and off (by solenoid valve) to maintain the right pier at 30°C. An additional variable resistance is provided to adjust the "oncycle" output of the electric heater. A digital thermistor (not shown) is connected to three probes in water-filled Microfuge tubes for continuous monitoring of the temperature gradient. A Lucite acrylic resin tube-holding rack (G) is filled with Microfuge tubes and placed in position on top of strip (A).

Results

Figure 2 presents activity data for six of the enzymes that were assayed. MDH is notable for its biphasic curves; approximately 70% of the enzyme

354

WELL NUMBER

WELL NUMBER Fig. 2. Enzyme activity curves. The results obtained from six of the enzyme assays performed are shown (for the region where thermal denaturation occurred). The temperature increased as the well numbers decreased. Solid lines indicate the stripped sample; dashed lines, +NADH; dotted lines, +MgADP.

355 is present in a relatively heat-stable form.

We have also observed a

biphasic curve with MDH from human red blood cell lysate (1), where only 20% occurs as the heat-stable form.

Both forms are more stable in the

presence of NADH, which is a substrate with MgADP which is not.

for this enzyme and less stable

GOT is of interest as the most stable of the

enzymes assayed; the activity increased with increasing temperature until denaturation occurred. The basic portions of the 2-D gels from this series are shown in Figure 3. In all of the gels shown, molecular weight increases from the bottom up, pi increases from left to right and temperature increases from left to right across the series.

Samples in the middle and bottom series were

supplemented with 5 m M NADH and 5 m M MgADP, respectively.

The numbered

spots are identified in Table 1 where the results are summarized.

The

locations of these proteins in the gel pattern were determined earlier

(5),

with the exception of MDH which was tentatively identified from this experiment and subsequently confirmed with the nearly pure protein. the only enzyme not yet located in the pattern. correlates well with the GOT activity curves.

GOT is

The behavior of spot #9 The molecular weight of

spot #9 is approximately one-fourth of that reported for non-denatured GOT; however, pig and chicken heart s-GOT are believed to contain only two subunits (18).

It should be possible to confirm, or eliminate, spot #9 as

GOT by supplementing with its cofactor (pyridoxal mal denaturation.

phosphate) during ther-

For the rest of the proteins, spot disappearances cor-

relate well with loss of activity.

Figure 4 is an enlargement of the gel

pattern which presents, diagrammatically, the effects obtained with each of the cofactors.

Discussion

The enzyme activity curves obtained agree remarkably well with the corresponding 2-D gel spot abundances.

Although the point of 50% removal

for

each spot through the series was subjectively determined, it should be possible to obtain more accurate differentiation of enzymes using computerized quantitation of spot intensities (see paper by J. Taylor et a K

in

356

357 Fig. 3. Two-dimensional g e l s . The 2-D acrylamide slab gels were stained with Coomassie Blue, photographed, and arranged in order of increasing temperature for each of the three rows of samples. Each spot, or complex of spots, i s numbered in the upper left-hand g e l . Each number appears again where the spot abundance was judged (by visual inspection) to have decreased by 50%.

TABLE 1

Published MW 68,000

Complex # 1

Protein Albumin PGM

50% Denaturation in Well # 9 8

Change upon Addition of NADH

MgADP

Known to Bind

0

+2

?

0

+2

Mg++

65,000

2

56,000

3

PK

8

0

0

41,000

4

Enolase

11

-1

+2

40,500

5

CPK

11

0

0

ADP

39,000

6

Aldolase

ADP Mg++

9

0

0

-

+2

0

NADH

36,000

7

G3PDH

11

35,000

8

LDH

11

+2

-1

NADH

9

?

6

0

-1

?

? 26,600

10

TPI

9

0

-1

-

21,600

11

ADK

13

+2

+1

ADP

MDH

12

+3

-1

NADH

35,000

12

ARGÇJNNE NATIONAL LAB.OfUTOflY

The proteins are i d e n t i f i e d by spot, or complex, number (see Figures 3 and 4) and name, with molecular weight and pertinent binding information from the l i t e r a t u r e . The 50% denaturation and cofactor s h i f t data are from v i s u a l a n a l y s i s of the g e l s .

these proceedings).

In p a r t i c u l a r , t h i s might enable us to determine

whether the s i n g l e spot assigned to MDH includes both the thermostable and thermolabile forms ( i . e . , spot disappearance i s biphasic) or whether another, as yet undetected, spot accounts for the second of the two forms. I t i s evident from the cofactor s h i f t data that NADH generally causes larger s h i f t s than MgADP; in f a c t , the larger MgADP s h i f t s may be ascribed

358

10;, nfetD 3

F i g . 4. Enlargement of gel pattern. The r e s u l t s are d i a grammed on a s i n g l e gel pattern. The spot numbers correspond to those in Table 1 and i n Figure 3. S o l i d and dashed rectangles indicate s t a b i l i z a t i o n and d é s t a b i l i s a t i o n , respect i v e l y , by MgADP; s o l i d and dashed hexagrams indicate s t a b i l i z a t i o n and destabil i z a t i o n by NADH; the number of well s h i f t s i s noted within the boxes.

to Mg++ alone, leaving only 0±1 well s h i f t s caused by ADP or the MgADP complex.

This r e s u l t i s c o n s i s t e n t with the greater area of the protein-

NADH contact (and presumably t i g h t e r binding constant) compared to ADP. I t i s i n t e r e s t i n g to note that ADP causes d e s t a b i l i z a t i o n in two of the three enzymes known to bind (and be s t a b i l i z e d by) NADH (see Table 1), perhaps i n d i c a t i n g that ADP "unbalances" the dinucleotide binding s i t e by f i l l i n g only h a l f of i t . Of special i n t e r e s t i s the +2 unit s t a b i l i z a t i o n of ADK by NADH.

This

enzyme employs only ADP as substrate and needs no c o f a c t o r , making the NADH binding apparently superfluous.

Sequence a n a l y s i s has shown, how-

ever, that ADK i s homologous to enzymes possessing the c l a s s i c tide f o l d " of the dehydrogenases ( 1 9 ) .

"dinucleo-

I t therefore seems reasonable to

suggest that the thermostabilization caused by NADH binding i s evidence of a v e s t i g i a l

interaction of the protein with t h i s cofactor.

( I t should

359 be noted that the samples supplemented with ADP a l s o contained ATP and AMP as reaction products due to the presence of ADK.)

The remaining en-

zymes known to u t i l i z e ADP (PK and CPK) show no s h i f t with ADP or with NADH, for reasons not yet understood. Thermal denaturation a n a l y s i s , as described here, i s probably more l a b o r i ous than i s o l a t i o n of binding proteins us'ing a f f i n i t y chromatography on cofactor or substrate substituted s o l i d supports, and i t i s therefore appropriate to point out two peculiar advantages of the present technique. F i r s t , i t may be applied to e n t i t i e s too small to be attached to an a f f i n i t y support, i . e . , Mg++.

Second, i t gives thermodynamic information on

binding energy which i s d i f f i c u l t to obtain using a f f i n i t y absorption. These advantages have encouraged us to e x p l o i t the technique as a general s o l u t i o n to the problem of detecting i n t e r a c t i o n s between proteins and low molecular weight compounds and as an aid in spot i d e n t i f i c a t i o n .

Acknowl edgment This work was supported by the U. S . Department of Energy under contract No. W-31-109-ENG-38.

References 1.

Anderson, N. L . , E i s l e r , W. J . , Anderson, N. G.: 441-445 (1978).

Anal. Biochem. 91_,

2.

Anderson, N. G.:

Anal. Biochem. 28, 545-562 (1969). Z. Anal. Chem. 261, 257-271 (1972).

3.

Anderson, N. G.:

4.

O ' F a r r e l l , P. Z., Goodman, H. M., O ' F a r r e l l , P. H.: 1142 (1977).

5.

Giometti, C. S . , Anderson, N. G., Anderson, N. L.: Muscle protein a n a l y s i s . I . High r e s o l u t i o n two-dimensional electrophoresis of skeletal muscle proteins for a n a l y s i s of small biopsy samples. Clin. Chem., i n press.

6.

Anderson, N. L . , Anderson, N. G.:

Cell U ,

1133-

Anal. Biochem. 85, 341-354 (1978).

360 7.

S o t t o c a s a , G. L . , K u y l e n s t i e r n a , B . , E r n s t e r , L . , B e r g s t r a n d , A . : Methods i n Enzymology, V o l . X, E s t a b r o o k , R. W. and Pullman, M. E. ( e d s . ) , Academic P r e s s , New York 1967, p. 457.

8.

P i n t o , P. V. C., Kaplan, A . , VanDreal, P. A . : 360 ( 1 9 6 9 ) .

9.

Amador, E . , Massod, M. F . , Franey, R. J . : 419-428 ( 1 9 6 7 ) . I . T.:

Biochem. J . 61_, 116-122

C l i n . Chem. 1 5 ,

349-

Am. J. C l i n . Path. ^ 7 ,

10.

Oliver,

(1955).

11.

Bergmeyer, H. U., Gawehn, K . , G r a s s i , M.: Methods of Enzymatic A n a l y s i s , Second e d i t i o n , Bergmeyer, H. U. ( e d . ) , Academic P r e s s , New York 1974, p. 449.

12.

Bergmeyer, H. U., Gawehn, K . , G r a s s i , M.: Methods of Enzymatic A n a l y s i s , Second e d i t i o n , Bergmeyer, H. U. ( e d . ) , Academic P r e s s , New York 1974, p. 467.

13.

Wroblewski, F . , LaDue, J . S . : 213 ( 1 9 5 5 ) .

14.

M e h l e r , A . H., Kornberg, A . , G r i s o l i a , S . , Ochoa, S . : 174, 961-977 ( 1 9 4 8 ) .

15.

Beeson, M. A . , B l a c k , J . A . : (1976).

16.

Schwartz, M. K . , Bethune, V. G., B a c h i , B. L . , Woodbridge, J . E . : C l i n . Chem. 17, 656-657 ( 1 9 7 1 ) .

17.

Bergmeyer, H. U . , Gawehn, K . , G r a s s i , M.: Methods of Enzymatic A n a l y s i s , Second e d i t i o n , Bergmeyer, H. U. ( e d . ) , Academic P r e s s , New York 1974, p. 515.

18.

B r a u n s t e i n , A. E . : The Enzymes, I X , T h i r d e d i t i o n , Boyer, P. D. ( e d . ) , Academic P r e s s , New York 1973, p. 379-463.

19.

VonZaberb, I . , W i t t m a n - L i e b o l d , B . , Untucht-Grau, R . , S c h i r m e r , R. H . , P a i , E. F . : E u r . J. Biochem. 68, 281-290 ( 1 9 7 6 ) .

Proc. S o c . Exp. B i o l . Med. 90, 210J . B i o l . Chem.

Comp. Biochem. P h y s i o l . 53B, 489-493

THE

QUANTITATION

OF

DIMENSIONAL GELS:

RADIOACTIVELY

LABELED

PROTEINS

ON

TWO-

TESTS OF A METHOD FOR ANALYZING CHANGES IN

PROTEIN SYNTHESIS AND GENE EXPRESSION

George C. Stone, David L. Wilson, and G.W. Perry Department of Physiology and Biophysics University of Miami, School of Medicine Miami, FL 33101

Introduction The use of high resolution, two-dimensional polyacrylamide gel electrophoresis

for

the

separation of proteins

has become

a

popular research tool since its introduction by O'Farrell (l). The technique has been widely used for qualitative as well as quantitative comparisons of proteins.

Despite the usefulness

and popularity of the technique, the quantitative of

the

gel

instance,

system in

have

typical

not

samples

been

thoroughly

consisting

of

properties

examined. a

For

heterogeneous

population of proteins, the possible influence of one protein on the apparent abundance of other proteins in the gel has yet to be considered.

In light of studies that attempt determina-

tion of relative synthesis rates of specific proteins, such an analysis of quantitative properties of the gels is of obvious importance. adding

By

loading

purified,

examined

the

various

individual

quantitative

total

proteins

protein on

the

reproducibility

amounts,

and

gels,

we

have

of

the

gel

procedure. Most of the available procedures for quantitation of amounts of labeled protein species on two-dimensional gels have been expensive and complex (2,3). capable

of

yielding

an

Some of these are, in principle,

exhaustive

analysis

densitometric devices coupled to computers with

© 1980 Walter de Gruyter & Co., Berlin • New York Electrophoresis '79

and

utilize

sophisticated

362 software.

However, for the analysis of a more limited number

of protein species, direct determination of radioactive counts in

cut-out

pieces

of

the

gel

provides

a

simpler

and

less

expensive approach, as previously described (4). A most useful potential application of the enhanced resolution of protein species afforded by the two-dimensional gels is the detection gene

and

analysis

expression.

of

changes

in protein

This application

depends

synthesis

and

upon a number

of

assumptions made in relating the incorporation rates of radioactive amino acids to the synthesis rates of proteins. present

paper

counts

in

rates,

we

gel

can be

relative

explore

pieces,

the

question

reflecting

of

whether

precursor

used

to detect

and quantitate

of

synthesis

of

rates

results

indicate

coupled

with

that

the

different

two-dimensional

simple

measured

incorporation changes

in the

proteins.

gel

quantitation

In the

Our

electrophoresis

method

provides

a

reliable means for studying the regulation of gene expression.

Materials and Methods Sample

preparation.

ganglia

from

the

"^S-methionine

Seventh,

frog, Rana

eighth,

and ninth

catesbieana,

were

dorsal

root

labeled

with

(Amersham, 600-1,300 Ci/mmole) at 18°C in frog

Ringer's solution (5) supplemented with vitamins and essential amino

acids

except methionine,

as previously

described

(4).

Protein concentrations in the samples were increased by addition of unlabeled, desheathed frog sciatic nerve. homogenization

procedure

has

been

described

The tissue

previously

(4).

In brief, tissue was homogenized at room temperature in 14 yl of a solution containing II sodium dodecyl sulfate 10%

2-mercaptoethanol

in 8M urea

(ultra pure,

(SDS) and

Schwarz-Mann).

The homogenate was added to 4 mg of dry urea plus 5 pi of a mixture of 4 ml of 101 NP-40 (Particle Data Laboratories) plus 0.32 ml of pH 5-7 ampholine

and 0.08 ml of pH 3-10 ampholine

363 (BioRad or LKB).

Two rinses of 10 pi each of O'Farrell's

lysis buffer were added to the sample.

fuged at 100,000 xg for 1 hour at room temperature. centrifugation,

the

supernatant

was

mixed

divided into aliquots for electrophoresis. tion was determined

(l)

The sample was centri-

using, the Bradford

by

Following

vortexing

and

Protein concentra-

(6) assay with

bovine

serum albumin dissolved in the same homogenization buffer as a standard. Two

dimensional

tions,

gel

procedure

gel

electrophoresis.

preparation

and

With

electrophoresis

reported

isoelectric while

here,

focusing

acrylamide

the

diameter

gels was

(l).

of

was

the

modification

For some of the

the

increased

concentration

modifica-

followed

of Wilson, et a^. (4), itself a minor

of the procedure described by O'Farrell studies

minor

first-dimension

from

decreased

2.5

to 3.0 mm,

from

4%

to

31.

These changes may contribute to a reduction in the "clogging" of

proteins

at

the

molecular-weight lower

acrylamide

top

of

the

proteins

to

focus

concentrations,

gel

and

more

it

may

allow

sharply.

was

found

high-

With

the

necessary

to

support the gel with a finely meshed nylon screen attached to the bottom of the gel tubes following polymerization.

Other-

wise,

during

the

gels

isoelectric

tended

to

focussing.

slide

First

out

of

dimension

the gels

tubes were

run

for

19 hrs at 400V. The

final pH gradient

ranged from approximately

in figures) to 5 (right side). second

weights

from approximately

200,000

(top

of

proteins were not well Quantitation Following 25%

of

A 101 lower slab gel was used

15,000

gel).

(bottom of gel) to greater

Above

150,000

daltons,

most

focused.

radioactively

staining, with

trichloroacetic

side

to separate proteins with molecular

for the than

dimension

8 (left

acid

0.11 for

labeled

proteins

Coomassie

on

Brilliant

0.5 hr, gels were

the

gels.

Blue R

in

destained

in

36^ 7.51

acetic acid.

The acetic acid solution used for

initial

destaining was recycled many times, with clearing of the stain by charcoal adsorption.

Gels were finally destained overnight

in fresh 7.51 acetic acid.

Gels were dried under vacuum with

mild heating (50-60°C) onto Whatman No.3 filter paper. Twelve stained proteins spots (Fig. la) were selected for the quantitative

analysis.

These

were

cut

from

each

dried

gel

with a scalpel and placed in scintillation vials with 6 ml of scintillation fluid containing, per liter of toluene, 4 g PPO, 0.05 g

POPOP,

NH^OH.

After

transparent

100 ml

NCS

shaking

for

and

swollen

(Amersham-Searle), 30-48

in

the

hours,

the

fluid.

and

20 ml

4M

gel

had

become

of

the

radio-

Most

activity had leached from the gel by 30 hours, and after 48 hours counts remained constant for days (4).

This procedure

has previously been demonstrated to give reasonable reproducibility for major species of protein from rat nervous tissue in two separate studies (7,8).

Samples were counted in a Packard

2425 liquid scintillation spectrometer at 801 efficiency, with a background of 20 cpm. Purification brain

by

of proteins.

the

method

of

Tubulin was

Shelanski,

isolated

£t al_.

from

(9).

bovine

Myosin

was

isolated from rabbit skeletal muscle by slight modification of the method

of Szent-Gyorgyi

(10), and Mommaerts

and

Parrish

(11). Normalization. selected

to

In

cover

the

a range

present of

study

relative

twelve

spots

abundances,

from

were the

most abundant (including actin and tubulin) to those proteins incorporating abundant

1%

spots.

or

less

of

the

Normalization

counts was

found

in

performed

following equation for each gel: Icpm in spot =

cpm in spot cpm in 12 spots

x 100

the

most

using

the

365 Standard

deviations

are

included

whenever

means

have

been

ganglion

from

calculated. Aplysia

neuron

Aplysia

californica

seawater

experiments. (250-500g)

containing

Ci/mmole)

for

15

procedure

was

Dissected

neurons

The

abdominal

was

incubated

40 pCi

of

^S-methionine

minutes

at

18-19°C.

similar

to

were

that cut

of Giller

in

halves

in

Neuron and or

50 yl

of

(Amersham,

800

dissection

Schwartz

quarters

(12).

with

a

scalpel and the neuron parts were separately homogenized (13). The proteins gels

(10%

(14,15), brief

from

the cell parts were separated on SDS

polyacrylamide), prior

labeling

to

drying.

period

dimensional analysis. x-ray

film

procedures described

or for

to

and

PPO

The

counts

were

not

imbedded

in

the

incorporated

sufficient

slab

for

gels

in a

the two-

The dried gels were exposed to Kodak NS

LKB

Aplysia

Ultrofilm neuron

for

1-2

experiments

months. were

Other

previously

(13).

Results and Discussion A

typical

two-dimensional

shown in Figure 1 (a § b).

gel,

and

its

autoradiograph,

Most of the heavily-stained

are spots

are also being synthesized at a significant rate as indicated by

the autoradiograph.

In contrast,

Figure 1 (c § d)

shows

gel and autoradiograph of frog skeletal muscle proteins, where actin and other prominantly abundantly

stained proteins were not

being

synthesized.

Figure 1 a and b. Two-dimensional patterns of stained (a) and labeled (b) proteins from frog dorsal root ganglia. The eighth and ninth ganglia were labeled with ^ S - m e t h i o n i n e in vitro, and the extracted proteins were separated in two dimensions. a) The ganglion staining pattern. The 12 spots selected for analysis are indicated, b) The autoradiographic pattern of the same gel (Kodak NS x-ray film). Photographs of dried gels and autoradiographs were taken with high contrast copy film.

366

367 ««H

Figure 1 c and d. Two-dimensional patterns of stained (c) and labeled (d) proteins from frog skeletal muscle. c) Muscle staining pattern. d) Autoradiographic patterns of the same gel. Notice that most of the major muscle proteins are not highly labeled.

368 Day-to-day

and

sample-to-sample

reproducibility.

Table

I

presents the results from gels of nerve and ganglia proteins of tests for gel-to-gel, day-to-day, and sample-to-sample producibility

of

the

technique

in

our

hands.

The

re-

counts

present in a typical spot (relative to others on the same gel) will the

vary by only same

101

sample.

increased, standard

with

(standard deviation)

Occasionally the

counts

deviation.

on gels run from

variability

in

a

typical

is

considerably

spot

On the more variable

showing

30%

sets of gels, the

staining patterns frequently indicate streaking or some other abnormality. more

Interestingly,

variation

proteins cause

at

of

different

some

than others.

the

top

of

some

of

days,

even

gel

Comparing

different

showed

in the clogging

first-dimension

scatter.

with

consistently

Variability

the

the

spots

control

is a

runs

of

likely

made

samples,

on

shows

that the counts in a typical spot had a standard deviation of 351

(Table l).

With this level of variation, a doubling

relative

synthesis

detected

as

rate

for

significant

a

(p^.02)

typical with

3

protein

of

would

experimental

be

and

3

control gels. Effect

of

protein

containing

amount

loaded

onto

gels.

Fifteen

varying amounts of the same sample were

gels

compared.

Figure 2 shows the effect of loading from 75 to 320 ug protein on

the

total

selected

counts

spots.

regression analysis. from

zero.

Thus,

recovered

from

increased

by

ug

drawn

loaded)

line

recovered

is the

result

in of

the a

12

linear

The slope is not significantly different there

the a

(per

The

12

was

no

significant

loss

spots

as

the

of

factor

of

4.

amount

Notice

that

of

counts

protein

this

was

analysis

emphasizes the contribution of the more abundantly synthesized proteins among the 12 selected for analysis. comparing

the

open

and

filled

circles

As can be seen by in

Figure 2,

the

recovery also was independent of whether sample volumes were proportional or

constant

to the amount of protein loaded (open circles); sample

volumes

were

used

(closed

circles;

369

o

n • p-j co i-i

00

C • rH

i/> to

to

e re e m a S u

/—\

dP di' to VO \o LO r j r j O to >—1 II II II

O o o ex & &

O z PS t—( Ph S ft a, o u

2.

After the

incubation is completed, the medium is removed and placed into a separate tube, and the tissue is transferred from the grid to a conical glass tube and washed three times by centrifugation in sterile balanced salt solution.

The tissue is then

homogenized in 400 yl of 0.25% deoxycholate in balanced salt solution using a model W185F sonifier (Heat Systems Ultrasonics, Inc.) adjusted to an output of 4. performed using three 10-second bursts.

Sonification is The homogenate as

well as the culture medium are then centrifuged at 30,000 g for 25 minutes in a Beckman 65 rotor, and the supernatants obtained in this fashion are used for one and two-dimensional

kkG gel analysis before and after immunoprecipitation of the newly synthesized apoproteins.

To immunoprecipitate newly synthe-

sized apoA-I, carrier HDL and anti-HDL antibodies are added to the supernatants.

The optimal amount of anti-HDL antibody was

determined as follows: 8.5 yg of HDL protein was mixed with different amounts of anti-HDL antibody and the mixture allowed to immunoprecipitate at 4°C for four days.

The immunopreci-

pitate was collected by centrifugation for two minutes in a Beckman microfuge, washed two times with 20 mM potassium phosphate buffer pH 7.0 and once with water.

The immunopre-

cipitate was then dissolved in SDS sample buffer and subjected to one-dimensional SDS polyacrylamide gel electrophoresis on 12% slab gels.

The proper ratio of antibody to HDL for maxi-

mum apoA-I precipitation was estimated from the intensity of the Coomassie brilliant blue stained apoA-I band.

After the

proper ratio of antibody to apoA-I was determined, the organ culture extracts and the culture media were mixed with 8.5 yg of carrier HDL protein and immunoprecipitated by the predetermined optimal volume of anti-HDL antibody.

The immunopreci-

pitates were then dissolved in SDS sample buffer or lysis buffer and used for one-dimensional SDS polyacrylamide gel electrophoresis or two-dimensional polyacrylamide gel electrophoresis, respectively.

Polyacrylamide gel electrophoresis

reveals the carrier proteins by protein staining of the gels and the newly synthesized apoproteins by autoradiography of the same gel. RESULTS AND DISCUSSION A.

Two-Dimensional maps of human VLDL and HDL apo-

proteins .

Figures 1 and 2 show the overall two-dimensional

maps of VLDL and HDL apoproteins, respectively.

It is

apparent that most of the apoproteins of VLDL and HDL are composed of several isoproteins.

ApoA-I and apoE isoproteins

will be presented in detail later in the text.

kk 7

FIGURE 1. Two-dimensional gel electrophoresis of 100 yg of normal human VLDL. Two-dimensional gel electrophoresis was performed as described in the Materials and Methods section. The molecular weight protein markers are bovine serum albumin 67 K. ovalbumin 43 K, bovine purine nucleoside Phosphorylase 30 K, apoA-I 28 K, trypsin inhibitor 19 K, egg white lysozyme 14 K, apoA-II 9 K. The position of known apoproteins (B,C, E) is indicated. IN THIS AND ALL SUBSEQUENT TWO-DIMENSIONAL GELS THE SECOND DIMENSION WAS RUN ON A SLAB OF 12% POLYACRYLAMIDE. IN THIS AND ALL SUBSEQUENT TWO-DIMENSIONAL GELS AND AUTORADIOGRAMS, THE BASIC SIDE (CATHODE) IS ON THE LEFT AND THE ACIDIC SIDE (ANODE) IS ON THE RIGHT OF THE PHOTOGRAPH.

k k 8

-XI

c

All

I PH

6

5

55

ff i

5

19 K -

r

4 5

FIGURE 2. Two-dimensional gel electrophoresis of 340 ug of normal human HDL. The pH gradient of the gel is indicated. The molecular weight protein markers are bovine serum albumin 67K, ovalbumin 43K, bovine purine nucleoside phosphorylase 30K, trypsin inhibitor 19K. The position of known apoproteins is indicated (A-I, A-II, C, E). Minor individual proteins or groups of proteins are indicated (XI, X2...X8). The gel shown in Figure 2 was loaded with excess protein to reveal minor apoprotein components thus the apoA-I isoproteins are not distinct. The VLDL apoproteins appear to be those previously described: apoB, apoE, and the apoC's. However, in the HDL fraction we observe, in addition to the previously described apoproteins, apoA-I, apoA-II, apoC's and apoE, a number of other protein components. These have been named X-l to X-8. Most of the HDL apoproteins can be immunoprecipitated with

449 anti-HDL antibodies.

All of the VLDL apoproteins can be

immunoprecipitated with anti-VLDL antibodies.

The apparent

isoelectric points of the VLDL and HDL apoproteins are given in Table IIA. TABLE IIA Apparent Isoelectric Points (Pi's) of Plasma Apolipoproteins Apoprotein

Apparent Isoelectric Point

A-I A-11 B CII CIII-0 CIII-1 CIII-2 E X-l X-2 X-3 X-4 X-5 X-6 X-7 X-8

See Table IIC 5.0 > 6.8 4.7 4.8 4. 55 4.3 See Table IIB 5.3 5.45 5.50 6.50 6.25 >6.8 6.15 5.10

The approximate molecular weights of the X protein components of plasma HDL are:X-l = 33K, X-2 = 60K, X-3 = 90K, X-4 (X4') and X-5 (X5') = 38K, and X-6 to X-8 = 20K.

Protein

spots X-l to X-8 were cut from the gel, iodinated, and rerun on a 16% polyacrylamide gel in the presence of proteolytic enzymes.

The one-dimensional staphylococcal protease peptide

maps of proteins X-l, X-2 and X-3 produced one peptide with molecular weight 2 8K and smaller peptides homologous to those of apoA-I (Figure 3A).

In contrast, there was no homology of

X-4 and X-5 peptides with those of either apoA-I or apoE (Figure 3B).

450

FIGURE 3. Panel A: Autoradiogram of one-dimensional staphylococcal protease peptide maps of HDL proteins X-l, X-2, X-3 and apoA-I. Iodination and one-dimensional peptide mapping of protein spots cut from two-dimensional gels was performed according to published procedures (92-94). Lane a, protein X-l, lane b, protein X-2, lane c, protein X-3, lane d, apoA-I. The position of the undigested apoA-I is indicated. The film was exposed for two days. Panel B: Autoradiograms of onedimensional chymotryptic peptide maps of HDL and VLDL apoE isoproteins and of proteins X-4 and X-5. Lane a, protein X-4, lane b, protein X-5, lane c, HDL apoE isoprotein 3, lane d, HDL apoE isoprotein 4, lane e, HDL apoE isoprotein 5, (see Figure 2), lane f, VLDL apoE isoprotein 3 (see Figure 5A). The undigested apoE is indicated. The film was exposed for 4 days. Proteins X-6, X-7 and X-8 did not have any .peptide homology with apoA-I or apoE.

However, their molecular weight,

approximately 20K, is close to the molecular weight range of apoprotein D, a previously described minor apoprotein component of HDL (50-52) .

Proteins X-2 and X-3 have a molecular

451 weight of approximately 60K and 90K Daltons, rexpectively, and may represent aggregates of apoA-I.

Such aggregation arti-

facts after SDS gel electrophoresis have been described previously (97).

However, proteins X-l, X-4, and X-5 with

molecular weights in the range of 33K to 38K and isoelectric points as described in Table IIA are not aggregation artifacts and do not match the description of any of the known apoproteins and therefore they represent new minor apoproteins of HDL. The two-dimensional polyacrylamide gel electrophoresis maps of apoCIl and CIII isoproteins are shown in more detail in Figure 4 and their isoelectric points are listed ii) Table IIA.

I

CIII-2

CIIN

Oil

FIGURE 4. Two-dimensional gel electrophoresis of 200 yg of VLDL. The panel shows magnified section of the gel in the vicinity of apoC. The apoC proteins are indicated. Note that apoCIl and the CIII peptides are composed of two or more isoproteins with differences in their molecular weight. In this study, we observe the previously described polymorphism of apoCIII (34, 46-48).

The isoproteins of apoCIII

have been shown to be the result of an O-glycosidic linkage

between threonine-7 4 and a polysaccharide composed of galactose, galactosamine and either 0, 1 or 2 molecules of sialic acid.

However, our studies suggest that isoproteins of apo-

CIII with the same charge have two or more components that differ in their molecular weights.

The existence of these

forms of apoCIII has not been reported previously and their molecular explanation requires further investigation.

A

similar molecular weight heterogeneity of apoCII is also seen in Figure 4. B.

Isoproteins and genetic polymorphism of apoE. Two-

dimensional gel electrophorfetic analysis of VLDL apoE revealed two major isoprotein patterns designated as class a and class f3 (Figure 5A, 5B, and 5C) .

Class a consists of at

least seven isoprotein groups designated 1 to 7.

Each group

contains one or more isoproteins all of which have the same isoelectric point but slight differences in their molecular weight.

Class B consists also of at least seven isoprotein

groups with similar characteristics to those of class a but has a diminished concentration of one of the major isoproteins present in class a.

The apparent isoelectric points and

charge differences of the apoE isoproteins are given in Table IIB.

All apoE isoproteins were found to have the same one-

dimensional chymotryptic peptide maps which suggest that they have a similar but not necessarily identical primary amino acid sequence.

It is unlikely that all of these isoproteins

are products of different genes but rather that they are caused by post-translational modifications of one or a few closely related genes that specify the primary sequence of apoE.

Post-translational modification of proteins is a wide-

spread phenomenon in nature

(92,98-101) and these modifica-

tions of apoE may play an important but as yet undefined role in plasma lipoprotein physiology. C.

Four apoE subclasses in normal humans and a unique

subclass in type III hyperlipoproteinemia. Two-dimensional gel electrophoretic analysis of mixtures of VLDL obtained

^53

A i

i

•

t

• 1

*

7

6

i i ' #

*

a

B ; J

C

1

• I ! ^ <


5 m l glycerin and 85 ml water and dried

at 115°C for 10 minutes. Yellow-brown zones could be seen in visible light and strongly fluorescent zones at 360 nm. For visualization with ninhydrin the paper was first

impregnated

with 0,3 M citrate buffer, pll 5>5. The dry, buffered paper was dipped into a 0,2 "fa solution of ninhydrin in methanol

contai-

ning 2 °/o glycerin. After drying for less than 20 minutes at room temperature the paper was used for printing. The ninhydrin print was dried at 60°C for 1 0 - 2 0

minutes. The

carrier

ampholytes appear as blue-grey-violet zones stained with varying intensities.

Results and Discussion For the first step (i) of recycling, the separation of carrier ampholytes from proteins, we employed membranes with retentions ranging from 5 000 to 20 000. Membranes with cut-off levels of 500 - 5 000 were tested for the second step (il), the

558 Table I: Ultrafiltration membranes Membrane PA 20

Manufacturer Kalle

UM 20

Amicon

UM 10

Amicon

DM

5

Amicon

2

Amicon

CA UM

Kalle

UO CA 70

Kalle

UM 05

Amicon

Nominal MW cut-off

Material

20 000 20 000 10 000 5 000 4 000 2 000 700 500

polyamide synth. polymer synth. polymer synth. polymer cellulose

acetate

synth. polymer cellulose acetate synth. polymer

recovery of carrier ampholytes (Table i). Membranes of noncellulosic material and cellulose-type membranes of different origin were chosen to overcome problems possibly raising from noticeable adsorption on some membranes. Commercially available carrier ampholytes are claimed to have average molecular weights of 300 - 700 with a maximum molecular weight of less than 1 000. Difficulties in the removal of carrier ampholytes from proteins (10), results of gel tography (7)11) and staining artefacts

chroma-

(12) suggest that the

actual molecular weight of carrier ampholytes deviates

from

the values reported by diverse manufacturers. This was strongly supported by our findings. Typical results obtained with broad pll-range carrier ampholytes, pH 2 - 1 1

'Servalyt', ana-

lytical grade, and pll 3>5 - 10 'Ampholine', analytical grade, are shown in Fig. 1. The PA 20 membranes proved best for the removal of carrier ampholytes from proteins. Less than 1 °/o of carrier ampholytes of different origin and of different pliranges were retained on these membranes. In experiments with 0,2 - 1 °/o solutions of a mixture of sperm whale myoglobin (MW 17 000) and bovine serum albumin (MW 67 000) protein rejections^ 90 °/o were observed. High retentions of ampholytes were noted for the UM 10 membranes with a nominal cut-off of 10 000 indicating

that the separation of low molecular weight

559

100-i % :

100 %

r

I— oI"

50

(

50

'Servalyt',pH 2-11

0 o

0

'Ampholine',pH 3,5-10

50

u

)

in —1

. u.

100

100

-J-

1

F i g . 1: D i s t r i b u t i o n of 'Servalyt', pH 2 - 11 and

'Ampholine',

pH 3>5 - 10 carrier ampholytes on different membranes in absence of proteins. R - retentate, U - ultrafiltrate proteins

(MW

10 000) from carrier ampholytes could prove

difficult with this type of membrane. The data of Fig. 1 and the results of additional experiments have shown that by all types of membranes

'Ampholine' and

'Pharmalyt'

carrier

lytes are consistently retained more effectively 'Servalyt' carrier ampholytes suggesting higher

ampho-

than the molecular

w e i g h t s . C o m p a r i s o n of the results of the non-ionic D M membranes w i t h the UM membranes carrying some free indicates

ionic

sites

that ion-exchange processes and concentration

rization may contribute

to the higher

R e c o v e r y of carrier ampholytes

retention.

(step II) from

ultrafiltrates

of step I is best achieved w i t h the U M 05 membranes, covery b e i n g u s u a l l y better

the re-

than 85 - 90 °/o for all types of

carrier ampholytes. Several membranes retained

the

'Ampholine'

carrier ampholytes, whereas substantial portions of the valyt'

carrier ampholytes passed some of these membranes

indicating a lower molecular weight or a low adsorptive action w i t h the membranes

pola-

'Seragain inter-

(Fig. 1). Average flow rates of 1 -

56o

Myoglobin (17 800) Cytochrome C (12 kOO) Aprotinin (6 500) Polymyxin B (1

150)

Azocarmin B (700)

Starting line

Fig. 2: Thin—layer gel chromatography on Bio-Gel P-10. Sample application 10 jil (5

+ 5

myoglobin as internal marker.

Print stained with Amido Black 10 B, marked spots - visualization with fluorescamine. 1 - MW-markers, 2 -

1

Servalyt' con-

trol, 3 - 'Servalyt' retentate on PA 20, ^ - 'Servalyt'

recyc-

led, 5 — 'Ampholine' control, 6 - 'Ampholine' retentate on PA 20, 7 - 'Ampholine' recycled, 8 - 'Pharmalyt'

control,

9 - 'Pharmalyt' retentate on PA 20, 10 - 'Pharmalyt'

recycled,

11 - MW-markers. 2 °/o solutions of carrier ampholytes without and with 1 "fo added protein were 1 , 5 ml/cm

2 ml/cm

x h for the PA 20, and 0,3 - 0,6

x h for the UM 05 membranes.

These findings of ultrafiltration were corroborated by thinlayer gel chromatography (Fig. 2). The prints were

stained

first with fluorescamine followed by staining with Amido

Black

10 B, a dye which has the highest affinity for carrier ampholytes (3»9). Visualization of carrier ampholytes with fluorescamine, ninhydrin or lactose ( 7 )

does not parallel the stai-

ning with Amido Black 10 B, which stains more intensively and preferentially the high molecular species. The lowest apparent

561 molecular weights, namely 800 - 1 200 and a relatively uniform distribution were repeatedly found for the 'Servalyt 1

carrier

ampholytes. High molecular weight species are virtually absent in these carrier ampholytes with only traces appearing in retentate I. 'Ampholine' carrier ampholytes have apparent molecular weights in the range of 1 000 - 6 000 and appear less uniform than 'Servalyt' ampholytes. The patterns of retentate I and retentate II differ distinctly and show that by ultrafiltration through a PA 20 membrane some high molecular weight species can be removed. The

'Pharmalyt' carrier ampholytes are

composed of a particularly heterogeneous population of ampholyte species with apparent molecular weights ranging from 1 000 - 15 000. Comparison of the retentate I and II patterns suggests that a substantial portion of the high molecular weight species of 'Pharmalyt' can be separated by ultrafiltration on a PA 20 membrane. The results with the carrier ampholytes are puzzling since a gel

'Pharmalyt'

chromatographic

step is claimed to be involved in the production of these carrier ampholytes and a uniform size distribution

therefore

could be expected. Most results described in this report were obtained with BioGel P - 1 0 or P-6 gels. Additional experiments on Sephadex G-50 layers confirmed these results indicating that the observed differences are not due to an interaction with the gel matrix. Commercially available

1k

led with C

'Ampholine' carrier ampholytes,

label-

were previously shown to have much lower appa-

rent molecular weights on Sephadex G-25 columns

calibrated

with polyethylene glycols of known molecular weight (13). A possible explanation for the observed discrepancies could be that peptides, resembling more closely the carrier ampholytes, were employed as markers in this work. In thin-layer isoelectric focusing on Bio-Gel P-60, followed by visualization of the carrier ampholytes with ninhydrin, formaldehyde or fluorescamine, the commercial and recycled

562

Control

FLU FOR

recycled

NIN

FLU FOR NIR

Fig. 3: Thin-layer isoelectric focusing of commercial and recycled

'Servalyt' pH 2 - 1 1

carrier ampholytes on Bio-Gel P-6o.

Visualization of the Whatman No. 1 prints with fluorescamine (FLU), formaldehyde

(FOR) and ninhydrin

(NIN)

ampholytes were very similar both with respect to distribution and number of ampholyte species. The ninhydrin and formaldehyde patterns closely resembled those previously found

(7)-

In conclusion, commercially available carrier ampholytes can be recycled in a two-step procedure by employing membranes with different retention characteristics. Reasonable flow rates were obtained already in stirred cells; hollow fiber systems with the recently introduced fibers with low molecular weight cut-off (H1P2 cartridge type from Amicon) would afford the advantage of operation at higher rates than can be expected with conventional ultrafiltration systems. The properties of recycled carrier ampholytes are even better than those of the commercial material: the cathodic drift is reduced and the size distribution is more uniform because high molecular weight components, occasionally present in commercial ampholytes, are removed in the first step. Recycling of carrier ampholytes is

563 inexpensive and likely to improve considerably the economy of preparative isoelectric

focusing.

References 1. Martin, A.J.P., Hampson, F.: J. Chromatog. V59, 101 - 110 (1978) . 2. Righetti, P. G.: Separat. Purificat. Methods 4, 23 - 72 (1975). 3. Vesterberg, 0.: in 'Isoelectric Focusing' (N. Catsimpoolas, Ed.) pp. 53 - 76, Academic Press, New-York, 1976. 4. Callaham, M.F., Poe, W.E., Heitz, J.R.: Anal. Biochem. JO, 542 - 5^6 (1976). 5. Delincle, H., Radola, B.J.: Anal. Biochem. ¿ 0 , 609 - 623 (1978). 6. Radola, B.J.: J. Chromatog. _38, 61 - 77

(1968).

7. Radola, B.J., Tschesche, H., Schuricht, H.: in 'Electrofocusing and Isotachophoresis' (B.J. Radola and D. Graesslin, Eds.) pp. 97 - 110, Walter de Gruyter, Berlin, 19778. Hung, C. H., Strickland, D.K., Hudson, B.G.: Anal. Biochem. 80, 91 - 100 (1977). 9. Radola, B.J.: Biochim. Biophys. Acta 295, 412 - 428 (1973). 10. Fawcett, J.S.: in 'Isoelectric Focusing' (j.P. Arbuthnott and J.A. Beeley, Eds.) pp. 23 - 43, Butterworths, London (1975). 11. Baumarm, G., Chrambach, A.: Anal. Biochem. 64-, 530 - 536 (1975) . 12. Otavsky, W.I., Drysdale, J.W.: Anal. Biochem. 6_5, 533 536 (1975). 13» GaspariS, V. Rosengren, A.: in 'Isoelectric Focusing' (j.P. Arbuthnott and J.A. Beeley, Eds.) pp. 178 - 181, Butterworths, London (1975)«

REMOVAL OF SALTS AND CARRIER AMPHOLYTES FROM BILIPEPTIDES OF LOW MOLECULAR WEIGHT

H.-P. Köst and E. Köst-Reyes Botanisches Institut der Universität München, Menzingerstr. 67 D-8 München 19

Introduction The phycoerythrins and phycocyanins of red algae, blue-green algae and cryptophytae have been identified as biliproteins with covalently linked bile pigment chromophores (1»2,3). In order to elucidate the nature of this linkage(s), the preparation of small bilipeptides was required. The main subject of our investigations (3»^»5»6) has been B-phycoerythrin from the marine red alga Porphyridium cruentum. B-phycoerythrin was isolated in pure form from crude P. cruentum biliprotein by a carrier-free electrophoretic separation on LKB columns (5»6). Small bilipeptides with intact native phycoerythrobiline chromophore were obtained proteolytically (5>6). After several purification and separation steps, an ammonium sulphate precipitation of bilipeptide containing fractions was carried out. Since a subsequent separation by isoelectric focusing was im portant, it arose the general problem to remove contaminating salts. After completed electrofocusing, the carrier ampholyte R\ R (Ampholine or Servalyt ) had to be removed.

Results 1) Preparation of low molecular weight bilipeptides from B-phycoerythrin of Porphyridium cruentum. Ammonium sulphate precipitation and salt removal. For isolation and purifi-

1980 Walter de Gruyter & Co., Berlin • New York Electrophoresis '79

566 cation of B-phycoerythrin, see r e f e r e n c e ( 6 ) P u r e

B-phycoery-

thrin was first denatured with TCA. A enzymatic degradation with trypsin (1:10) and pepsin (1:10) followed. Small bilipeptides could be obtained, exhibiting a mol. weight between ca. 1000 and 3000 Daltons. For their purification the steps given in the following fig. 1 were applied.

Preparation of Low Molecular Weight Bilipeptides from Porphyridium cruentum B-Phycoerythrin.

B-phycoerythrin

i

TCA-denaturation trypsin digest f1i10J pepsin digest (1:10)

1

chloroform extraction.

1 I

.denatured protein ¡.chloroform phase

water solution n-pentanol extraction-

water solution ammonium sulphate precipitation (80$ sat.) salt removal isoelectric focusing and subsequent spectroscopy

•denatured protein •n-pentanol solution addition of petrol ether/ water bilipeptides in water solution ammonium sulphate precipitation (80$ sat.) salt Removal*

i

isoelectric focusing removal of carrier ampholytes EDMAN-degradation etc.

567 The degradation mixture was first extracted with chloroform in order to remove enzyraatically split chromophore.- Upon extraction with n-pentanol, a separation into bilipeptides that were extractable into the organic phase and bilipeptides staying in the water phase was achieved. By addition of the threefold volume of petrol ether and water to the organic phase, the bilipeptides were transferred back into water solution. They were precipitated by addition of solid ammonium sulphate to 80$s saturation

( 5 , 6 ) . In order to carry out isoelectric focu-

sing with the redissolved

(NH^)^SO^-pellets a salt removal

procedure was required. Dialysis was difficult because of the low molecular weight. A desalination procedure that avoids dialysis is given in the following (5)« A LKB 8101 column (110 ml) was filled with a Tricine Hydroxy-1.1-bis(hydroxymethyl)-ethyl

(=N- 2-

-glycine) solution with

superimposed density gradient (33 mM Tricin, 7 mM sodium sulphate, pH=5.3; sucrose ). A solution of the bilipeptides to be desalinated was first adjusted to an appropriate specific density (with solid sucrose) and column temperature

(10°C). The

isoelectric point of the peptides to be desalinated was near 5.3. The solution of the peptides was applied to the middle of the column with the aid of a small PTFE-tubing. Electrophoresis was than carried out first at 100 V , finally at 50 V, 3.2 W

were never exceeded. The anode was at the top of the

column. After about 300 minutes, the bilipeptides were

suffi-

ciantly salt-free to carry out electrofocusing. The volume of the desalinated peptide solution was 6.2 ml.

(fig. 2, fig. 3)

2) Removal of carrier ampholytes. Removal of carrier ampholytes from bilipeptides that \iere not extractable into n-pentanol was difficult. For n-pentanol extractable

bilipepti-

des, the following procedure was used: Bilipeptide samples, 4 - 1 0 ml each, containing carrier ampholyte and sucrose, were adjusted to pH=1 with concentrated hydrochloric acid. They were extracted with 3x10 ml

568 Anode

A

A

A A

O O O O

OK 0 KOAKO I A „A OK OK 0 AO •

K

K K

K K

K

f i g

K K

K K K

K K

K K

K

Cathode

Cathode

3.

f i g

t i m e 6

50

109

a

200

250

!Mn) 300

569 n-pentanol. If necessary, the amount of acid was doubled and the extraction was repeated, The combined organic colored

frac-

tions were washed with 0.1 N HC1 (3 * 10 ml) and 90 (180) ml of petrol ether were added, followed by 10 ml of water. Upon intense shaking, the bilipeptides were extracted into the water phase. Repetition of the procedure with another 10 ml of water led to nearly quantitative recovery of bilipeptides from the organic phase. The product obtained was tested by TLC for freedom from carrier ampholytes.- The procedure described is most suitable for acidic bilipeptides (P^=3.81). Bilipeptides with PT«i6

tend to give low yields, however (< 50$).

Legends for figs. 2 and 3 fig. 2 Salt Removal by Electrophoresis near The Isoelectric

Point

1. Schematic drawing of electrophoresis column after application of the sample to be "desalinated" (usually obtained by redissolving low molecular weight ammonium sulphate precipitates) 2. Column during salt removal process. Partially the anions and cations have migrated out of the bilipeptide middle zone. 3. Column after salt-removal. Cations and anions have completely been removed from the bilipeptide middle zone. The bilipeptides are now suitable for subsequent electrofocusing. 0 = bilipeptide molecule = zero) K = cation (e.g. NH^ ^ A = anion (e.g. SO^ )

(net charge at the isoelectric point

Power/ Time Plotting: Salt Removal by Electrophoresis near The Isoelectric Point (pH=5.3) Electrophoresis was first carried out at 100 V, than at 50 V (upper part of the graph). The values for constructing the first (lower) part of the graph have been obtained by multiplication of the original values with 0.25 (power increases with the square of the voltage). The peaks indicate arrival of the ions to be removed at the electrodes (NH^, SO^ and 4 4 others)

570 3) Test for LKB carrier ampholyte "Ainphollne" (acidic i:H range) Either 1 ml of the fraction to be tested or a solution of the carrier ampholyte (0.03 ml + 0.97 ml dist. water) was used.. 0.03 ml conc. hydrochloric acid was added, 'flie mixture was shaken with 0.5 ml n-pantanol. The organic upper phase and the lower water phase were chromatographed on silica gel GFgj.^ coated plates. Solvent sytem used: n-butanol/' glacial acetic acid/ water = 3 5 3 1 1 (v:v:v). Carrier ampholine constituents were detected as blue spots with the chlorine/ benzidine reagent. In the organic phase, trace amounts of carrier ampholyte only or none at all could be detected, whereas the water phase yielded several blue spots when carrier ampholytes were present

(7).

References 1

1. Rüdiger, Vi.: Hoppe-Seyler s Z. Physiol. Chem. 350, 1^91— 1300 (196S). 2. Rüdiger, W., 0 Carra, P. s Eur. J. Biochem.

509-516

( 19^9)

3. Köst, H.-P., Rüdiger, W., Chapman, Ü.J.: Liebigs Ann. Chem. 1975, 1582-1593. 4. Köst-Reyes, E., Köst, H.-P., Rüdiger, Vi.: Liebigs Ann.Chem. 1975. 1594-1600. 5. Köst-Reyes, iC.: Dissertation, Universität München(1978). 6. Köst-Reyes, E., Köst, H.-P.: Eur. J. Biochem, in print.

7. Köst, Ii.-P.: Dissertation, Universität Hünchen (1974).

PREPARATIVE ELECTROPHORESIS IN GEL-BLOCKS WITH DISCONTINUOUS ELUTION

H. Stegemann Institut für Biochemie, Biologische Bundesanstalt, Messeweg 11, D-3300 Braunschweig, West-Germany

General Considerations The term "preparative" is a matter of definition in protein and nucleic acid separation: fractionation by salts and solvents or by gel filtration can be done with kg or even tons, electrophoretic methods, however, deal with milligrams or a few grams. That implies the application after a first and different separation step or with limited material. On the other hand the quality of the electrophoretic separation in gels is nearly unsurpassed and it is the final goal to maintain the excellent discrimination between macromolecules in analytical gels also in the preparative procedure.

Abbreviations AA BIS PAA

= Acrylamide = N,N 1 -Methylene-bis-AA = Poly AA gel, mostly in the sense of 95% AA and 5% BIS PAGE = PAA-Gel-Electrophoresis PrepPAGE = Preparative PAGE PoroPAGE = PAGE in porosity gradient PAGIF = PAA-Isoelectric Focusing SDS = Sodium dodecyl sulfate ME = Mercapto ethanol Mapping = PAGIF in first dimension, PAGE in the second SDS-Mapping = after PAGIF incubation with SDS, then SDS-PAGE

© 1980 Walter de Gruyter 8. Co., Berlin • New York Electrophoresis '79

572

One may choose the more static separation "focusing" in gels (I believe density gradients as a stabilyzing medium are a little outdated except for few cases) and the dynamic "zone electrophoresis". The focusing can take a good load (up to five times that of electrophoresis), is easy to perform in granulated or solid gels and resembles the cut-and-squeeze procedure, when applied to electrophoresis. Using the same load, the former separation is somewhat sharper and more informative, since it depends on a well defined isoelectric point. Taking advantage of five times the load, the separation quality is similar. In contrast, electrophoresis depends on charge and size. In SDS-PAGE, where mainly size governs the separation, the low load (about a 1/50 of the load in PAGE) is a very limiting factor. All the advantages of preparative focusing will be discussed elsewhere, certainly as will the disadvantages of high cost and the sensitivity to small ions, which make necessary a cumbersome dialysis step with all its drawbacks. There is one point that speaks in favor of electrophoresis: the loss of enzyme activity during focusing. Points less important are the ease of recovery in solution and the negligible impurities from the carrier when the separated fractions migrated through the gel and were collected discontinuously. We will give examples later. The most appropriate procedure must be checked analytically beforehand. For isolation of a few milligrams in a relatively pure state and if oligomers of AA can be tolerated one should neither use a preparative scale of focusing nor electrophoresis with elution, but should stick to a two-dimensional mapping (PAGIF in 5 x 16 x 120 mm gel, PAGE in 16 x 120 x 250 mm) as described in ( 1 ) ( 2 ) . Geometry

of the Apparatus

A few years after the introduction of PAA the cylindrical gel was favored in PrepPAGE (3,4 and many others) and is s ti 11 used with more or less success. Another ingeniuos approach with

573 some practical difficulties not yet completely solved is the separation in a thick slab, its 90°-turn, the perpendicular electrophoretic extraction and the collection due to higher density at the bottom ( 5 ). it was our experience that it works well for fast migrating proteins and a short elution time,

but not so good

for slow proteins after a long ex-

traction. The diffusion is too prominent to get a good concentration and even in cases where the extracted proteins concentrated a later dilution by diffusion was found. Comparing thoroughly cylindrical ( 6 ) and rectangular

gels for Prep-

PAGE ( 7 ) W e can conclude that the rectangular design is far more recommendable due to better cooling, more efficient eluo tion and good yield. Blocks with 20 cm area give no problems with up to 3 g proteins or 400 mg of any individual protein. For economic reasons and for a routine use in courses for students we use the cuvette for PrepPAGE and the auxiliary parts as optional equipment of the basic apparatus PANTA-PHOR, which can deal with all other electrophoretic methods as well. The rectangular cuvette from glass holds PAA-gels firmly; with few buffers a first treatment with methacryloxypropyltrimethoxysilan ( 8) is advisable to avoid a slipdown. Many instruments have been developed, among them Nees (9), Foissy (10), Ernst and Niedner (11) and Chrambach and Nguyen (12) where also part

574 Gel For PAA in PrepPAGE pure chemicals are recommended and sulfite should be added to the catalysts for faster polymerisation (13) ( 14 ). A spacer gel of 3% PAA should be used for loads above 2 g or above 300 mg for individual compounds or for sample volumes above 3 ml. This spacer gel may contain the same buffer as the separation gel or may be diluted 1:2. Except in very few cases the discontinuous system does not contain any advantage. Boiled water should be used for overlayering. No supports for the gel. To achieve a good adherence the glass cuvette should be completely free from impurities. Any other handlings are described in detail in our 32-pages-instruction for the use of the PANTA-PHOR apparatus which is sent on request (15). For nucleic acid separation a block of agarose-PAA-gel is formed, the bottom of the cuvette consists of a 1 mm thick plexiglas® sheet whose sides are 0.5 mm

bigger than the in-

side of the cuvette. This will prevent the agarose-gel from slipping down. Pumping in and pumping out should be done at the same speed to avoid any change of pressure in the elution chamber. The membrane has to be tested beforehand ( 16 ) that it does not adsorb macromolecules. For proteins, sap from potato tubers either dialyzed or non dialyzed - is a very good test material since it contains about 1% proteins and glycoproteins between the isoelectric points 3 and 10.5 in an even distribution. It must be worked up under reducing conditions ( 17 ). The pattern should be determined in analytical PAGE before and after ad2

ding about 100 cm

of the respective wet dialysis tubing to

5 ml sap. When a part of one lot is checked, it applies to the whole lot. Most of the lots are very dependable. In case of insisting adsorption the current may be timed to flow in the opposite direction for one minute just before flush elution is started.

575 Samples Either raw or partially purified samples are applied which have a salt content below that of the buffer. For instance raw juice from potato tubers can be applied directly, thus minimizing the time of unwanted reactions. For nucleic acid preparations the complete absence of nucleases is essential. Since usually sucrose (rock candy) is added to samples (2 to 20 ml or 0.3 to 3 g and not more than 400 mg per individual compound per 2 20 cm ) the sucrose also has to be checked for degrading enzymes in spite of the fact that small rock candies are one of the purest organic compounds. All this does not apply to SDS treated samples.

Those loads

must be reduced to about a

1/50 as mentioned before in agreement with the findings by (18). See the review ( 13 ), compiled in late 1977.

Elution The primitive form is the cut-and-squeeze version. A guide band of the gel is stained and according to that the rest is cut out, squeezed through a fine mesh, e.g. nylon stockings (on a support) put into a disposable syringe followed by the extraction of the slurry ( 2). The yield is in the order of 80% but the extract contains much more oligomeric AA compared to the elution from intact gels. The oligomeric material should be removed by passage through columns with Sephadex-G 100 if proteins are further used for e.g. trypsinolysis and finger printing. For re-electrophoresis the extracted material can be used directly. A far more advanced procedure is the elution of fractions which had been passed through the gel after separation and are collected in a continuous or discontinuous stream of buffer, the latter also monitored by a special program. No streaming buffer at all but replacement from the lower reservoir is

576 used by ( 18). There are too many designs in existence to discuss here and it is true that most of them did not succeed in solving a practical problem ( 12 ). This latter does not hold for our elution device applied to isolate prominent bands of equal migration rate in different potato cultivars and vice versa (19) and to isolate a protein found only in the immature tuber ( 17 ). For results see chapter "Application". Ten years experience with this device and comparison with others may result in some general conclusions: Rectangular cuvette, no support for a continuous or possibly discontinuous gel, efficient flushing, reliable membrane. These are not only drawn from working with various designs but from working as well with students not experienced in gel techniques, who successfully performed the PrepPAGE after a few hours of instruction. The eluted proteins have the native configuration and are not digested by trypsin unless prior heating. Timer (Type DCSW) For programed elution a timer (Fig. 2) is useful to flush out at increasing intervals the material collected in the elution chamber to counteract the decreasing migration rate of the "slow" molecules. No success could be seen with retarded flow rates since the eluting stream becomes so slow that complete rinsing is not achieved. A flush with 2 ml per minute sweeps out nearly 90% of the collected material; a further 4 ml in 2 minutes gets most of the remainder. So, a rinsing time of 3 minutes is usually sufficient. The time set between 0.5 and 6 minutes, can be programed for this purpose in addition. The intermission between flushing is governed by punching a black Super-8-film. The speed of the film is 25.4 cm/h, any interval can be choosen.

This timer is commercially available

(Labor-Miiller KG, D-3510 Hann. Miinden) and has proved to be reliable since 1969. The total program may run up to 10 days (depending on the film lenght) for any given interval not

577 shorter than 2 minutes. The rinsing time is set independently. Details of the instructions are described in (15)« see fig. 2.

Concentration The ratio of the concentration of one compound in the original sample to that in the collected fraction depends on the starting volume, the volume of the fractions and the intermission time between sweepings. The schedule can be calculated

(20).

We have seen no dilution at all with 6 ml samples and 6 ml fraction.

More conventional is a dilution of 1 : 1 or of 1 : 2,

which is still concentrated enough to use e.g. 50^ul of a 6 ml fractions for an analytical re-electrophoresis without any need for concentration. If reduction of the volume cannot be circumvented, samples are filled into unknotted dialysis tubing in a tray with dry high-molecular polyethyleneglycol ( 21 ) for re-electrophoresis in PAA. For SDS-PAGE this procedure is not good since low-molecular impurities from PEG interfere with SDS. Another method of concentration almost as easy, and applicable in any case, is the use of tested collodium bags, in which 100 ml or more can be reduced to a few ml overnight ( 16 ). Never use freeze-drying or procedures removing the water layer around the protein and exposing it to oxidation, as they will certainly lead to diffuse bands

in re-PAGE, odd results in mapping

and diminished enzyme activity except with very stable molecules .

Fig. 2

Programm-Geber Timer Typa DCSW

Timer and puncher for programing stepwise elution

578

Applications PAGE patterns of proteins in potato tubers are characteristic for a cultivar or a variety ( 21 ) ( 22 ) as they are for other plant or animal tissues. The pattern change in potatoes is mainly due to a change of charge and - since by calculation only a quarter of mutation will bring about a charge change (see page 326/327 in reference (13))- it was of interest to determine the amino acid composition of PAGE-purified proteins of the same migration rate but from different cultivars. Also the composition was analyzed for bands of different speed within one cultivar to learn something about the nutritional properties of single potato proteins. This was discussed at the FEBS meeting and partially published (19 ). Since the prints were taken from color slides with meager results in identifying the fractions, a new sketch was done, fig. 3. The close similarity of fractions among cultivars is seen except in one case belonging to a band which can be split further by mapping.

p1 3—#*— 3—'

Amino acid composition of protein fractions (potato tuber); ^^^^^ isolated by prep. PAGE C.V. MARITTA ( ES335SI ) t C.V. VORAN ( ) , C.V. HYDRA ( ).

J|IIIP

a-

11leu I Leu | Tyr [ Phe j His | Lys | Arg | Asp | Thr | Ser | C.lu | Pro | Gla yl^|MetAla

579 ©

Fig.V; PrepPAGE of 5 ml raw potato sap in 5% Cyanogum, buffer pH 7.9, fractions 6 ml, re-electrophoresis of 50 pi, emphasis on slow proteins

3>

Fj

-g•

5

PrepPAGE as in Fig.4: but 2 5% sucrose added. Emphasis on faster proteins. Re-PAGE without sucrose

Whether one has slowly (Fig. 4. ) or faster (Fig. 5. ) migrating proteins, the separation can be achieved. Conversion of an isolated band was discovered (Fig. 2 in publication ( 17 )) in basic buffers which confused the interpretation before it was isolated and could have led to the conclusion of a badly separating PrepPAGE. All isolated fractions were not attacked by Trypsin unless they were boiled, showing the native state. Since some of the faster bands represent aggregating acidic glycoproteins, this PAGE was done with 25% sucrose in the gel to weaken or to prevent the aggregation. Compared with PAGE without sucrose the pattern appears much clearer. This is a problem often encountered in plant proteins and was discussed already in 1959 for preparation of extracts in the presence of polyhydroxy compounds ( 23 ). The application to PAGE is very promising.

580 Fig. 6 PoroPAGE of rice proteins stained for esterases as well. Fast migrating strong esterase is seen

i o

f

Fig. Mapping of rice esterases as in Fig. 6. Strong esterase not seen 3 after focusing Another problem was the purification of a protein complex found in immature tubers (24). It is homogenous in PoroPAGE and splits in PAGIF (24, 17) or with urea. Its property to migrate as one band at pH 7.9 or pH 8.9 in PAGE with almost the same speed makes it easy to separate the complex from all other proteins by consecutive PrepPAGE for antigen production a monovalent antiserum. A third example is the isolation of an esterase from rice which cannot be purified by PAGIF since it disappears when focused (Stegemann and Park, in preparation). Fig. 6

shows

the PoroPAGE pattern, first stained for esterases, followed by Supranolcyanin staining for proteins. The arrow marks the disappearing esterase. A check is done by mapping where this dominant esterase is not seen, fig. 7 . On the left there is the unfocused sample in the same slab. A last experiment not yet fully "student-course proof" is the isolation of intact viruses from raw material. With mixed gels of PAA and agarose in blocks we had some success with "easy" virus preparations and would like to extend it to hard-topurify material like the cucumber mosaic virus and other virus strains of

economic importance.

The aim is to purify them

directly from raw sap without time lag to such an extend as to get the pure virus for antibody production. The antiserum is than used for identification of virus in plants to maintain healthy crops. This work is done in cooperation with R. Koenig (Braunschweig).

581 Conclusions The results demonstrate that even slowly migrating bands can be separated very well by PrepPAGE. The

sharp

separation of

those bands is seen in fig.4 . In fig. 5 separation is shown of fast

aggregating

proteins which consist mainly of neutral

and acidic glycoproteins. The quality of separation is enhanced if runs are performed in PAA with 25% sucrose. PAGE in sucrose is superior to PAGE in urea since it diminishes especially the interference with carbohydrate constituents and the full enzyme activity is left. Sharpness of bands increased in the protein patterns of potatoes and all cereals tested. Good separations in PrepPAGE depend on an already clear pattern in analytical PAGE, the performance of the instrument (which was found best with rectangular gels and discontinuous flushing), the membrane and finally the selection of the appropriate intervals between flushings. They can be calculated (20) and punched into the black super-8 film of the timer type DSCW for up to 10 days or even set by hand when a more limited time is sufficient. Most of the separations were done within 30 hours. Detailed descriptions are found in the leaflet on using the PANTA-PHOR apparatus by students and technical assistants, which will be sent free of charge upon request.

References 1. Macko, V., Stegemann, H.: Hoppe-Seylers Z. physiol. Chemie 350, 917-919 (1969) . 2. Stegemann, H.: Z. anal. Chemie 252, 165-169 3. Hjerten, S.: J. Chromatogr.

66-

(1970).

(1963).

4. Altschul, A.M., Evans, W.J., Carney, W.B., McCourtney, E.J., Brow, H.D.: Life Sciences 3, 611-615 (1964). 5. Raymond, S., Jordan, E.M.: Sep. Science

95-1 1 1 (1966).

6. Stegemann, H., Rien, W.: Report Med. Forschg.-Anstalt d. Max-Planck-Gesellschaft (1963). 7. Stegemann, H.: Z. anal. Chemie 261, 388-391

(1972).

582 8. Radola, B.J.: personal communication. 9. Nees, St.: Theoretical and Practical Aspects of High Resolution Preparative Gel Electrophoresis Using a Continuous Elution System, p. 189-198, in "Electrophoresis and Isoelectric Focusing in Polyacrylamide Gel", ed. by R.C. Allen and H.R. Maurer, W. de Gruyter, Berlin - New York 1 974 . 10. Foissy, H.: Laboratory Practice 25, 839-844

(1976).

11. Ernst, W., Niedner, R.: J. Chromatogr. 130, 331-335 (1977). 12. Chrambach, A., Nguyen, N.Y.: Preparative Electrophoresis, Isotachophoresis and Electrofocusing on PAA Gels, p. 337368, in Electrokinetic Separation Methods, ed. by Righetti/ Van Oss/Vanderhoff, Elsevier, Amsterdam 1979. 13. Stegemann, H.: SDS-Gel-Electrophoresis in PAA, Merits and Limits, p. 313-336 in Electrokinetic Separation Methods, ed. by Righetti/Van Oss/Vanderhoff, Elsevier, Amsterdam 1979 . 14. Stegemann, H.: Hoppe-Seylers Z. physiol. Chemie 348, 951952 (1967). 15. Stegemann, H.: Electrophoresis and Focusing in Slabs, Using the PANTA-PHOR, Lab-Manual 1968/1979. Sent on request. 16. Francksen, H., Garadi, R.: Z. anal. Chemie 271, 340-344 (1974). 17. Stegemann, H., Francksen, H., Macko, V.: Z. Naturforschung 28 c, 722-732 (1973). 18. Koziarz, J.J., Köhler, H., Steck, Th.L.: Anal. Biochem. 86, 78-89 (1978). 19. Stegemann, H.: Properties and Use of Proteins from Potatoes and other Sources, p. 11-20, in Biochemical Aspects of New Protein Food, ed. by J. Adler-Nissen et al., FEBSMeeting Copenhagen, Vol. 4_4, Symposion A 3, Pergamon-Press, Oxford 1977. 20. Rohloff, H.: in preparation. 21. Loeschcke, V., Stegemann, H.: Phytochemistry 5, 985-991 (1966) . 22. Stegemann, H., Loeschcke, V.: Potato Research 20, 101-110 (1977). 23. Heitefuss, R., Buchanan-Davidson, D.J., Stahmann, M.A.: Arch. Biochem. Biophys. 8J5 , 200-208 (1959). 24. Stegemann, H. Characterization of Proteins from Potatoes, and the "Index of European Varieties", p. 279-284, in "The Biology and Taxonomy of the Solanaceae", ed. by Hawkes/Lester/Skelding, Birmingham-Symposium 1976, Linnean Soc. Symp. Series No. 7, 1979.

F U R T H E R D E V E L O P M E N T S IN P R E P A R A T I V E

DISPLACEMENT

E L E C T R O P H O R E S I S IN G E L

F.

Hampson

Angewandte Physikalische Chemie, Universität des S a a r l a n d e s , 6600 S a a r b r ü c k e n

Introduction

T h e r e c e n t l y d e v e l o p e d m e t h o d of d i s p l a c e m e n t e l e c t r o p h o r e s i s in a t h i n a n n u l a r g e l w i t h v e r y e f f i c i e n t c o o l i n g , ( 1 ) , m a k e s it p o s s i b l e to r e a l i z e t h e m a j o r t h e o r e t i c a l a d v a n t a g e s of d i s p l a c e m e n t e l e c t r o p h o r e s i s o v e r zone e l e c t r o p h o r e s i s and i s o e l e c t r i c focusing, a s a p r e p a r a t i v e technique. When the method was developed the only supporting m e d i u m a v a i l a b l e , h a v i n g a low e n o u g h e l e c t r o e n d o s m o s i s , w a s c r o s s - l i n k e d

polyacrylamide.

T h i s m e d i u m h a s p r o v e d t o b e s u i t a b l e f o r u s e in p r e p a r a t i v e d i s p l a c e m e n t e l e c t r o p h o r e s i s b u t it e x h i b i t s t w o p r o p e r t i e s w h i c h a r e l e s s t h a n o p t i m a l . F i r s t l y , its p o r e s i z e is l i m i t e d . T h i s m e a n s not only that the s i z e of t h e p r o t e i n s , w h i c h c a n b e s e p a r a t e d , i s l i m i t e d b u t a l s o t h a t s m a l l a m o u n t s of o v e r s i z e d m a t e r i a l , ( l a r g e p r o t e i n s , d e n a t u r e d m a t e r i a l , c e l l w a l l a n d m e m b r a n e f r a g m e n t s ) p r e s e n t in t h e s a m p l e a n d h a v i n g t h e s a m e c h a r g e a s t h e p r o t e i n s of i n t e r e s t , c a n , b y b l o c k i n g t h e p o r e s a t t h e g e l s u r f a c e , h i n d e r o r m a k e i m p o s s i b l e t h e l o a d i n g of t h e s a m p l e i n t o t h e g e l . F o r t h i s r e a s o n , in t h e o r i g i n a l w o r k , t h e s a m p l e w a s a p p l i e d a s a c o n c e n t r a t e d s o l u t i o n s o a k e d into f i l t e r p a p e r , a m e t h o d w h i c h m i n i m i z e s t h e d i f f i c u l t i e s of s a m p l e a p p l i c a t i o n b u t w h i c h i s n o t a p p l i c a b l e to m a n y s a m p l e s a n d w h i c h d o e s n o t m a k e u s e of t h e n a t u r a l c o n c e n t r a t i v e p r o p e r t i e s of d i s p l a c e m e n t e l e c t r o p h o r e s i s .

Secondly

c r o s s - l i n k e d p o l y a c r y l a m i d e , having a l a r g e p o r e s i z e , is s o f t and not

© 1980 Walter de Gruyter & Co., Berlin • New York Electrophoresis '79

584 e a s y to cut a c c u r a t e l y into f r a c t i o n s . Both these d i f f i c u l t i e s would be r e m o v e d if it w e r e p o s s i b l e to u s e , f o r example,

1 % a g a r o s e as the supporting medium but the amount of e l e c -

t r o e n d o s m o s i s , caused by the n o r m a l l y a v a i l a b l e a g a r o s e , ( — M ^

>0.1),

is l a r g e enough to make d i s p l a c e m e n t e l e c t r o p h o r e s i s v i r t u a l l y i m p o s s i b l e . H o w e v e r , v e r y r e c e n t l y , a g a r o s e of v e r y low c h a r g e

e . g . (— M ^

0 . 0 0 5 ) has b e c o m e a v a i l a b l e . T h e only m a n i f e s t a t i o n of e l e c t r o e n d o s m o s i s with this m a t e r i a l , is that the gel b e c o m e s p r o g r e s s i v e l y thinner a s its r e s i s t e n c e i n c r e a s e s , so that the portion of the gel containing t e r minating e l e c t r o l y t e is thinner than the portion of the

gel containing

leading e l e c t r o l y t e . T h e portion of the gel containing the s a m p l e is m o r e o r l e s s thinned depending upon the r e s i s t a n c e of the s a m p l e . T h i s d i f f e r e n t i a l thinning of the s a m p l e - b e a r i n g g e l m a y be of use in locating the various separated proteins. A s i m p l e d e v i c e has been constructed ( F i g .

1) which m a k e s it p o s s i b l e

to load the s a m p l e onto the gel in up to about 3 m i s of solution. It c o n s i s t s of a s h o r t g l a s s tube, a little l a r g e r in d i a m e t e r than the g e l annulus and having a s e r i e s of s m a l l h o l e s p i e r c e d in its c i r c u m f e r e n c e r a t h e r c l o s e to the top. It is held in p o s i t i o n , c o n c e n t r i c to the top of the g e l , by m e a n s of a p l a s t i c s l e e v e attached to its upper end.

In u s e , the

bottom of the tube is a l l o w e d to dip a f e w m i l l i m e t e r s below the t e r m i nator - o - d i c h l o r o b e n z e n e i n t e r f a c e . T h e s a m p l e is placed in the gap between the g l a s s tube and the g e l . T h e r e s t of the apparatus is f i l l e d with t e r m i n a t o r . T h e s a m p l e can be t r a n s p o r t e d e l e c t r i c a l l y into the g e l . A n e l e c t r i c a l connection with the upper e l e c t r o d e is ensured by the h o l e s in the upper part of the g l a s s tube.

Experimental M e t h o d of o p e r a t i o n of the a p p a r a t u s . T h i s was e s s e n t i a l l y a s p r e v i o u s l y

585

F i g u r e 1. C r o s s section through upper part of column showing sample application device. (1) Outer glass vessel; (2) Glass cooler which supports the gel; (3) The g e l , containing leading e l e c t r o l y t e , in which the separation is performed; (4) Plastic support; (5) Cylindrical glass sample container with holes ( 5 a ) f o r the passage of e l e c t r i c a l current; (6) Terminating electrolyte; ( 7 ) Sample solution; (8) o-dichlorobenzene F o r c l a r i t y the coolant d e l i v e r y tube, in the centre of the glass c o o l e r , has been omitted. described ( 1 ) , except that instead of the sample being soaked as a concentrated solution into a piece of f i l t e r paper, which was then applied to the gel before the apparatus was assembled, the whole apparatus, including the sample loading chamber, was assembled and f i l l e d with o - d i c h l o robenzene to a f e w m i l l i m e t e r s above the level of the bottom of the a p p l i cation d e v i c e . S e e F i g . 1. The space above and outside the loading d e v i c e was then filled with terminating e l e c t r o l y t e . As this was being done the sample, (in water, lead or terminating electrolyte and made denser than the terminator by the addition of 5 - 10 % w . v . of sucrose), was i n jected into the space, between the glass sample container and the g e l ,

586 u s i n g a h y p o d e r m i c s y r i n g e , through one of the holes d e s i g n e d f o r the p a s s a g e of e l e c t r i c a l c u r r e n t . A f t e r the s a m p l e had been i n j e c t e d , the r e s t of the s p a c e between the g l a s s s a m p l e - c o n t a i n i n g tube and the gel w a s f i l l e d by l a y e r i n g t e r m i n a t i n g e l e c t r o l y t e c a r e f u l l y onto the top of the s a m p l e . T h e liquid l e v e l s inside and o u t s i d e the tube had to be r a i s e d a t a p p r o x i m a t e l y the s a m e r a t e , s o that the position of the s a m p l e r e l a tive to the gel w a s not g r e a t l y a l t e r e d when the liquid l e v e l s r e a c h e d the h o l e s , provided f o r e l e c t r i c a l t r a n s p o r t , and the h y d r a u l i c c i r c u i t w a s c o m p l e t e d between the liquids inside and o u t s i d e the g l a s s s a m p l e c o n tainer . It would a l s o be p o s s i b l e to fill the s p a c e above the o - d i c h l o r o b e n z e n e c o m p l e t e l y with t e r m i n a t i n g e l e c t r o l y t e and then to inject the s a m p l e s l o w l y into the s a m p l e c h a m b e r s o that the s a m p l e would sink to the bottom by v i r t u e of its higher d e n s i t y . H o w e v e r , it proved v e r y d i f f i culté to do this with an o r d i n a r y h y p o d e r m i c s y r i n g e without c a u s i n g c o n s i d e r a b l e mixing of the s a m p l e and the o v e r l y i n g t e r m i n a t i n g e l e c trolyte . T h e s a m p l e w a s driven e l e c t r i c a l l y f r o m the s a m p l e c h a m b e r and into the g e l . T h i s had to be done using a r a t h e r low c u r r e n t , 5 mA o r l e s s a t the b u f f e r concentration u s e d , o t h e r w i s e convection c u r r e n t s c a u s e d mixing of the s a m p l e and the o v e r l y i n g t e r m i n a t i n g e l e c t r o l y t e . \Ahen a l l the s a m p l e had been driven into the gel and the top of the s a m p l e zone w a s a t l e a s t 5 m m below the bottom of the application d e v i c e , then the level of the o - d i c h l o r o b e n z e n e - t e r m i n a t o r i n t e r f a c e w a s lowered until it w a s below the bottom of the a p p l i c a t o r but above the top of the sample. T h e s e p a r a t i o n w a s then p e r f o r m e d a s p r e v i o u s l y d e s c r i b e d ( 1 ) . T h a t i s , the potential drop a c r o s s the a p p a r a t u s w a s r a i s e d to the d e s i r e d level f o r s e p a r a t i o n (in this work 1200 V) and a s the s a m p l e zone m i g r a t e d

587 down the tube the c u r r e n t was kept constant by allowing o - d i c h l o r o b e n zene to run out of the a p p a r a t u s whilst the level of the t e r m i n a t i n g e l e c trolyte w a s maintained by the addition of m o r e t e r m i n a t o r . When the p r e p a r a t i v e s e p a r a t i o n w a s c o m p l e t e the cooling tube and gel w e r e r e m o v e d f r o m the a p p a r a t u s . S a m p l e s f o r a n a l y t i c a l s e p a r a t i o n w e r e obtained by cutting a n a r r o w s t r i p of gel (2 m m wide) along the length of the g e l . T h e s t r i p w a s then divided into s m a l l s e g m e n t s which w e r e a n a l y s e d by a n a l y t i c a l i s o e l e c t r i c f o c u s i n g . T h e p r e p a r a t i v e s e p a r a t i o n could be v i s u a l i z e d by dipping the gel on its cooling tube into 10 % a q u e o u s t r i c h l o r o a c e t i c a c i d , when the p r o t e i n s precipitated. M a t e r i a l s u s e d in P r e p a r a t i v e E l e c t r o p h o r e s i s . T h e gel w a s 1 % w . v . a g a r o s e having a v e r y low e l e c t r o e n d o s m o s i s M ^ — 0 . 0 0 5 , ( L K B A g a r o s e - E F B a t c h N 00001). T h e leading e l e c t r o l y t e pH 8 . 1 w a s 0 . 0 0 7 6 M T r i s half n e u t r a l i z e d with phosphoric a c i d . T h e leading e l e c t r o l y t e pH 8 . 9 w a s 0 . 0 0 7 6 M diethanolamine half n e u t r a l i z e d with p h o s p h o r i c a c i d . T h e t e r m i n a t i n g e l e c t r o l y t e w a s 0 . 115 M 2 - a m i n o p r o p i o n i c a c i d containing 0 . 0 3 7 M T r i s . T h e s a m p l e of bovine s e r u m a l b u m i n , B S A (Cohn F r a c t i o n V) and of c r y s t a l l i n e bovine h a e m a g l o b i n Hb w e r e o b tained f r o m the S i g m a C h e m i c a l C o m p a n y , Munchen. A n a l y t i c a l i s o e l e c t r i c f o c u s i n g w a s p e r f o r m e d using an L K B 2117 M u l tiphor s y s t e m a c c o r d i n g to the m a n u f a c t u r e r ' s i n s t r u c t i o n s .

The

p i e c e s of p r e p a r a t i v e gel w e r e laid onto the a n a l y t i c a l p l a t e , without p r i o r elution. A f t e r f o c u s i n g the h a e m a g l o b i n bands w e r e f i x e d by s o a king the p l a t e in 10 % w . v . a q u e o u s t r i c h l o r o a c e t i c a c i d .

588 R e s u l t s and D i s c u s s i o n T h e s e p a r a t i o n of s u b s t a n c e s p r e s e n t in g r e a t l y d i f f e r i n g p r o p o r t i o n s . T h i s m a y b e i l l u s t r a t e d b y t h e s e p a r a t i o n of a n a r t i f i c i a l m i x t u r e of bovine s e r u m albumin and bovine haemaglobin. A

trivial separation

e x c e p t f o r t h e f a c t t h a t t h e h a e m a g l o b i n r e p r e s e n t s o n l y 1 . 8 % of t h e p r o t e i n in t h e s a m p l e . T h e s e p a r a t i o n i s s h o w n in F i g . 2 , w h i c h i l l u s t r a t e s t h e c o n c e n t r a t i v e p r o p e r t i e s of d i s p l a c e m e n t e l e c t r o p h o r e s i s . T h i s i s e m p h a s i s e d b y c o m p a r i n g t h e c o n c e n t r a t i o n s a n d v o l u m e s of t h e p r o t e i n s in t h e s a m p l e a n d in t h e g e l a f t e r s e p a r a t i o n , a s i s d o n e in T a b l e 1. Table

1

C o m p a r i s i o n s of V o l u m e s a n d C o n c e n t r a t i o n s of B S A a n d Hb b e f o r e a n d a f t e r S e p a r a t i o n it

Sample

Separated Bands

Volume (ml)

Cone, (mg/ml)

Volume (ml)

BSA

1.0

65

2.9

22

Hb

1.0

0.12

10

1.2

Cone,

(mg/ml)

£ C a l c u l a t e d f r o m t h e m e a s u r e d z o n e l e n g t h a n d t h e o r i g i n a l w e i g h t of protein. I t i s i n t e r e s t i n g to n o t e t h a t t h e h a e m a g l o b i n o c c u p i e s l e s s t h a n o n e e i g h t the v o l u m e a f t e r s e p a r a t i o n than b e f o r e . H a e m a g l o b i n h a s a m u c h h i g h e r i s o e l e c t r i c point than bovine s e r u m a l b u m i n and h e n c e , when anionic

at

pH 8 . 9 , it f o r m s a d i s p l a c e m e n t e l e c t r o p h o r e t i c z o n e w h i c h t r a v e l s b e hind the bovine s e r u m a l b u m i n z o n e . One would e x p e c t the p r o t e i n c o n c e n t r a t i o n in t h e h a e m a g l o b i n z o n e to b e t h e h i g h e r . In f a c t i t s a p p a r e n t c o n c e n t r a t i o n ( c a l c u l a t e d f r o m t h e z o n e l e n g t h a n d t h e o r i g i n a l w e i g h t of p r o t e i n ) is the l o w e r , b e c a u s e the h a e m a g l o b i n zone is p r o p o r t i o n a t e l y longer than e x p e c t e d . T h e m a i n r e a s o n f o r this is undoubtedly that the bovine s e r u m albumin s a m p l e contains s o m e i m p u r i t i e s which have about

589

F i g u r e 2 . D i s p l a c e m e n t e l e c t r o p h o r e s i s o f a m i x t u r e of B S A ( C o h n F r a c t i o n V ) 6 5 m g a n d b o v i n e h a e m a g l o b i n 1 . 5 m g in 1 m l w a t e r . L e a d e l e c t r o l y t e pH 8 . 9 . L o a d i n g t i m e 2 5 m i n , r u n n i n g t i m e 10 m i n . T h e r e a r e n d o f t h e s a m p l e h a d m i g r a t e d 8 0 m m d o w n the g e l . the s a m e m o b i l i t y a s h a e m a g l o b i n . H e n c e the h a e m a g l o b i n z o n e i s m i x e d a n d d o e s n o t c o n t a i n p u r e h a e m a g l o b i n . It m a y a l s o b e t h a t t h e s e p a r a t i o n t i m e o f 10 m i n u t e s w a s t o o s h o r t a n d t h a t if t h e s e p a r a t i o n h a d b e e n a l l o w e d to c o n t i n u e , t h e b a n d w o u l d h a v e n a r r o w e d . It s h o u l d b e n o t e d t h a t the c o n c e n t r a t i o n of r e s o l v a b l e m i n o r c o m p o n e n t s is a g e n e r a l of d i s p l a c e m e n t

property

electrophoresis.

A t t e m p t e d s e p a r a t i o n of h a e m a g l o b i n c o m p o n e n t s . A s e p a r a t i o n o f c r y s t a l l i n e h a e m a g l o b i n ( 2 5 m g ) w a s a t t e m p t e d a t pH 8 . 1 . T h e s a m p l e r a t e d into t w o o b v i o u s p r o t e i n z o n e s . O n e c o l o u r l e s s a n d of l o w e r

sepamobi-

l i t y , b e i n g a b o u t 5 % o f t h e w h o l e , a n d the o t h e r r e d b r o w n in c o l o u r . T h e r e d z o n e w a s d i v i d e d into n i n e f r a c t i o n s , w h i c h w e r e a n a l y z e d b y g e l i s o e l e c t r i c f o c u s i n g ( F i g . 3 ) . T h e two m o s t m o b i l e f r a c t i o n s c o n t a i n o n l y d e n a t u r e d m a t e r i a l . In the r e m a i n i n g f r a c t i o n s t h e f o c u s a b l e

compo-

n e n t s a r e p a r t i a l l y s e p a r a t e d . When the e x p e r i m e n t w a s r e p e a t e d with l e a d e l e c t r o l y t e a t pH 8 . 9 a l m o s t no s e p a r a t i o n o c c u r e d , s o it i s p o s s i b l e t h a t , with a l t e r e d c o n d i t i o n s , the s e p a r a t i o n m a y be

improved.

590

F i g u r e 3 . I s o e l e c t r i c f o c u s i n g a n a l y s i s of p r e p a r a t i v e s e p a r a t i o n of Hb (25 m g ) . T h e s a m p l e s at e i t h e r end a r e the o r i g i n a l s a m p l e . T h e f r a c tions a r e a r r a n g e d with the m o s t m o b i l e on the l e f t . Conditions of p r e p a r a t i v e s e p a r a t i o n ; pH of lead 8 . 1; distance t r a v e l l e d 180 c m ; zone length 1.8 c m ; total a n a l y s i s t i m e 45 m i n .

Acknowledgements

I g r a t e f u l l y a c k n o w l e d g e r e c e i p t of an A l e x a n d e r von Humboldt F e l l o w ship and of the hospitality of P r o f . I . Halcisz.

References

1. H a m p s o n , F . , M a r t i n , A . J . P . : J . C h r o m a t o g .

174, 61-74 (1979)

VELOCITY GRADIENT STABILISED CONTINUOUS ELECTROPHORESIS

A.R. Thomson, P. Mattock, and G.F. Aitchison Biochemistry Group, Engineering Sciences Division, Harwell, Oxfordshire, England. 0X11 ORA.

Introduction The development of electrophoresis for use in the production of labile biologically active materials on a substantial scale (> lOg/hr) has been an attractive goal for decades for biochemists and biochemical engineers.

If combined with

continuous processing (with its anticipated reduction in equipment size and processing time) it could offer substantial benefits in the production of high value and labile biochemicals (e.g. from human blood plasma).

The recent trend

in food and drug legislation which emphasises the side effects of contaminating compounds and the spectacular advances in molecular biology (particularly in the use of recombinant DNA), emphasise the need for improved separations technology usable on an industrial scale. Removal of Joule heat or the prevention of convection caused by heating are the fundamental problems to be solved in scaling up electrophoresis.

The solution to this problem used on an

analytical scale (anti-convective media, usually gels) is not feasible on a large scale because of poor heat conduction.

The

factors involved and the solutions tried so far have been summarised succinctly by Bier(l).

Apart from free-flow

electrophoresis, which has been used for the industrial production of purified rubber latex, none of these techniques has found widespread application.

© 1980 Walter de Gruyter & Co., Berlin • New York Electrophoresis '79

592 Velocity Gradient Stabilised Electrophoresis At Harwell, we have been developing an electrophoretic separation system based on the concept

proposed by J. St. L.

Philpot (2,3) in which electrophoresis is carried out in an annulus.

Laminar flow in the annulus is stabilised against

convective disturbance due to heating, by rotation of the outer cylinder (rotor) while keeping the inner cylinder (stator) stationary. theorem.

This is an application of the Taylor-Proudman

In this way, a gradient of angular velocity is

generated and maintained across the annulus, and perpendicular to the long axis of the separator (see Fig. 1).• The cylindrical electrodes are separated from the inner walls of the annulus by electrode chambers bounded on the annulus side by membranes mounted on cylindrical porous supports.

Concentrated

electrolyte is pumped continuously through these electrode chambers.

With this arrangement, electrical conductivity and

a voltage gradient can be

maintained across the annulus,

while electrolysis products and in particular gas, caused by electrolysis, can be removed effectively.

Electrophoresis is

carried out in a carrier buffer of defined pH and conductivity, which is passed continuously through the annulus.

The solution

to be fractionated (migrant) is injected through a thin slit immediately below the electrode section.

This injection system

enables migrant to be introduced around the whole circumference of the stator.

Thus, ionic components in the sample stream

migrate defined distances away from the stator, depending on net charge.

At the top of the electrode section, the fluid

flow is split into 30 streams via a "disc stack" assembly mounted vertically on top of the stator.

The stack consists of

30 plastic discs each of diameter equal to that of the stator. Each disc communicates via a series of channels with the annulus, and via a single outlet tube passing through the upper part of the stator assembly to the outside of the separator.

Because

of the design of the maze-like channels in the disc, fluid

Migrant in Outer electrolyte in

Inner electrolyte in ^ / r Inner electrolyte out To collection manifold

Outer electrolyte out

(steel)

Rotor

Continuous Electrophoretic Separator

Stator

594 collected uniformly around the whole circumference of each disc.

Provided laminar flow is maintained in the annulus, the

off-take stack will "strip" off successive cylindrical

layers

of fluid so that the layer nearest the stator wall emerge via the lowest disc, the next layer via the disc immediately and so on.

above

In this way the carrier buffer together with the

components of the injected migrant solution can be collected in a total of 30 fractions each representing laminae from the annular flow.

successive

The alignment and

concentricity

of the stator and rotor are maintained by a suitable arrangement of bearings and seals the rotor being driven from below by a motor

(for more details see References 3 and 5).

There is no internal cooling of the separator, thus all the heat generated is deposited in the carrier and leads to a temperature rise.

This we normally limit to ~15-20°C, for the

separation of biological materials.

Nevertheless,

the

temperature rise causes minimal inactivation since the residence time is very short

(20-60 sec.).

Further, we

normally cool carrier, migrant and electrode compartment solutions to 2-4°C before injection so that the maximum temperature of outlet fractions is ~20°C. Laminar flow stability is maintained by rotating the rotor assembly at -150 r.p.m., some stabilisation occurs at low rotational speeds, but band dispersion is minimal between -80 and 2 40 r.p.m. with turbulence and mixing occurring -300 r.p.m. in the separator. Grunow

above

It has been shown by Schultz-

(6) that stabilised annular flow can be maintained at

higher rotational speeds, but only if an annulus with

rotating

outer wall is machined to a very high degree of concentricity and is balanced very accurately. in more detail in Ref. 5.

The system will be described

595 Theoretical Considerations The hydrodynamic and electrophoretic aspects of the system are rather complex, and many of the design and operating variables are interactive.

Following detailed investigations of the

system, a mathematical model has been developed.

This takes

into account the effect of parabolic flow, migrant zone width at injection, carrier and migrant flow rates, temperature rise, applied voltage and particularly of diffusion, on band width and resolving power.

The behaviour of a variety of low and

high molecular weight molecules (e.g. fluorescein, a range of proteins) as well as whole cells have been investgated in detail.

Overall, the data shows reasonably close agreement

with the predicted behaviour, particularly with low molecular weight solutes such as fluorescein (Fig. 2).

There is more

divergence with macromolecules such as albumin, and with mammalian red blood cells, although with the latter, as expected, relatively low absolute dispersion values are obtained because of the low diffusion coefficient.

The

divergence seen with albumin and other proteins may be due to microheterogeneity due to ion-binding, partial modification or to association.

The effect of diffusion, a major cause of band

spreading, is difficult to quantify accurately since it is temperature dependent, and although input solutions are preequilibrated to ~2°C, the temperature profile is not regular across the annulus because of the conductivity profile.

Given

these difficult-to-quantify factors, the agreement between predicted and experimental data is encouraging.

Experiments

carried out at nil as well as a range of voltages suggest that there is minimum mixing at the migrant inlet zone, and only minor turbulence in the annulus.

Modelling of the off-take

system is clearly much more difficult and is being investigated, but experiments in which dye has been injected into the top of the annulus via specific off-take discs, suggest that there is only minimal re-mixing at least at the lower part of the off-

596 FIG 2

A COMPARISON OF EXPERIMENTAL AND CALCULATED O.D, VALUES FOR SODIUM FLUORESCEIN AND B . S . A . AT VARIOUS LOCATIONS ACROSS THE ANNULUS

597 take system. investigated

These aspects of the design are being further.

With separations based on differential zone migration rates, the resolution achieved is generally related to the path available.

length

With a reasonably sharp starting zone, more is

gained in terms of resolution the greater the path length than is lost due to diffusion, the main inconvenience being diffusional band spreading.

In the separati.on system described

here, the path length possible

(i.e. the annulus width) is

constrained by hydrodynamic factors since only a limited range of dimensions and flow rates are compatible with maintaining stable flow.

Further, the usable annulus width is also

limited

by the temperature rise acceptable for the range of applications envisaged

(for biological applications probably a At of fi45

the

(d) required for 95% ((J

+

3.

}

D

598 In most conventional systems therefore, d should be maximised since d is proportional to time, while a is proportional to /time.

However, in the velocity gradient stabilised system,

because of parabolic flow, it can be shown that there is a rapid increase in resolution towards the rotor with little to be gained beyond

0.7 of the annulus width.

Therefore, we

normally electrophorese so that the fastest component in a mixture emerges in outlets 20-21.

Applications In analysis, the main criteria in determining the impact of a new technique are resolution and precision.

In large scale

production, other factors become much more important.

Thus

throughput, cost, ease of operation, reproducibility and in particular, yield are often paramount.

The performance of the

separator has been investigated with a wide range of materials. The examples below illustrate the particular characteristics and advantages of the system.

A.

Proteins

1)

Intracellular enzymes.

Fig. 4 shows electrophoresis of

an ammonium sulphate fraction from rabbit muscle.

The salt

precipitate was dissolved in a minimum volume of carrier buffer and was dialysed before electrophoresis.

Under the

conditions used, pyruvate kinase, the more mobile of the two enzymes assayed, had a relatively low mobility yet is well resolved from phosphoglucomutase (peak maximum at off-take 4). Recovery of enzymic activity was virtually quantitative for both enzymes.

Similar results have been obtained for other

intra-cellular enzymes such as lactate dehydrogenase and the two malate dehydrogenase isoenzymes in Tris buffer extracts

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«

>1 >1 kl M 3 3 •rvn C C •H -H

3 oi »•a ai -h 3 3 01 rH oi >n ai •H en •P O 10 •H X! » -p ft M e o o 3 -H Vi ai n e u > a) e ra •H oi nj a

IT) •H e a> g 10

t ai c 0 rH 1 a

rH rH a> o

•Ö -r| 3

- rH

01 IH rH rH ai 01 G o •rl e M 3 -a ai M ai •p ai a 3 Ol

01 rH rH ai 01 01 o rH rH rH rH a. ai a) ai 6 m 3 Ü 0 3 G 01 n •H 01 •a •0 ai 1-1 -H s 01 3 EH « ai

•0 H N W

ft

•O •ri ü


1 •P 0) O C i

»

H H ai 01 10 •a •H •p o. ai CM 0 c •rl e id rH >1 TI •H 4-1

ft a ft •ri Q

i EH

ai 01 id TJ •H X o IH ai eu

ai 01 id tí ai Ul o> ai ai 01 01 o o 10 m x : vi ftTS G -O •rl •H >i 4J ai x: 01 O g ai •rl -d ai ai ai 01 4-> rH G •rl O id id o 4-1 -rl ai > 3 10 -P 4-1 3 55 aj o 0 M rH VI vi id U iJ p< Ai m

>.

637 shown in Table

3.

TABLE 3. PROTEINS /¡ND ENZYMES COMMONLY EMPLOYED IN FORENSIC MEDICINE BEST SEPARATED J ' Y PAC-IF ENZYME

SOURCE

METHOD

SUBSTRATE

Acid erythrocyte phosphatase

RBC, .semen, prostate

Direct

4-methyl-umbelliferyl phosphate, Phenophthalene-phosphate FAD MTT

Adenosine Deaminase RBC Adenylate Kinase RBC Alkaline placental Placenta phosphatase Alpha-l-Fucosidase Leukocytes Carbonic Anhydrase Methernoglobin reductnse-Erythrocyte NADH-Diaphorase Esterase D

Direct

Direct RBC Leukocytes, RBC, Direct Thrombocytes, Placenta, liver

10. Glycxalase 1 RBC 11. PhosphoglucoLeukocytes, RBC, Thrombocytes mutase 12. 6-Phosphygluconatedehydrogenase 13. Sperm-specific Diaphorase

SOURCE

1. Alpha-1-Antitrypsin Serum 2. Ceruloplasmin Serum 3. G-C Proteins

Serum

4. Haptoglobin

Serum

5. Hemoglobin

RBC

6. Transferrin

Serum

NADP MTT B-Naphthyl phosphate 4-methyl-umbelliferyla-L-fucoside DCIP MTT

Indirect

4 -me thy 1 -umbo 11 i f e ry 1 acetate NADU2 MTT

Indirect Indirect

NADP MTT

RBC

Glutamate pyruvate RBC transaminase

PROTEINS

Indirect Indirect Direct

Direct Indirect

DCIP MTT

NADP MTT DCIP MTT

DETECTION Coomassie Blue, Immunoprint Coomassie Blue, Immunoprint Coomassie Blue, Immunoprint Diaminobenzidine, Immunoprint Direct, Diaminobenzidine Coomassie Blue, Immunoprint

638 These techniques have greatly aided in the discovery of many homozygous genetic deficiencies whose prognoses are now well established. Furthermore , the consequences of previously unrecognized homozygous or heterozygous states alone or in combination with other genetic characteristics, are swelling our catalogue of human afflictions. The potential complexities of this area are illustrated by the unexpected relationship found between protease inhibitor (Pi) phenotypes, blood type, and periodontal disease as seen in Figure 1. P E R I O D O N T I T I S M A R K E D BY A L V E O L A R LOSS

PATIENT

AGE

RANGE

20-55

CREST YEARS

1009080-

13

?7 9

7079

z

60-

(J

50-

LU OC

K

53 13

37 "49

403020-

MS

MZ

37

13

11

AL L

ALL

M 0

M A, B

MlMi M] Mi 0 |A.B

M2M2

M] M;

A.B 1

10TOTAL

m2m2

117 127 P, 86-90, (1 978) .

ISOELECTRIC ENZYME PATTERN

: A NEW CONCEPT FOR

TUMOUR

CLASSIFICATION

H. J. R a d z u n , M. R. P a r w a r e s c h and D.

Schmidt

I n s t i t u t e of P a t h o l o g y at the U n i v e r s i t y 2300 Kiel, West Germany

of K i e l

Introduction A r e l i a b l e c l a s s i f i c a t i o n of n e o p l a s i a s r e q u i r e s

an

a p p r o p r i a t e r e c o g n i t i o n of the u n d e r l y i n g n o r m a l cell, w h i c h the t u m o u r d e r i v e s . This p r i n c i p l e

is the b a s i c

from guide-

line of the m o d e r n c l a s s i f i c a t i o n of m a l i g n a n t n o n H o d g k i n ' s l y m p h o m a s . The l a t t e r has b e e n b a s e d o n c y t o l o g i c a l ,

histo-

logical, electron microscopical and immunological criteria The v a r i o u s types of m a l i g n a n t

l y m p h o m a s have b e e n

to the s e q u e n c e of the p h y s i o l o g i c a l

lymphocyte

(1).

attributed

transfor-

mation. C o n s i d e r i n g the c y t o c h e m i c a l d i s t r i b u t i o n p a t t e r n of a c i d phosphatase

(EC 3 . 1 . 3 . 2 ) in n o r m a l l y m p h o c y t e

(2) a n d in cases of m a l i g n a n t

lymphomas

subpopulations

(3) the

of this l y s o s o m a l e n z y m e s e e m e d p r o m i s i n g as an c r i t e r i o n for the c l a s s i f i c a t i o n of d i f f e r e n t

polymorphism additional

lymphoma

entities.

Materials and Methods F r e s h tumour tissue s p e c i m e n s f r o m c h r o n i c leukemia cells

(B-CLL), f o l l i c u l a r

(CB/CC), i m m u n o c y t o m a

B-lymphocytic

l y m p h o m a of g e r m i n a l (IC) a n d p l a s m a c y t o m a

u s e d ; all these l y m p h o m a s c a n be r e l a t e d to the

center (PC) w e r e

intermediate

cells of B - l y m p h o c y t e m a t u r a t i o n o v e r g e r m i n a l c e n t e r and l y m p h o i d p l a s m a cells into m a t u r e p l a s m a c e l l s .

© 1980 Walter de Gruyter & Co., Berlin • New York Electrophoresis '79

cells

The

680 tissue specimens were homogenized and the protein related activity of

lysosomal acid phosphatase(AcP) was measured in the

supernatants after solubilization with Triton-X-100 and ultrazentrifugation (1 hr, 100 000 x g , 4° C). A minimum of 8 mU of AcP was subjected to isoelectric focusing (IEP) on Polyacrylamide thin layer slabs with a pH-range from pH 3-5 to pH 10. The AcP was visualized with naphthol-AS-BI-phosphate as substrate and hexazotized pararosaniline as coupler (4) and the isoelectric points of the isoenzymes were determined in gels. For comparison normal human B-lymphocytes from surgically removed fresh tonsils and monocytes from human blood were separated and subjected to the same procedure (5)-

Results and Discussion The lowest enzyme activity was found in B-CLL, the highest in cases of PC (Table 1).

B-CLL

CB/CC

IC

PC

2.1 + 1.0

4.6 + 1.4

8.7 + 2.3

29.7 + 6.1

(n=9)

(n=10)

(n=28)

(n=8)

Table 1: Protein related AcP activity in four major B-cell lymphoma types. On IEP all four lymphomas showed the basic pattern of fourteen bands arranged in three distinct regions (A-C) as in figure la .

indicated

68l

p H -

6.1

6.0

6.1

6.1

5.85 5.45

5.8 5.45 1

B

5.0-

4.0a

4.95

4.95

4.6

4.6

3.9

3.9 b

—

—4.95

4.0 c

Fig. 1: IEP pattern of AcP basically common for B-cell lymphomas (a) as compared with separated normal human B-lymphocytes (b) and monocytes (c). In cases of IC two additional bands between region A and B and in cases of PC five additional bands between region 3 andC could be detected. Comparing the basic pattern found in lymphomas with the polymorphism of AcP in purified human Blymphocytes a close similarity became evident (Pig. lb). Blymphocytes showed one solitary band at pH 6.3 and eleven bands between pH 5.3 and pH 3.8. The solitary band of B-lymphocytes at pH 6.3 was always prominent in all lymphomas investigated. The two additional bands of region A in the analysed

682 lymphoma entities could be attributed to mild contamination with monocytes or macrophages, which showed three bands of equal activity in region A (Pig. lc). Our results confirm the immunobiological classification of malignant non Hodgkin's lymphomas recently introduced by Lennert (1). The similarity between the IEP pattern of AcP in normal B-lymphocytes and the investigated lymphomas provides an additional proof for the origin of these lymphomas from the different cells of B-lymphocyte maturation line. The stepwise increase of protein related enzyme activity from B-CLL to PC, which was partly combined with the appearance of new isoenzymes , reflects the physiological sequence of B-lymphocyte transformation into mature plasma cells. Our results demonstrate that the high resolution capacity of IEP represents an appropriate device to apply enzyme polymorphism to the identification and classification of cell lines and of their functional or neoplastic derivatives. In this respect IEP surpasses all other analytical techniques.

References 1. Lennert, K., in collaboration with Mohri, N., Stein, H., Kaiserling, E., Muller-Hermelink, H.K.: Malignant Lymphomas other than Hodgkin's Disease, Handbuch der speziellen pathologischen Anatomie und Histologie Vol. I, 3B, (Ed. Uehlinger, E.), Springer, Jerlin-Heidelberg-New York 1978 2. Catovsky, D., Galetto, J., Oskos, A., Miliani, E., Galton, D. A. G.: J. clin. Pathol. 27., 767 (1974) 3. Yam, L. T., Li, C. Y., Lam, X. W. : N. Engl. J. Med. 284, 357 (1971) 4. Barka, T., Anderson, ?. J.: J. Histochem. Cytochem. 10, 741 (1962) 5. Radzun, H. J., Parwaresch, M. R., Xulenkampff, Ch., Stein, H.: Lysosomal acid phosphatase: Activity and isoenzymes in separated normal human blood cells; in preparation

MONOCYTE-SPECIFIC ISOELECTRIC FOCUSING PATTERN OF LYSOSOMAL ACID ESTERASE

: A NEW POSSIBILITY

HISTOCHEMICAL

IDENTIFICATION

J. W. W i t t k e , H. J. R a d z u n , M. R.

FOR CELL-SPECIFIC

IMMUNO-

Parwaresch

I n s t i t u t e of P a t h o l o g y at the U n i v e r s i t y of K i e l 2300 Kiel, West Germany

Introduction The c y t o c h e m i c a l a p p l i c a t i o n of the s o - c a l l e d acid esterase

nonspecific

(EC 3.1.1.6) r e a c t i o n h a s a t t r a c t e d

special

i n t e r e s t in h e m a t o l o g y , since it e n a b l e s a s e l e c t i v e

dis-

t i n c t i o n a n d clear i d e n t i f i c a t i o n of h u m a n m o n o c y t e s

and

T-lymphocytes

(1). I n b l o o d smears s u b j e c t e d to this

enzyme

c y t o c h e m i c a l m e t h o d only m o n o c y t e s a n d T - l y m p h o c y t e s

show a

s t r o n g a c t i v i t y , w h e r e a s a l l o t h e r cells show a w e a k

diffuse

r e a c t i o n , w h i c h is h a r d l y

visible.

The c o n s t a n c y and the s p e c i f i c i t y of the c y t o c h e m i c a l

pattern

w i t h i n the b l o o d c e l l p o p u l a t i o n s has m a d e this e n z y m e a h e l p f u l d e v i c e for i d e n t i f i c a t i o n of b l o o d cells a n d for f i c a t i o n of l e u k e m i a s o t h e r w i s e u n c l a s s i f i a b l e

classi-

o n the b a s i s

of c o n v e n t i o n a l m e t h o d s . W i t h these f i n d i n g s i n m i n d we e n d e a v o u r e d to see, w h e t h e r this c y t o c h e m i c a l

difference

b e t w e e n v a r i o u s b l o o d cells w a s m i r r o r e d also i n b i o c h e m i c a l t e r m s . S p e c i a l a t t e n t i o n w a s g i v e n to the q u e s t i o n , if the encountered cytotopographic difference between monocytes

and

T - l y m p h o c y t e s w a s a s s o c i a t e d w i t h d i f f e r e n c e s i n the p o l y m o r p h i s m of the e n z y m e

subfractions.

© 1980 Walter de Gruyter & Co., Berlin • New York Electrophoresis '79

684 Materials and Methods Monocytes, granulggytes, T- and B-lymphocytes were isolated from human venous blood using gradient centrifugation (2). The separated blood cells were controlled for viability and purity. Lysosomes were separated after cavitation and gradient centrifugation. Lysosomal enzymes were solubilized by TritonX-100 treatment and after ultrazentrifugation (1 hr, 100 000 x g, 4° C) the cell- and particle-free supernatants were subjected to di r ect measurement of the acid esterase (AcE) activity (3)- A minimum of 40 mU was used for isoelectric focusing (IEP) on polyacrylamide thin layer slabs with a pHrange from 3-5-10, which were stained with Oc-naphthyl acetate as substrate and hexazotized pararosaniline as coupler at pH 5-8 (4).

Results and Discussion The results show that in accordance with the cytochemical experience blood monocytes exhibit the highest enzyme 7 activity as related to 1 x 10 cells. In contrast to the cytochemical results a major difference was not found between Tand B-lymphocytes. Using IEP monocytes showed within a pH-range from pH 5-5 to pH 6.5 four enzyme bands with an intensely stained anodal band (Pig. 1). This pattern of enzyme variants and specially the typical monocyte derived anodal band did never occur in the other blood cells investigated. This finding stresses our initial impression that the high enzyme activity detectable in cytochemical preparations of monocytes was due to one special enzyme fraction.

685

«i

Pig. 1: I E F p a t t e r n of l y s o s o m a l A c E f r o m m o n o c y t e s .

The a v a i l a b l e d a t a s t r o n g l y s u g g e s t that the a n o d a l m o n o c y t e derived band could also reveal antigenically

s p e c i f i c for

cell line. I n g e l s s u b j e c t e d to n o n s p e c i f i c p r o t e i n

this

staining

there w e r e no other v i s i b l e p r o t e i n b a n d s b e t w e e n pH 5 - 5 a n d p H 6.5 except the one c o r r e s p o n d i n g to the m o n o c y t e AcE a n o d a l s u b f r a c t i o n

(Fig.

2).

Since the i s o l a t i o n of this e n z y m e s u b f r a c t i o n by a n a l y t i c a l or p r e p a r a t i v e m e t h o d s to d i f f i c u l t i e s

specific

other

seems h a r d l y f e a s i b l e

in the a v a i l a b i l i t y

due

of v i a b l e h u m a n m o n o c y t e s ,

we w i l l try to cut out the a n o d a l b a n d p r i o r to s t a i n i n g

and

686

Pig. 2: AcE (top) and protein (bottom) staining of the lysosomal fraction from separated human monocytes.

apply it to rabbits for immunization. In such a procedure the polyacrylamide will work as an immune adjuvant. Gaining a specific antibody we would have the possibility of detecting monocytogenic or histiocytic tumours, which might derive from monocytes and still resist a reliable classification, using immunoperoxidase technique.

References 1. Ranki, A.: Clin. Immunol. Immunopathol. 10,47 (1978) 2. Boyum, A.: Scand. J. Clin. Lab. Invest. 21,77

(1968)

3. Radzun, H.J., Parwaresch, M.R., Kulenkampff, Ch., otaudinger, M., Stein, il.: Lysosomal acid esterase¡Activity and isoenzymes in separated normal human blood cells. In preparation 4. Miiller, J., B^run del Re, G., Juerki, H., Keller, H.-U., Hess, M..*/., Gottier, H.: Surop. J. Immunol. 5,270 (1975)

ISOELECTRIC FOCUSING IN CELLULOSE ACETATE MEMBRANE AND FLATBED GRANULATED GEL: APPLICATION TO THE STUDY OF HUMAN ALDEHYDE DEHYDROGENASE ISOZYMES

S.

H a r a d a , D. P.

Agarwal,

and H. W. Goedde

I n s t i t u t e o f Human G e n e t i c s , D 2 0 0 0 Hamburg 5 4 , Germany-

U n i v e r s i t y o f Hamburg,

Introduction V a r i o u s k i n d s o f s u p p o r t i n g m e d i a , s u c h as s a c c h a r o s e ( 1 ) , polyaerylamide gel (4)

and c e l l u l o s e

a c e t a t e membrane

applied for a n a l y t i c a l (IEF)

of p r o t e i n s ,

( 2 ) , sephadex g e l

(3),

(CA-membrane)

and p r e p a r a t i v e

acids.

focusing

The c h o i c e

a p a r t i c u l a r method and medium depends on t h e p u r p o s e s t u d y and t h e p r o t e i n t o be

and r a p i d i t y

preparative resolution step

of

of

analysed.

A n a l y t i c a l I E F on CA-membrane h a s a d v a n t a g e s plicity

gel

( 5 ) have been

isoelectric

i s o z y m e s and n u c l e i c

gradient

agarose

of s e p a r a t i o n

concerning

compared t o o t h e r

IEF in a f l a t - b e d g r a n u l a t e d g e l o f f e r s

sim-

media,

high

o f crude e x t r a c t s w i t h t h e a d v a n t a g e s o f a one

procedure.

Using b o t h t h e s e t e c h n i q u e s , we have s t u d i e d a l d e h y d e drogenase

(ALDH, E C:

1.2.1.3)

isozymes

in d i f f e r e n t

dehyhuman

tissues. F o u r ALDH i s o z y m e s were s e p a r a t e d from e a c h o t h e r by preparative flat-bed

IEF.

A c o m p a r a t i v e study on isozyme p r o f i l e w e l l as d e t a i l e d methods employed a r e

© 1980 Walter de Gruyter & Co., Berlin • New York Electrophoresis '79

in various presented.

tissues

as

688 Materials

and Methods

Sample p r e p a r a t i o n .

Human a u t o p s y s p e c i m e n s

lung, h e a r t ,

s t o m a c h , muscle and s p l e e n ) were

brain,

(liver,

kidney, collected

a t t h e d e p a r t m e n t o f L e g a l M e d i c i n e , U n i v e r s i t y o f Hamburg. Washed p o s t - m o r t e m t i s s u e s were homogenized w i t h an e q u a l volume o f d i s t i l l e d w a t e r u s i n g an u l t r a - t u r r a x and t h e homogena'tes w e r e c e n t r i f u g a t e d a t 3 4 , 0 0 0 g f o r 20 min. The n a t a n t was used f o r i s o e l e c t r i c Isoelectric 1.

Cellulose

focusing.

focusii>g a c e t a t e membrane.CA-membrane was s u p p l i e d by F u j i

P h o t o Co. , J a p a n

(Separax-EF,

110 mm x 2 0 0 mm x 140 ^ i m ) . I E F

was c a r r i e d o u t u s i n g a c o m m e r c i a l l y with a cooling system. prepared in

f o r about

p l a t e . The s u r p l u s

available

T h r e e % ampholine

1.5 % sucrose s o l u t i o n .

in the s o l u t i o n

F i l t e r paper

% ethylendiamine

strips

10 % s u c r o s e

containing

and

3 % am-

10 were used as a b r i d g e f o r a n o d i c

c a t h o d i c end o f t h e membrane, r e s p e c t i v e l y .

or

Before

sample

p r e - I E F was c a r r i e d o u t f o r 15 min t o

estab-

a pH g r a d i e n t i n t h e membrane.

directly

glass

ampholyte s o l u t i o n on t h e membrane was

3 % ampholine o r 0 . 5

application,

10) was

The membrane was s o a k e d

% phosphoric acid containing

p h o l i n e , pH 3 . 5 -

apparatus

(pH 3 . 5 -

1 min and p l a c e d on a c l e a n

removed c a r e f u l l y w i t h a f i l t e r p a p e r . soaked in 0 . 1

lish

super-

The samples were

a p p l i e d c l o s e t o t h e c a t h o d i c end o f t h e membrane

using a microsyringe

( 3 - 5

A constant voltage of

500

v o l t was a p p l i e d f o r 9 0 min. Staining;

A f t e r I E F on CA-membrane, ALDH was

u s i n g a s p e c i f i c enzyme s t a i n lay system.

The s t a i n i n g s o l u t i o n

MTT, 0 . 5 mg M e l d o l a B l u e , 2 0 0 y.1 p r o p i o n a l d e h y d e 8.6tcontaining

i n 25 ml o f 0 . 1

formazan.

30 mg p y r u v a t

and

t r i s / H C l b u f f e r , pH

After incubation

3 7 ° C f o r 30 - 60 m i n , ALDH i s o z y m e s zones f o r

over-

c o n t a i n e d 25 mg NAD, 3 mg

10 mg p y r a z o l ,

1 % agarose.

visualized

a p p l i e d i n an a g a r o s e

i n t h e dark

at

a p p e a r e d as dark b l u e

689 2.

P r e p a r a t i v e IEF i n granulated g e l .

Experimental

equipment

and g r a n u l a t e d g e l from LKB were used f o r p r e p a r a t i v e f i c a t i o n o f ALDH i s o z y m e s . tical

details

P r a c t i c a l procedures

puri-

and p r a c -

a r e g i v e n i n LKB a p p l i c a t i o n n o t e

198

(6).

Three ml o f s u p e r n a t a n t p r e p a r e d from l i v e r o r stomach homogenate was i s o e l e c t r i c a l l y t a i n i n g 2 . 5 % ampholyte of 950 v o l t f o r

focused in the g e l

(pH 3 . 5 -

15 h o u r s .

10)

at constant

convoltage

A f t e r t h e r u n , t h e g e l was

s e p a r a t e d i n t o 30 zones f o l l o w e d by e l u t i o n w i t h two volumes o f p h y s i o l o g i c a l Enzyme a s s a y .

Two b u f f e r s y s t e m s o f d i f f e r e n t

t o measure t h e a c t i v i t y : pH 7 . 5 -

10 and 0 . 1

containing

saline. 0 . 1 M sodium p y r o p h o s p h a t e

glycine/NaOH b u f f e r pH 10 -

1 mM NAD, 10 mM p r o p i o n a l d e h y d e

The a c t i v i t y

pH were used 12,

buffer, both

and 1 mM p y r a z o l .

i s e x p r e s s e d as change i n o p t i c a l d e n s i t y

340 nm a t 2 5 ° C p e r min/mg p r o t e i n .

A reaction

at

mixture

w i t h o u t s u b s t r a t e was used as a b l a n k . P a r t i a l l y p u r i f i e d isozyme p r e p a r a t i o n s were used t o d e t e r m i n e t h e Km v a l u e s . The a s s a y m i x t u r e NAD i n 0 . 1 M p y r o p h o s p h a t e b u f f e r , concentrations

and v a r y i n g

o f p r o p i o n a l d e h y d e o r 10 mM p r o p i o n a l d e h y d e

and v a r y i n g c o n c e n t r a t i o n s

Results

pH 9 . 5

c o n t a i n e d 2 mM

o f NAD.

and D i s c u s s i o n

As shown i n f i g u r e

1 a , f o u r main isozyme bands w i t h

p i s were o b s e r v e d from crude e x t r a c t s

different

o f human t i s s u e s

CA-membrane I E F . The i l l u s t r a t i n g scheme and p i v a l u e s given in f i g u r e (ALDH I

1 b . Two i s o z y m e s w i t h p i i n t h e a c i d i c

and ALDH I I )

other tissues

were found mainly i n

t h e s e i s o z y m e s were v a r i a b l e

using are range

l i v e r and k i d n e y . in t h e i r

intensity.

A t h i r d isozyme band w i t h p i

(ALDH I I I )

was o b s e r v e d mainly i n stomach and lung

staining

in t h e n e u t r a l

range

extracts.

In

690 a

b 4

t

•a

m

mmm

1»

m m

-

m

•




°>9 indicate IIS) 0,9

Q20,009

A1

36

36 (100)

30 (83)

29 (81)

16 (44)

36 (100)

30 (83)

2 (6)

A2

13

0 (0)

5 (38)

8 (62)

7 (54)

13 (100)

1 (8)

A3

6

0 (0)

3 (50)

6 (100)

0 (0)

A4

2

2 (100)

0 (0)

2 (100)

4 (67) 2 (100)

3 (23) 0 (0)

0 (0)

0 (0)

Group A B AB

12

Group B

- not confirmed

p r e l i m i n a r y d i a g n o s i s of MS 0 (0)

2 (17)

8 (67)

1 (17) 0 (0)

6 (50)

0 (0)

0 (0)

4 (33)

other d i a g n o s e s

B1

8

8 (100)

2 (25)

7 (88)

3 (38)

7 (88)

5 (62)

4 (50)

B2

95

0 (0)

5 (5)

48 (5D

35 (37)

26 (27)

4 (4)

24 (25)

The respective p e r c e n t a g e s are i n

parentheses.

I n the minor groups( A3 and A 4 ) there was either a s i m u l t a n eous absence of the b o t h m a i n parameters ( A 3 ) or presence of M

10 i n the absence of IIS ( A 4 ).

Group A B comprises samples w i t h not confirmed

preliminary

diagnosis of M S . No A T 10 and no signs of IIS were found i n this group.Moreover,almost no additional fractions of the r e g i o n s 7,8 and 9 were f o u n d i n contrast to the h i g h

699 frequence of AF 7,8,9 in the group A 1. One third of samples exhibited the signs of damaged HEB. Group B

comprises the patients with diagnosis other than MS.

103 samples of this group were divided in group B1 with positivity of AF 10 and IIS( 8 samples ). Both positivities characterise also the group A 1,but group B1 displayes additionaly the highest percentage of HEB damage.The main diagnoses of this group were lues of

CNS,tumors and tumor

metastases of CNS and polyradiculitis. Group B2 ( 95 samples) was without AF 10,but 27 per cent of samples showed the The MS and

IIS.

non MS samples differed not in the frequence of

the occurrence of double fraction of the region double(or triple) fraction of the region

5 and of

No correlation

of the presence of the highly alkaline fraction (HAF) with the diagnosis was found.

Discussion The additional fractions of the region 10 have diagnostic value particularly in combination

proved their with the

quotients.For their identification,however,a close comparison of CSF and

corresponding

were not able to

additional fractions of the

serum pattern,is necessary.We

confirm the diagnostic value of the of the regions 4 and 5-The occurrence

HAF depends upon the

technique of the application

of the samples on the gel.The recommended use of pieces of paper for the application ( LKB ) decreases the concentration of HAF in the

isoelectric focusing pattern.

References 1. Delmotte, P.: Z. klin. Chem. klin. Biochem. 9, 334-336 (1971) 2. K J e i l m , K.G.,Siden, A.: Eur. Neurol. 1j3, 40-50 (1977) 3. iUellin, K.G.,Vesterberg, 0.: J. neurol. Sci 23,199-213)

BINDING AFFINITIES BETWEEN VDBP AND VITAMIN D 3 , 25-(0H)-D 3 , 24-25-(0H) 2 -D 3 AND l-25-(0H) 2 -D 3 STUDIED BY ELECTROPHORETIC METHODS (PAGE-IEF, COMBINED IEF-ELECTROPHORESIS) AND PRINT-IMMUNOFIXATION.

J . Constans, M. Viau, C. Gouaillard, C. Bouissou and A. Clerc Centre d'Hémotypologie - CNRS - CHU Purpan, 31052 Toulouse Cedex, France

Introduction

Vitamin D metabolism has long been a subject of interest in pharmacology and biochemistry. Many steps in this process are now well known. To become an active metabolite, vitamin D 3 must be hydroxylated in the tissues to compounds such as 25-0H-D 3 , 24-25 ( 0 H ) 2 - D 3 a n d l-25-(0H) 2 -D 3 (1, 2). Other metabolites probably exist. Vitamin D 3 and its three main derivatives are simultaneously present in the serum at varying levels (3), vitamin D 3 being the most abundant, followed by 25-(0H)-D 3 . 24-25-(0H) 2 -D 3 and l-25-(0H) 2 -D 3 are present only at very low concentrations. All these ligands are specifically bound to a single protein called vitamin D-binding-protein (VDBP (4, 5), which is also known as Groupspecific-Component (Gc) to geneticists (6, 7). In the serum most of this protein is free (apoprotein). It shows a wide polymorphism (3 major genes : G c 1 F , Gc , and G c 2 ; 26 known variants) (8). Recently we showed that different affinities exist between the different proteins synthetised by the Gc^ and G c 2 genes and 25-(0H)-D 3 (9, 10). In this paper we describe the affinity of the VDBP for the four vitamin D 3 compounds named above. In order to avoid the tedious step of protein purification (11), we worked with the native serum protein. We used analytical electrophoresis and specific immunofixation procedures.

Material and Methods

Electrophoretic techniques - P.A.G.E. : Electrophoresis on polyacrylamide gel is performed according to the technique of Kitchin (12) using a system of discontinuous buffers (Tris,EDTA, boric acid), a running buffer pH 8.28 and a gel buffer pH 8.91. This technique is the most appropriate for separating the protein bands of the VDBP from albumin and other globulins. The vertical electrophoresis is performed on acrylamide plates 13x8x0.3 cm (Pharmacia GE 4). The spacing gel is monoacrylamide 2,5 %, bis-acrylamide 0,62 % (Eastman Kodak), the migration gel is monoacrylamide 6,6 %, bis-acrylamide 0,2 % ; b o t h gels are polymerised in the presence of TEEMED (EASTMAN KODAKj and ammonium persulfate (Merck). The migration lasts 3 hours at 102C with 15 V/cm. I.E.F. : The isoelectrofocusing is performed on a polyacrylamide gel (acrylamide, bis-acrylamide , Eastman Kodak) 1 mm thick (Multiphor LKB). The gel contains an ampholyte solution pH 4-6 (Ampholine LKB) 2 % and a riboflavine

© 1980 Walter de Gruyter & Co., Berlin • New York Electrophoresis '79

702 solution which allows the p h o t o p o l y m e r i s a t i o n . T h e conditions o f the electrophoresis are V . max. 1200 V . , P. m a x . 15 W , I max. 20 m A . during f o u r and a half hours. T h e temperature o f the gel is maintained at 10 -C. T h e strip-bands are soaked at the anode in a 1M solution o f H g P O ^ and at the cathode in a 0,2 M solution o f N a O H . T h e serum samples are applied o n the gel 1 c m f r o m the cathode (Whatman papers 3 M M ) . Bidimensional electrophoresis : T h e first dimension is p e r f o r m e d o n P A G E , the second on I E F . A f t e r the first dimension, using as reference the position o f the serum albumin, a band o f 4x0,5 cm is cut out o f the gel. It is placed o n the surface o f the I E F gel 1 cm f r o m the cathode. I m m u n o f i x a t i o n procedure : Electrophoresis o n agarose gel f o l l o w e d b y an immunof i x a t i o n revelation was described firstly b y A l p e r ( 1 3 ) . Successive modifications o f the m e t h o d made it m o r e adapted t o other techniques o f e l e c t r o p h o r e s i s ( 1 4 , 15). T h e technique w e used is an i m p r o v m e n t o n the ones described so far. A cellulose acetate strip (Millipore cellotate) 3 c m w i d e was soaked w i t h a 50 % dilution o f m o n o s p e c i f i c anti V D B P (anti G c globulin Behring) and then air dried. A f t e r migration the cellulose acetate strip was laid o n the gel in the protein focusing z o n e . T h e strip was left in contact w i t h the gel f o r 1 t o 2 minutes, then r e m o v e d and washed in saline solution f o r at least three hours and stained w i t h Coomassie Brilliant Blue ( R 2 5 0 ) T h e principles o f this m e t h o d can be m o d i f i e d according t o the electrophoretic

gel (% o f

acrylamide and bisacrylamide), the protein level and the nature o f the antigen ( m o r e than o n e serum protein m a y b e revealed o n the same gel) this procedure enabled us t o k e e p the sharpness o f the protein bands acquired during isoelectrofocusing. During the i m m u n o f i x a t i o n the antigen present in the gel moves t o the dry strip o f cellulose acetate. T h e antigen-antibody reaction is c o m p l e t e in o n e or t w o minutes. This m e t h o d is better adapted t o show up t w o distinct bands, proteins w i t h isoelectric points o f o n l y 0.01 p H unit d i f f e r e n c e . This m e t h o d w i t h some minor modifications can b e applied t o agarose or p o l y a c r y l a m i d e gel

C o m b i n e d IEF-Electrophoresis : T h e technique o f this useful procedure was described first b y Rosengren ( 1 6 ) and adapted b y Righetti and L a b i e D . ( 1 7 ) t o the comparison o f H b variants. During the first dimension ( I E F ) a linear p H gradient is obtained. I n the second electrophoresis (right angle) the proteins migrate in the p H gradient according t o the aminoacid ionisation. A specific protein titration curve is obtained. W e applied this original procedure t o the protein-ligand reaction which is p H dependent, W e tried t o f o l l o w the dissociation o f the h o l o p r o t e i n f o r m w i t h the d i f f e r e n t ligands in a p H gradient. A w i d e p H range can be obtained as described ( 1 6 , 18). N a r r o w e r p H ranges can also be adapted t o the specific p r o b l e m under study. A 2 m m thick 7 % p o l y a c r v l a m i d e gel ( 1 2 , 5 x 1 5 cm dimensions) is prepared as described above w i t h f o r the second electrophoresis run, a narrow trough cast in the middle o f the gel 9 cm long 1 m m w i d e 1mm d e e p which is filled w i t h about 100 j i l o f sample.

703 T h e I E F is carried out with constant power ( 1 0 W) during 9 0 minutes t o 1 0 5 minutes in a cooling system. In order to follow the pH gradient a Whatmann paper 3 M ( 3 x 5 mm) soaked in an Hb solution is laid on the cathodic side o f t h e gel. T h e Hb band focusing zone is a good reference o f the pH gradient. T h e second electrophoresis is run at right angles, 6 0 V / c m during 2 0 minutes. T h e anodic shift o f the Hb bands is also a useful reference for the stability o f the pH gradient. At the end o f the experimentation a print i m m u n o f i x a t i o n is carried o u t . On t h e cathodic side 1 c m from the central trough, across the gel, strips are cut ( 0 ' 5 c m wide) which are eluted in 0 . 0 0 1 M K CI solution. A f t e r 2 hours quilibration t o 2 0 ° temperature the pH o f the solutions are read on a pH meter (Philipp digital) with a c o m b i n e d electrode (Ingold) pH measures were also done with Multiphor microelectrodes ( L K B ) directly on the gel on t h e anodic and cathodic sides and near to the central trengh. Similar values were obtained b y b o t h procedures.

Results and Discussion T h e V D B P is present in t h e serum in two forms : o n e is ligand-free, the a p o f o r m , the second is linked t o the ligands, the h o l o f o r m . With electrophoretic methods it is possible to separe the two forms. T h e h o l o f o r m presents a more anodic mobility than the a p o f o r m . Its antigenicity remains unchanged after binding with the ligand ( 4 , 1 9 ) . T h e use o f radiolabeled compounds C 1 4 vitamin D 3 or H 3 2 5 - ( 0 H > D 3 , H 3 l - 2 5 - ( 0 H ) 2 - D 3 is another way to detect the holoprotein. A f t e r i m m u n o f i x a t i o n (apoprotein m o b i l i t y ) and radiofilm c o n t a c t ( h o l o f o r m mobility) we were able t o show the anodic shift o f the h o l o f o r m after P A G E o r I E F ( 1 9 ) we observed after bidimensional electrophoresis ( P A G E - Polyacrylamide Gradient G e l Electrophoresis), that this shift is not only due t o charge difference b u t to confornational modification. It was also demonstrated that the binding o f t h e ligand b y t h e protein is equi molecular. T h e mean serum level o f V D B P is about 0 , 3 mg/ml which corresponds t o 2 jig o f ligand/ml o f serum t o completely saturate the V D B P . In the course o f this work we have used the same serum sample (phenotype G c 2-2) which is characterized after electrophoresis b y a single protein b a n d . T h e difference in affinity for 2 5 - ( 0 H ) - D g o f the various genetic forms o f V D B P was described previously (9) Figure 1 shows the pattern obtained in P A G E for the V D B P holoprotein with 2 5 - ( 0 H ) - D 3 , 2 4 - 2 5 - ( 0 H ) 2 - D 3 , l - 2 5 - ( 0 H ) 2 " D 3 and vitamin D g . T h e apoprotein mobility used as reference confirms the anodic shift o f all the holoproteins (Fig. 1 A - B ) . I t can b e seen that the 25-(0H)-Dsj, 2 4 - 2 5 - ( 0 H ) 2 " D g and vitamin D 3 holoforms present similar electrophoretic mobility (Fig. I B - samples 2 , 3 , 4 ) while the l - 2 5 - ( 0 H ) 2 - D 3 h o l o f o r m has a m o b i l i t y intermediate between that o f the apoprotein and the h o l o f o r m o f the other ligands (Fig. I B - sample 5 ) . I m m u n o f i x a t i o n indicates that no apoprotein remains after a saturating dose o f ligand.

704

VDBP

} HOIOfOMM( «ranter ti* 1

I

3

4

S

Figure 1 : Electrophoretic pattern obtained after polyacrylamide gel electrophoresis A and immunofixation B with the serum VDB protein. A : Samples 1 and 3 correspond to the serum sample without ligand. The vitamin D binding protein is in the apoform. Its electrophoretic mobility is in theo( globulin region Sample 2 : sample 1 after addition of 3 yg 25-(0H)-D 3 /ml serum. The VDBP migrates as holoprotein. B : Samples 1 and 6 correspond to the free protein (apoprotein) present in a serum belonging to the Gc 22 phenotype. In this paper all the experiments are conducted with the same serum. Samples 2, 3, 4 and 5 correspond to the different holoprotein forms obtained after addition to sample 1 of an excess of ligand (5 jig/ml of serum) Sample 2 : holoprotein with 25-(0H)-Dg Sample 3 : holoprotein with 24-25-(0H) 2 "D3 Sample 4 : holoprotein with vitamin Dg Sample 5 : holoprotein with l-25-(0H)2"Dg Figure 2 is the IEF pattern obtained with the same samples. The apoprotein focused to its isoelectric point around pi 5.10. The holoform had a lower pi of around 4.95. After IEF the pattern is more heterogeneous than after PAGE. It can be seen that the 25-(0H)-Dg and 24-25-(0H)2"Dg present a similar mobility (pi 4.95) (sample 4 and 5) while the vitamin Dg holoform (sample 6) is partly dissociated into the apoform (pi 5.10). N o l-25-(0H)2"Dg holoform is present ; only its apoform is observed (sample 1). On the immunofixation pattern the blurred zones between the holoform position and the apoform seen in samples 1 and 6 show that a substance with immunological reaction of VDBP is present, corresponding to the redissociation of the holoform as discussed latter. Figure 2 : IEF patterns obtained after immunofixation,-the same samples as studied by PAGE (Figure 1) are submit-

®

f

p"

ted to IEF.gel slab 1 mm thick, ampho-

m

HOLOPROTEIN line pH 4-6 range 2 %. 1 : VDBP l-25-(0H) 2 -D 3 holoform

S.10

2 : standard serum Gc 1F-1S

© 2

3

4

s

-«APOPROTEIN ^ " s t a n dard serum Gc 2-2.VDBP apoform reference mobility 4 : VDBP 25-(0H)-D 3 holoform 5 : VDBP 24-25-(0H) 2 -D 3 holoform 6 : VDBP vitamin Dg holoform

705 VDBP attached to its four ligands shows the same IEF mobility (pi 4.95) but when the pH is near the pi there is a 50 % dissociation of the vitamin Dg holoform and a complete dissociation of the l-25-(0H)2-Dgholoprotein. These different electrophoretic behaviors are not artefactual as we demonstrated by two dimensional PAGE-IEF in figure 3. PAGE

®

©

pH U t

VDBP i •

at**-

HOLOFORMS

IfF

Q> u o ©

*

B

C

D

Figure 3 : Immunofixation pattern obtained after bidimensional polyacrylamide gel electrophoresis (first dimension) and isoelectrofocusing (second dimension). The samples studied are the same as in figure 1 A : VDBP - 25-(0H)-D 3 holoform B : VDBP - 24-25-(0H) 2 -D 3 holoform C : VDBP - Vitamin Dg holoform D : VDBP - l-25-(0H) 2 - Dg holoform Sections A and B (figure 3) correspond to the migration of the 25-(0H)-Dg and 24-25-(0H)2" Dg holoforms. After the first dimension these holoforms present an anodic mobility. They are preserved in the course of the second dimension. Figure 3 C shows that the vitamin Dg holoform dissociates during the second dimension into apoprotein. The holoform with l-25-(0H)2' Dg is enterely dissociated after the second dimension into the apoprotein (figure 3 D). The holoproteins with the different ligands do not present the same stabilities at their isoelectric points. In this pH range there are other serum proteins such as albumin and lipoproteins which could compete with VDBP to bind vitamin Dg and l-25-(0H)2"Dg. The electrophoretic behaviors (PAGE and IEF) of the differents holoforms confirm that only one specific binding site is present on the VDBP. Stable and unstable links are generated between the ligand and the binding site. These links differ according to the structure of the ligands. These results show that the pH is an important factor in the binding of the ligand by the protein. The combined IEF-electrophoresis method described by Rosengren (16) and applied by Krishnamoorthy (18) and by Righetti to macromolecule-ligands interactions (17) should be a valuable tool to follow these pH holoprotein dissociations. Figure 4 shows the titration curve obtained with purified VDBP. An identical curve is obtained with the serum VDBP after immunofixation. From pH 3.5 to 6.5 the slope of the curve is high which corresponds to a rapid change of the ionisation of the aminoacids around pH 5. This observation suggests a high pourcentage of acid aminoacids in the molecule as described by Bowman (20) and Svasti (21).

706

Figure 4 : Titration curve of the 25-(0H)-Dg and 24-25-(0H) 2 -Dg holoforms. A - Vitamin D binding purified protein in presence of a half-saturating dose of 25-(0H)-Dg. IEF : ampholine final concentration 2 %. The ampholine mixture used contained 70 % ampholine pH 3.5-10, 10 % pH 3,5-5, 10 % pH 7-9, 10 % pH 9-11 gel slab 2 mm thick (12,5x8 cm). The migration time for the electrophoresis is 10 minutes. All other conditions are described in the text. The two titration curves of the VDBP holoform and apoform are only observed in the pH zone 7 to 10. B : Titration curve of the 25-(0H)-Dg holoform after immunofixation. IEF : ampholine final concentration 2 %. Ampholine mixture : ampholine pH 4-6, pH 5-7 and pH 3.5-10 volume-volume. No dissociation of the protein-ligand complex occurs. C : Titration curves of the VDBP 25-(0H)-Dg holoform and of the apoprotein. The serum sample had received 1,5 jig of ligand/ml of serum. The VDPB is in the holoform and apoform. IEF-EI conditions are the same as in fig. 4 B. In this pH gradient, the two curves are clearly separated. This observation was not possible in a same pH zone in figure 4 A. At p H > 6.5-7 the charge variation of the protein is very little modified. A flatenning of the curve is observed until pH 9„The 25 and 24-25-(0H)2"Dg holoproteins in the pH 3.5-10 range are distributed as a single curve. No protein-ligand dissociation occurs. At half saturating dose of 25 or 24r25-(0H) 2 -D 3 the VDBP is in both holo and apoproteins (Fig. 4 A) which is illustrated by the two curves observed. Because of the important slope between pH 5 to 7 the two curves are not seen. Fig. 4 C shows that when the slope is reduced the two curves are clearly present. Saturating dose of vitamin Dg was used to study the dissociation curve of the VDBP holoform The apoprotein generated during the second electrophoresis by the unstability of the proteinligand complex could be detected by the appearance of its specific curve.

707 According to the technical difficulties observed in the former experiment (titration curves of the 25-(0H)-Dg holo and apoforms) the combined IEF-EI was developed using narrow pH range solutions in IEF. This procedure enables us to separate the holo and apoprotein curves. The electrophoretic patterns in figure 5 show the different curves obtained after combined IEF-EI using pH 4 to 7 ampholine mixture In the course of this experiment we have observed non-reproducible results after combined IEF-EI. Other factors can influence the dissociation of the holoform. In the vitamin Dg holoform dissociation the main factor is the time of sample application. If the second electrophoresis is started immediatly after the sample application the dissociation observed is not reproducible. During the migration (20 minutes) the dissociation equilibrium does not have enough time, to be formed in the pH gradient, only the apoprotein curve in the acidic zone of the pH gradient is observed. It corresponds to the slower mobility of the VDBP near to its isoelectric point.

Figure 5 : Influence of the time on the pH equilibrium on the dissociation of the vitamin D j holoform. IEF conditions as described in Fig. 4 B and C. The second electrophoresis is started after sample application in the trengh during : A : 30 minutes B : 90 minutes C : 120 minutes For pH values higher than 6.5 the holoform is not dissociated. From pH 6.5 to 5 it is noticed that the apoprotein proportion is favoured. Whan the second electrophoresis is started 30, 90 or 120 minutes after the sample application the holo-apoform-ratio decreases (Fig. 5 A,B,C). During this period a best equilibrium with the pH gradient is realized between the holoform and the apoprotein. In these conditions^pH and time of sample application as described in figure 5 C) from pH 5 to pH 6.5 the holoform proportion increases. Above pH 6.5 the protein-ligand complex does not dissociate. The holoform remains stable in the basic part of the pH gradient. The more the pH decreases, the more the proportion of the apoform increases. Below pH 5 only the apoprotein is present.

708 The importance of the contact time is also observed when we compare the dissociation results obtained in figure 3 and those presented in figure 5. Dissociation occurs in a shorter time after combined IEF-EI than after PAGE-IEF (migration time 5 hours for IEF) these differences can be explained b y the different procedures used. After PAGE-EIF albumin and /or lipoproteins of the serum are separated from the VDBP holoproteins.the dissociation of the complex is slow. During combined IEF-EI and in the acidic pH zone the influence of these serum proteins are important. They are able to compete with VDBP in binding vitamin Dg (22). Temperature is also an active factor in the stability of the holoprotein as observed by Righetti (17). The pH equilibrium in the trengh and the kinetic of the dissociation are influenced by the temperature of the gel. In combined IEF-EI it is difficult to analyse separately the influence of the time, temperature and pH conditions because they are acting simultaneously in the protein-ligand dissociation. These factors can determine the results obtained after electrophoresis, mostly when the kinetic of the dissociation are very slow as in the vitamin Dg holoprotein dissociation. Combined IEF-EI presents also some limits in particular the difficulty to obtain a good separation in the second electrophoresis. The l-25-(0H)2"Dg holoform dissociation cannot be followed because the two holo and apoprotein have mobilities too close as shown in figure 1.

Conclusions

The affinities of the VDBP for the four ligands are analysed on the native serum protein using different electrophoretic procedures. The immunofixation procedure allows us to follow the behavior of the holoform and their dissociations. The binding of the ligands by the VDBP induces charge modification and changes in the conformation. The stability of the complex with vitamin Dg and l-25-(0H)2"Dg is pH dependent. At p H > 6.5 the vitamin Dg holoform is favoured. At pH

x -o

X-

3

cr

Pig. 1.

/ "

O

Interaction of PHA with monoclonal IgM in Immunoelectrophoresis (a and b) and in PHA selection electrophoresis.

a.

Visualization of pentameric IgM with specific antiserum (in upper antibody trough) and with PHA.

b.

Absence of a PHA precipitation line with monomeric monoclonal IgM.

c.

Simultaneous demonstration in macroglobulinemic serum of pentameric IgM and of accompanying monomeric IgM (arrow).

mers. Accordingly, particularly in Waldenstrom's macroglobulinemia, presence of free IgM monomers accompanying the bulk of pentameric monoclonal IgM could be demonstrated by PHA selection electrophoresis, i.e., Immunoelectrophoresis of whole serum proteins in agar gel containing 0.2$ PHA (fig. 1c). During electrophoresis the rnacromolecular proportion of the paraprotein was selected by PHA and formed a symmetrical precipitate in the central part of the slide. In contrast, monomeric IgM migrated freely and was visualized as a separate

763 precipitation line by subsequent exposure to specific antibody (fig. 1c). Normal or increased polyclonal IgA did not noticeably react with PHA in Immunoelectrophoresis. However, marked precipitation lines appeared with monoclonal IgA2 in all of 35 myeloma sera (4). Among 279 IgA1 myeloma sera, PHA did not react with the monoclonal proteins in 244 cases (87.4$); mostly faint precipitation lines occurred in 35 cases (12.6$) because, contrary to IgA2, only the polymeric moieties of these IgA1 myeloma proteins were precipitated by PHA (fig. 2).

2 2 - 2 5 26-29

32-36

37-42

3D

b 8

4 0

Pig. 2. a.

^

Interaction of PHA with monoclonal IgA1 of the PHA-precipitable type. Gel filtration (Sephadex G 200) of myeloma serum. Proteins from shaded elution fractions were immunoelectrophoretically tested.

b.

PHA-precipitable dimeric (9.3S) IgA1 in tubes 26-29.

c.

PHA-nonprecipitable monomeric (6.5S) IgA1 in tubes 32-36.

764 In h u m a n colostrum and saliva dimeric "secretory" IgA is largely of subgroup IgA2. In Immunoelectrophoresis, PHA precipitated w i t h (protective) "secretory" IgA but not w i t h coexisting m o n o m e r i c (nonprotective)

IgA1

(4).

-

In all tests

interpretation of immunoeiectrophoretic findings w e r e

facili-

tated by the fact that PHA precipitated n e i t h e r w i t h normal or m o n o c l o n a l IgG nor w i t h free immunoglobulin L chains.

In

IgM and IgA molecules, PHA-receptor sites are located in the P c r e g i o n of }i and a chains. A b s o r p t i o n studies w i t h red cell ghosts, and w i t h monoclonal IgM or IgA2, respectively,

sug-

gested structural similarity of the receptors on proteins w i t h the P H A - s p e c i f i c glycopeptide on erythrocyte

membranes

(2).

Conclusions No findings w e r e made on the basis of the PHA

precipitin

r e a c t i o n that could not have b e e n obtained also w i t h other analytical m e t h o d s which, however, are often too

demanding

or time-consuming for routine use in a clinical

laboratory.

Under the above indications the a p p l i c a t i o n of PHA as a novel diagnostic tool in electrophoretic differentiation of humoral immunoglobulins offers the practical advantages of simplicity of equipment, rapidity and economy.

References 1. Weber, T., Nordman, C.T., Grasbeck, R.: 4, 77-80 (1967).

Scand. J. Haemat.

2. Spengler, G.A., Weber, R.-M., v o n Wyttenbach, H., Huser, H.-J.: Schweiz. med. Wschr. H>8, 1605 (1978). 3. Spengler, G.A., Weber, R.-M.: (in press).

J. Immunol. Methods

4. Spengler, G.A., Weber, R.-M.: Protides of the Biological Fluids, 2 7 t h Colloquium (1979). P e r g a m o n Press (in press).

FEASIBILITY OF STUDYING THE FUNCTIONAL NATURE OF HUMAN IgG SUBCLASS RESPONSES BY MEANS OF ANALYTICAL

ISOTACHOPHORESIS

K. W. Hedlund, R. Wistar, Jr. and D. Nichelson U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, M D , USA, and Naval Medical Research Institute, Bethesda, M D , USA

Introduction

The human immune response is divided into humoral and cellular phases.

The

humoral phase is mediated by immunoglobulin (Ig) molecules w h i c h specifically combine w i t h homologous antigens.

The role of these

immunoglobulins

in host defenses involves not only physical binding of the variable regions of the light and heavy chains to the homologous antigen, but also the presentation of the offending immune complex to the rest of the host's defense armamentarium.

Edelman (1) and Dorrington et al. (2) pointed out that cer-

tain domains within the effector or constant region of the immunoglobulins mediate physiological reactions of immunity, such as complement placental transport and phagocytosis by mononuclear cells.

fixation,

The number and

diversity of the Ig classes and subclasses are generally felt to be an evolutionary attempt to enhance the survival of the host beleaguered by a w i d e range and variety of infectious antigens. Of the 5 classes of Ig, class G (IgG) is of particular interest.

It com-

prises 75 % of the total serum antibodies directed against bacteria, viruses and toxins.

Table 1 demonstrates some of the physical

characteristics

of the human IgG subclasses. Although a complete understanding of the relationship of structure to b i o logical function is still evolving, an ever-expanding body of data supports this association.

A list of partial functional activités can be seen in

Table 2.

© 1980 Walter de Gruyter 8. Co., Berlin • New York Electrophoresis '79

766 TABLE 1 PHYSICAL PROPERTIES OF HUMAN IgG SUBCLASSES

Serum concentration % of total IgG mg/ml

IgG-1

IgG-2

IgG-3

IgG-4

64 - 70 6.7 - 10.5

23 - 28 2.5 - 4.3

4 - 7 0.55 - 1.0

3 - 4 0.36

Half-life (days)

21

21

Papain sensitivity

2+

Resistant

3+

Resistant

2

4

>12

2

Inter-heavy chain S-S bonds MW of heavy chain

54,200

54,200

7-8

60,950

21

54,200

TABLE 2 BIOLOGIC ACTIVITIES OF IgG SUBCLASSES IgG-1

IgG-2

IgG-3

IgG-4

Reversed passive cutaneous anaphylaxis

+

0

+

+

Cytophilic antibodies

+

0

+

0

Placental transport

+++

+

++

+

Complement fixation

+++

+

+++

0

Staphylococcal protein A binding

+

+

0

+

Monocyte binding

+

0

+

0

Hopefully having made the point that physical as well as functional differences do exist between the subclasses of human IgG, we would like to tie this to the concept that studies of the patterns of subclass responsiveness to specific antigens may be important in assessing the appropriateness of the host's humoral response.

In humans, it is generally assumed that the

secondary or anamnestic response to protein antigens produces IgG subclass responses roughly proportional to their serum concentrations.

It is evi-

dent, however, that a variety of antigens evokes restricted IgG subclass responses.

Some of these responses are shown in Table 3.

767 TABLE 3 RESTRICTED IgG SUBCLASS RESPONSES Antigens or disease states

*

Clotting factors VIII and IX

Ref. 4, 5

IgG-1

IgG-2

-

-

-

Platelets

6

-

Polysaccharides, teichoic acids

5

-

*0n cells in periodontal associated disease

7

+4+

*"Resistant" childhood allergies

8

-

IgG-3 -

IgG-4

+++

+++

-

+++

-

-

_

+++

+

-

+++

-

Rh factors

9

+++

+

Diphtheria and tetanus toxoids

5

+++

+

Blood group A

5

+++

+

DNA

3

+++

+

+++

+ -

-

-

-

+++

+

Although Schur (3) pointed out that many studies of IgG subclasses have focused on antibodies in autoimmune disorders, it is reasonable to assume that there are certain "normal" responses to particular antigens and that the appropriateness of the response pattern can be inferred in some situations, such as infectious diseases, by the level of protection conferred by immunization with homologous antigens.

In this paper, a method recently

described by Hedlund et al. (10) demonstrates that the individual subclasses of human IgG can be identified by means of analytical isotachophoresis.

The technology presently exists to assay either total serum IgG or

the IgG subclass antibodies after dissociation from their homologous antigens (Vandvik et al., 11). The basic principles of electrophoretic separation techniques and of isotachophoresis in particular have been described in excellent reviews by Haglund (12) and Everaerts et al. (13).

Briefly, in isotachophoresis, ions

are separated on the basis of differences in the effective mobilities. Once a steady state has been established, all the zones of interest, from the sample to the terminating ion, move with a velocity equal to that of the leading ion. Our present studies demonstrate that each subclass of IgG migrates with its

768 own unique mobility, when analyzed by isotachophoresis.

An uncharacterized

IgG peak can be identififed readily by its superimposition upon one of an already known pattern of peaks.

Materials and Methods The LKB 2127 Tachophor (LKB-Produkter AB, Bromma I, Sweden) was equipped with a thermal detector and an UV detector set at 280 nm.

Electrophoretic

separation took place in a 20-cm Teflon capillary tube (0.5 mm, inside diameter) at a constant temperature (20°C).

The equipment was operated at a

constant current of 75 yA with a voltage increase from 4 kV at the beginning to 24 kV at the end of an experiment.

Analysis time was =15 min.

A

1.0-yl sample volume containing 5 yg of IgG was used for most separations.

Buffer systems The leading buffer contained 0.005 M HCl-ammediol, pH 8.4 (Eastman Kodak Co., Rochester, NY) and 0.04 % hydroxypropyl-methylcellulose A4M (Dow Chemical, Midland, MI).

The terminal buffer was 0.01 M e-amino caproic acid ad-

justed to pH 9.4 with BaiOH^.

Both buffers were prepared, degassed and

filtered immediately prior to use. Two protein spacer solutions were used.

The first consisted of 2.0 yl of

0.01 % ampholine, pH 3.5-10 (LKB) and was used in each analytical run.

The

second was composed of 2.0 yl of the first spacer solution supplemented with 1.0 yl of a 1:2:2 mixture of 0.01 M glutamine, leucine and B-alanine.

Immunoglobulins

Myeloma serum and plasma specimens were obtained from a number of cooperating physicians and from patients at the Walter Reed Army Medical Center and the National Naval Medical Center.

The specimens included 21 sera with

IgG-1 (8 kappa and 13 lambda), 8 sera with IgG-2 (6 kappa and 2 lambda), 3 sera with IgG-3 (2 kappa and 1 lambda), and 6 sera with IgG-4 (3 kappa and 3 lambda) proteins.

769 In most cases the myeloma proteins were first precipitated with (NH^^SO^ at 35 % saturation, pH 7.4.

The precipitate was dialyzed against 0.9 % sa-

line to remove the (NH^^SO^ and then equilibrated with the initial ion-exchange buffer, 0.01 M Tris-HCl, pH 7.4.

Ion exchange chromatography was

performed with DE-52 (Whatman, Clinton, NJ), using the Tris-HCl buffer with a conductivity of 1100 ymos.

If the myeloma IgG protein failed to elute

with the starting buffer, the conductivity of the buffer was increased in a stepwise manner until the desired protein was recovered.

With some of the

IgG, especially IgG-2 and IgG-4 proteins, gel filtration on Sephadex G-200 was used to remove contaminating proteins.

Before use, the proteins were

characterized by electrophoresis and Immunoelectrophoresis using antiserum to IgG and whole serum.

Assay for the IgG subclasses Antibodies to individual myelomas of known subclass were raised in rabbits, goats and sheep.

The resulting antisera were absorbed with proteins of the

"wrong" subclasses until they were specific as judged by their ability to agglutinate red cells coated with purified IgG of one subclass and not cells coated with IgG of the other 3 subclasses.

As a final test of suita-

bility, each antiserum was also tested by hemagglutination-inhibition (HI), using several IgG of each subclass and light chain-type, by methods described previously by Wistar et al. (14).

Reagents for typing the IgG-1

subclass were obtained through the courtesty of Drs. William Yount and Mel Shanfield.

Results After the homogeneity and subclass of the IgG myeloma protein samples were determined by Immunoelectrophoresis and HI assays, 5 yl of each preparation, containing 5 yg of protein was analyzed by isotachophoresis.

All samples

from a particular subclass had unique net mobilities characteristic for that subclass.

770 Representative mobility patterns and their localization by subclass are shown in Fig. 1.

In Fig. 1.1 through 1.4, characteristic isotachophero-

grams for human IgG subclasses 4, 2, 1 and 3 are shown in the decreasing order of mobilities.

Fig. 1.5 illustrates the mobilities of the individual

components in a sample mixture of IgG-4 and IgG-2.

When IgG-1 is added to

this mixture, the 3 characteristic and individual components are illustrated in Fig. 1.6.

Fig. 1.7 shows the fourth characteristic peak follow-

ing the addition of IgG-3 to the mixture of IgG-4, IgG-2 and IgG-1.

These

isotachophoretic analyses were done with the first protein spacer solution which consisted of ampholine, pH 3.5-10, alone.

To achieve greater separa-

tion, especially between the peaks of IgG-1 and IgG-3, the second protein spacer solution was used.

2

3

4

0.

s

UJ

(5 z 0 C1:

Fig.

unid.

I = °enzyme3 : Unidentified

Ar.E.

: Aromatic

band

esterase

1. Serum c h o l i n e s t e r a s e isozyme p a t t e r n in d i f f e r e n t human t i s s u e s f r o m i n d i v i d u a l s w i t h normal (UU) and a t y p i c a l (UA) phenotype

8oo tissue extracts. tissues

A t y p i c a l p a t t e r n of isozymes

in d i f f e r e n t

from i n d i v i d u a l s who had a normal p h e n o t y p e

serum c h o l i n e s t e r a s e i s p r e s e n t e d in F i g .

h e t e r o g e n e i t y i n t h e i s o z y m e c o m p o s i t i o n between t i s s u e s was n o t i c e d .

(UU)

for

1. A c o n s i d e r a b l e different

There was no v a r i a t i o n i n t h e

p r o f i l e of a p a r t i c u l a r t i s s u e from d i f f e r e n t Although i n d i v i d u a l s with normal o r g e n e t i c a l l y a t y p i c a l phenotype showed s i g n i f i c a n t i d e n t i t y

isozyme

individuals. determined among m a j o r

isozyme components l i k e C^ and C^, t h e r e were pronounced differences regarding their s u s c e p t i b i l i t y In F i g .

towards

inhibitors.

1 t h e isozyme p r o f i l e of v a r i o u s o r g a n s from an

atypical heterozygote

(UA) i n d i v i d u a l i s shown. The isozyme

p a t t e r n a f t e r p r e - t r e a t m e n t with i n h i b i t o r s

like

sodium f l u o r i d e o r d i i s o p r o p y I f l u o r o p h o s p h a t e information regarding genetic variations.

Fig.

dibucaine,

(DFP)

gave

2 gives

a

s c h e m a t i c d e s c r i p t i o n of the t y p i c a l i n h i b i t i o n p r o f i l e of i s o z y m e s from d i f f e r e n t

the

tissues.

S i n c e a l l t h e i s o z y m e bands c o u l d be i n h i b i t e d by p r e - t r e a t ment w i t h DFP, they must be only due t o e s t e r a s e

activity.

H e m o l y s a t e s showed an a d d i t i o n a l band i n t h e r e g i o n of C^ which was n e i t h e r i n h i b i t e d w i t h

acetylcholinesterase

i n h i b i t o r s nor w i t h s e r u m c h o l i n e s t e r a s e i n h i b i t o r s . i d e n t i t y of t h i s band s t i l l

remains

The

obscure.

Conclus ion LIDDELLet a l . ,

(6) i n v e s t i g a t e d s e v e r a l human o r g a n s

comparison with the phenotypes of c h o l i n e s t e r a s e the b a s i s dibucaine,

of a c t i v i t y

for

in serum. On

a s s a y as w e l l a s i n h i b i t i o n w i t h

they c o n c l u d e d t h a t both t h e normal and t h e

variant

enzyme a r e c o n t r o l l e d by t h e same gene i n a l l t h e o r g a n s . We h a v e c o n f i r m e d t h e s e f i n d i n g s by u s i n g PAGE. The i s o z y m e p r o f i l e of normal and v a r i a n t enzyme i n d i f f e r e n t o r g a n s i n the p r e s e n c e of d i f f e r e n t i n h i b i t o r s

showed t h a t a t

and least

8oi

no J. Ui b. NaF. Ro. £snr. DhT. Hon?.

* : : : • : :

without Inhibitor Dibucaine Sodium Fluoride Ho 2-o683 En'Tine Di if opropyl fluorophosphate Bon ".oyl cholinrhlorji=7)

[""H&l

50-

tf-

71 W E E K S OF GESTATION

IE"

N-AVM.-WT.

M

100 JH

90 -

ao -

72

7, W E E K S OF

GESTATION

X

F i g . 1, u p p e r p a r t : O s m o t i c e f f i c i e n c y of m a t e r n a l p l a s m a p r o t e i n s (i.e. C O P p e r g r a m p r o t e i n in 100 ml P l a s m a ) c o r r e l a t e d to w e e k s of g e s t a t i o n in n o r m a l p r e g n a n c y a n d c a s e s of s e v e r e e d e m a a n d EPH g e s t o s i s . F i g . 1, l o w e r p a r t : N u m b e r plasma proteins correlated clinical groups.

a v e r a g e m o l e c u l a r w e i g h t of to w e e k s of g e s t a t i o n in the

same

8o8

o PLASMA M ' ('normol) MW 80 000 a PLASMA 'S'lEPH] MW 102 500

RESULTS OF OSMOMETRIC DETERMINATION OF NUMBER AVERAGE MOLECULAR WEIGHT (NAMW) OF HUMAN PLASMA P R O T E I N S . CORRELATED TO THREE PROTEIN FRACTIONS (1,11.ID IN SDS-PAA ELECTROPHORESIS.

PROT CONC Ig°/o] NORMAL PREGNANCY: n=19; SYMBOL EPH-GESTOSIS : n=17; SYMBOL F E T A L PLASMA : n= U. SYMBOL

ELECTROPHEROGRAM [so % pagI

lo.l % SDSI

1 6 0 6 0 0

0 RFC/.REL

n

270 600 I-103] MOLECULAR WEIGHT

MOBILITY] x OV ALB » H ALR o TRANSF • POLYM 1601 IO^I

F i g . 2 : COP and p r o t e i n c o n c e n t r a t i o n i n two m a t e r n a l plasma s a m p l e s . F r a c t i o n s in t h e s e p l a s m a were c a l c u l a t e d by r e l a t i v e m o b i l i t y o f marker p r o t e i n s in SDS-PAGE.

Plasma

samples

different such

plasma

protein The

with

colloid

samples

fractions,

reliability

by r e s u l t s

values

of

of

of

increasing

concentration (see

fig.2).

revealed

which c o r r e l a t e

NAMW c o u l d

centrations

protein

pressure

by S D S - P A G E

of osmotic

proved

and

similar

osmotic

F i g . 3 : C o r r e l a t i o n o f NAMW and t h r e e p r o t e i n f r a c t i o n s ( I , 1 1 , 1 1 1 ) , c a l c u l a t e d by procentual f r a c t i o n area of the t o t a l e l e c t r o p h e r o g r a m .

(see

differences

to d i f f e r e n t

determination

SDS-PAGE

of

with

concentrations

low m o l e c u l a r of p r o t e i n s

exert

NAMW

in

of

three

values.

NAMW c o u l d

fig.3).

be f o u n d as an e f f e c t

proteins

can

Evaluation

be

Increasing of

decreasing

weight with

con-

(fraction

high

I)

molecular

809 weight The

(fractions

calculated

same

maternal

lity

(within

Serial The

II a n d

fraction plasma,

5X)

determinations

slope

of t h e

of

the

areas

run

of o u r

measurements

actions

of e l e c t r o p h e r o g r a m s

parallel,

SDS-PAGE

of N A M W

proteins line

( p < 0.001)

showed

within

5%

the

reproducibi-

reproducibility.

influenced (steric

in N A M W

with

a high

of

method.

were

of N A M W w e r e

plasma

regression

significantly

III).

by

intermolecular

exclusion

evaluation

increasing

NAMW

effect).

The

increased

value.

Comment The

properties

purpose, better run

although

band

all

of

homogeneous the

use

resolutions.

samples

of o n e

gel

were

sufficient

of a g r a d i e n t For a c c u r a c y ,

patient

double

gel

would

it w a s and

for

our

result

in

advantageous

in a p a r a l l e l

to

manner.

Conclusion SDS-PAGE

and m e m b r a n e

perinatal

research:

interpreted

by t h e

a criterium

results

analysis

SDS e l e c t r o p h e r o g r a m s . become

osmometry

The

proved

to

of N A M W

of m o l e c u l a r

Osmotic

efficiency

of t h e r a p e u t i c

and

be

useful

methods

determinations weight

fractions

of p r o t e i n s

prognostic

can

in be

in

should

value.

References 1. M e n d e n h a l l , H . W . : A m . J. O b s t e t . G y n e c o l . 1 0 6 , 3 8 8 ( 1 9 7 0 ) 2. S c h r o e c k , R . , e t a l . : In H u s s l e i n ,H. , (ed) , G y n ä k o l o g i e u n d G e b u r t s h i l f e , v o l . 1 , 2 0 3 , H. E g e r m a n n , W i e n 1977 3. W e b e r , K. , a n d O s b o r n , M . : J. B i o l . C h e m . 2 4 4 , 4 4 0 6 ( 1 9 6 9 ) 4. D e r g e , K.: G I T , F a c h z t s c h r . L a b . 12, 1 0 9 7 ] T 9 6 6 ) 5. v a n ' t H o f f , J . H . : Z. p h y s . C h e m . 1 7 4 8 1 ( 1 8 8 7 ) A c k n o w l e d g e m e n t s : The and technical support

t e c h n i c a l a s s i s t a n c e by M r s . I l o n a G r a s s l by D r . h a b i 1 . N . M e n d l e r is a p p r e c i a t e d .

SERUM PROTEIN ANALYSIS BY MOLECULAR INTERPRETATION OF CELLULOSE ACETATE ELECTROPHORESIS: EVALUATION OF ITS CLINICAL USEFULNESS.

e

i®

Qoo

F.Aguzzi, C.Petrilli, G.Merlini, * • •/ i• ' / < ". ;/ ;/

//

/

- 4 - 3 - 2 - 1

0

1

2

3

y

/ ? •4 - 3

/

/

. ! - 2 -1

r. r. " W x

• 0

T^. 1

Fig. 1 Frequency distributions of "normals" and three groups of disorders for the second component. Fig. 2 Frequency distributions of three subgroups of liver disorders for the second ccrnponent.

Results and discussion. The Table summarizes the main alterations in the different groups of disorders. The alterations in pA and Alb are highly correlated and from the Table it is evident that pA is more sensitive than Alb, probably due to the electrophoretic technique. The other proteins which seem to be informative are AT, Hp, Tf, C3 and the ¡i-t fusion. These conclusions are supported bv the Principal Component Analysis. The latter also in a certain sense summarizes these variations, taking into account the correlations between them.

815 The distribution of each of the first five principal component for the localized diseases and atherosclerosis are concentrated around the value 0 which indicates normality. On comparison to these, each component distinguishes at least one group of disorders and each group is different for at least one component. The first approximate component (-pA + AT +Hp - Tf ) has on the average a significantly raised value for inflanmations, neoplasms and liver disorders. Here we examine in some detail the second principal canponent (Hp + C3 fusion) . The frequency distributions for the three disorders mentioned and for the "normal" groups is shown in Fig. 1. Further, Fig. 2 shows the differences between subgroups within the group of liver disorders. In Fig. 1, both inflanmations and neoplasns give relatively higher values and liver disorders lower values with respect to the "normals". In several disorders, there is a modification in the level of one or more proteins as evaluated by cellulose acetate electrophoresis. The combination of modifications observed in any patient can be indicative of the kind of disorder. The principal components help in picking out patterns of variations. As can be seen from our results, some of the components are associated with particular disorders. References. 1) J.O. Jeppsson, C.B. Laurell, B.Franzen - Clin. Chan. 25,629,1979 2) C.A. Alper, A.M. Johnson - Vox Sang. 17,445,1969 3) F.Aguzzi, N.Foggi, T.Chiara - La Ricerca Clin.Lab. 8 (Suppl.n.1),171, 1978 4) F.Aguzzi, N.Poggi - Boll.1st.Sieroter.Milanese 56,212,1977 5) J.Kohn - Protides in the Biological Fluids (Peeters H. Ed.), 23rd Coll. Pergamon Press, Oxford, 1976, p.325

G E N E T I C A L AND B I O C H E M I C A L MOUSE L A C T A T E

CHARACTERIZATION

OF A D O M I N A N T M U T A T I O N

OF

DEHYDROGENASE

W. P r e t s c h and D.

Charles

G e s e l l s c h a f t für S t r a h l e n - u n d U m w e l t f o r s c h u n g , Abt. für I n g o l s t ä d t e r L a n d s t r . 1, D - 8 0 4 2 N e u h e r b e r g ( G e r m a n y )

Genetik,

Abstract

(101XC3H)F,J male mice were t r e a t e d with p r o c a r b a z i n e untreated test-stock

and caged

f e m a l e s . S e v e r a l e n z y m e s from the

were then a n a l y z e d by p o l y a c r y l a m i d e

offspring

gel i s o e l e c t r i c f o c u s i n g .

1,374 a n i m a l s of the c o n t r o l group, 1 a n i m a l with an e n z y m e was d e t e c t e d ; under

1,781

offspring

of the e x p e r i m e n t a l

a n i m a l s with an e n z y m e v a r i a t i o n were f o u n d . of the e x p e r i m e n t a l

group (spermatogonial

types we o b t a i n e d w i l d t y p e s ,

variants

g e r m - c e l l s t a g e ) we

(LDH) p a t t e r n . By c r o s s e s b e t w e e n the and h e t e r o z y g o t e

Among

variation

group, 5

In one of the

define a d o m i n a n t i n h e r i t a n c e of the b a n d i n g a l t e r a t i o n tate d e h y d r o g e n a s e

with

could

in the

lac-

variant

and h o m o z y g o t e

variant

t y p e s , as e x p e c t e d by M e n d e l ' s l a w s for d o m i n a n t i n h e r i t a n c e .

In

ad-

d i t i o n to l i v e r , the b a n d i n g a l t e r a t i o n was also d i s p l a y e d in k i d n e y , m u s c l e , h e a r t , b r a i n , s p l e e n and lung. The m u t a t i o n was f u r t h e r a c t e r i z e d by p o l y a c r y l a m i d e

gel e l e c t r o p h o r e s i s ,

t e r m i n a t i o n s and t h e r m o l a b i l i t y electrophoresis

char-

enzyme activity

s t u d i e s . It w a s d e t e c t a b l e by

gel

in all t i s s u e s e x a m i n e d and c o n c e r n e d the b a n d s

r e s p o n d i n g to L D H - A . In l i v e r , m u s c l e and s p l e e n , the s p e c i f i c vity of LDH d e c r e a s e d to a b o u t 50 % of the w i l d t y p e in the

de-

coracti-

heterozy-

gote m u t a n t and to a b o u t 9 % in the h o m o z y g o t e m u t a n t . In k i d n e y , h e a r t , b r a i n and lung, the d i m i n u t i o n of LDH a c t i v i t y in the was l e s s p r o n o u n c e d .

In t h e r m o l a b i l i t y

the liver l a c t a t e d e h y d r o g e n a s e

of the m u t a n t s was much more

s e n s i t i v e than that of the w i l d t y p e . Due to these b i o c h e m i c a l p e r t i e s , we assume that the i n d u c e d m u t a t i o n a f f e c t s the LDH w h i c h c o d e s for l a c t i c d e h y d r o g e n a s e

© 1980 Walter de Gruyter 8. Co., Berlin Electrophoresis '79

New York

mutants

e x p e r i m e n t s , we o b s e r v e d

of the m u s c l e

type.

that

heat prolocus

8l8 Introduction In a d d i t i o n to the c l a s s i c a l t i o n s in m a m m a l s , ted assay locus test

i. e. the d o m i n a n t l e t h a l

(2), the "in v i v o " c y t o g e n e t i c (4), s o m e b i o c h e m i c a l

o p e d : the p r o t e i n - m a p p i n g system

m e t h o d s for the d e t e c t i o n

method

(6) a n d p o l y a c r y l a m i d e

the d e t e c t i o n

of e n z y m e

The d e s c r i b e d

lactate

ding pattern

(5), the b i o c h e m i c a l

gel i s o e l e c t r i c f o c u s i n g

variants

tory by the P A G I F m e t h o d and b i o c h e m i c a l

Materials

and

(8).

muta-

host-mediaspecific

been

devel-

specific (PAGIF)

locus for

(7). mutant

dehydrogenase)

genetical

(1), the

(3) a n d the

methods have recently

dehydrogenase

of l a c t a t e

test

test

of g e n e

(alteration

in the

was d e t e c t e d in our

labora-

In t h i s p a p e r ue are p r e s e n t i n g

properties

of t h i s L D H

ban-

some

mutant.

Methods

Hybrid male mice ( 1 0 1 / E l x C 3 H / E l w e r e injected intraperitoneally w i t h 600 mg p r o c a r b a z i n e per kg b o d y w e i g h t at the age of 12 w e e k s . A f t e r i n j e c t i o n , e a c h m a l e w a s c a g e d w i t h an u n t r e a t e d t e s t - s t o c k f e m a l e , h o m o z y g o t e for 7 r e c e s s i v e m a r k e r s (4). W h e n the o f f s p r i n g r e a c h e d 8 w e e k s of age, 150 - 200 mg of l i v e r u a s r e m o v e d f r o m thB m o u s e by b i o p s y . The s a m p l e s w e r e i m m e d i a t e l y h o m o g e n i z e d in d i s t i l l e d w a t e r c o n t a i n i n g 5 ml*! d i t h i o t h r e i t o l ( D T T ) a n d c e n t r i f u g e d at 1 0 0 , 0 0 0 g for 45 m i n at 4 C. T h e o t h e r t i s s u e s w e r e p r e p a r e d in the s a m e w a y . I s o e l e c t r i c f o c u s i n g w a s p e r f o r m e d on L K B - 1 8 0 4 - P A G p l a t e s (pH 3.5 9 . 5 ) , w h i c h w e r e p r e f o c u s e d at 2 C for 30 min at 20 W a t t s w i t h the L K B - 2 1 1 7 - M u l t i p h o r a n d the L K B - 2 1 0 3 - P o w e r S u p p l y . A f t e r p r e f o c u s i n g , 10 - 20 p i of the s u p e r n a t a n t u a s a p p l i e d at the a n o d a l e n d on f i l t e r p a p e r . E l e c t r o f o c u s i n g w a s t h e n p e r f o r m e d for 70 m i n at 30 W a t t s . P o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s ( P A G E ) w a s c a r r i e d out in an 0 R T E C e l e c t r o p h o r e s i s a p p a r a t u s , u s i n g a 4 . 5 , 7 . 5 % s t e p w i s e g r a d i e n t gel in 1.5 CI T r i s - C i t r a t e b u f f e r pH 9 . 0 . 0 . 0 6 5 l»l T r i s - B o r a t e pH 9.0 w a s u s e d as the tank b u f f e r . B r o m o p h e n o l b l u e w a s u s e d as the t r a c k i n g d y e . E l e c t r o p h o r e s i s w a s p e r f o r m e d at 4 C at 300 V o l t s w i t h p u l s e r a t e s of 7 5 , 150, 225 p u l s e s / s e c f o r 5 m i n e a c h a n d t h e n 300 p u l s e s / s e c to the e n d of e l e c t r o p h o r e s i s . The P A G I F a n d PAGE g e l s w e r e s t a i n B d for L D H a c t i v i t y f o l l o w i n g the p r e v i o u s l y d e s c r i b e d p r o c e d u r e (9). LDH a c t i v i t y w a s m e a s u r e d u s i n g p y r u v a t e as the s u b s t r a t e ( 1 0 ) . The protein concentration was determined with Folin-Ciocalteu reagent(ll). The t h e r m a l s t a b i l i t y e x p e r i m e n t s w e r e c a r r i e d o u t in a c o n s t a n t t e m p e r a t u r e w a t e r b a t h at 55 C a n d 65 C for 5 a n d 15 m i n . T h e t i s s u e e x t r a c t s w e r e d i l u t e d in p r e w a r m e d 0.1 It T r i s - H C l , pH 7.6, c o n t a i n i n g

819 5 mfl D T T . The i n c u b a t i o n uas stopped by t r a n s f e r r i n g the tubes to an ice bath and the r e s i d u a l a c t i v i t y was i m m e d i a t e l y a s s a y e d .

R e s u l t s and

Discussion

The m u t a n t No. 1592 o r i g i n a t e d

in a study

sing was used for the d e t e c t i o n treatment with procarbazine. pattern

of LDH

compared

to the w i l d t y p e ,

closer

acidic

pole were less stained

to the basic

The m u t a n t was then paired offspring 1

(F^),

Plendelian g e n e t i c s , that the o r i g i n a l

©

in mice

exhibited

(Fig.

banding

i. e. a d d i t i o n a l b a n d s

(101XC3H)F^.

mouse

of u i l d t y p e s

that the m u t a t i o n

1 2 3 4

conclude,

is d o m i n a n t l y

1592 is h e t e r o z y g o t e .

5

6 7

8

ü i

•Pffj?

In

and m u t a n t types

I). We can t h e r e f o r e

m u t a n t Nr.

apthe

1).

uith a hybrid

(Table

focu-

after

an altered

pole of the gel and some b a n d s near

the p r o p o r t i o n

: 1, a p p r o x i m a t e l y

of gene m u t a t i o n s

The m u t a n t

peared

in which i s o e l e c t r i c

9 10

uas

following

inherited After

the

and

crossing

11 12

~

WHWm.

Fig. 1. S e p a r a t i o n of mouse liver l a c t a t e d e h y d r o g e n a s e by i s o e l e c tric f o c u s i n g (5 % P o l y a c r y l a m i d e gel, A m p h o l i n e pH 3.5 9.5). 10 ¿jl u n d i l u t e d t i s s u e e x t r a c t uas a p p l i e d on the gel. The gel was stained for LDH a c t i v i t y . ( 6 ) ( 7 ) ( 8 ) u i l d t y p e , (1 )(2)(3)(5)(9)(11 ) h e t e r o z y g o u s m u t a n t , ( 4 ) ( 1 0 ) ( 1 2 ) h o m o zygous mutant.

820 Table

I. D i s t r i b u t i o n of p r o g e n y in d i f f e r e n t c r o s s e s b e t w e e n types (li/t), h e t e r o z y g o u s m u t a n t s (Ht) and h o m o z y g o u s tants (Hm). No. of crosses

Cross

U t x Ht

No. of offspring

8

lilt

Ht

Hm

79

38

41

0

90

40

44

0

Ht x Ht

21

165

35

Hm x Wt

5

44

0

the h e t e r o z y g o t e s

one with a n o t h e r , ue c o u l d o b s e r v e a t h i r d k i n d of (Fig. 1, No.

b a n d i n g p a t t e r n in the h o m o z y g o t e s of the o f f s p r i n g ( 1 0 ) ( 1 2 ) ) , in a d d i t i o n to the w i l d t y p e (8)) and the h e t e r o z y g o t e

pattern

m u t a n t type p a t t e r n

(5)(9)(11)). After outcrossing type

(Fig. 1, No. (Fig. 1, No.

m u t a n t type p a t t e r n

(Table

(4)

(6)(7) (1)(2)(3)

the h o m o z y g o u s m u t a n t s with the

( 1 0 1 X C 3 H ) F ^ , we o b t a i n e d as e x p e c t e d only o f f s p r i n g

the h e t e r o z y g o t e

uildmu-

uild-

carrying

I).

PAGIF uas also p e r f o r m e d in k i d n e y , m u s c l e , h e a r t , brain, l u n g s p l e e n . The a l t e r a t i o n of the LDH b a n d i n g p a t t e r n c o u l d be

and

observed

in all the t i s s u e s e x a m i n e d and is a p p a r e n t l y e x p r e s s e d in the

same

manner. This alteration

( b a n d i n g s h i f t ) is c l e a r l y due to a charge

m o d i f i c a t i o n of the LDH

molecule.

PAGE u a s c a r r i e d out with the same t i s s u e s as a b o v e . In the ue c o u l d o b s e r v e that LDH-A

er and s p l e e n , w h e r e a s L D H - B p o l y p e p t i d e

p r e v a i l s in h e a r t ,

b r a i n and lung. F i g u r e 2 s h o u s the LDH e l e c t r o p h o r e t o g r a m er, m u s c l e , h e a r t and k i d n e y . It can be seen zyme c o r r e s p o n d i n g mouse types,

uildtypes,

p o l y p e p t i d e p r e d o m i n a t e s in m u s c l e ,

kidney,

from

(1) that the LDH-1

b a n d h a s the same s t a i n i n g i n t e n s i t y

in all

(2) that the b a n d s c o n s i s t i n g totally or partly

LDH-A p o l y p e p t i d e

have l e s s or no s t a i n i n g i n t e n s i t y in the

z y g o u s and h o m o z y g o u s

isothree

of hetero-

measured

(Table II). It a p p e a r s that the a c t i v i t y of LDH in the m u t a n t s c r e a s e s more in the t i s s u e s uhere LDH-A

predominates

and s p l e e n ) than in those w h e r e L D H - B p r e d o m i n a t e s lung).

liv-

mutants.

The s p e c i f i c a c t i v i t i e s of LDH from d i f f e r e n t t i s s u e s uere

b r a i n and

liv-

(liver,

(heart,

de-

muscle

kidney,

821

3 4

5

6

7 8

3

5

6

7

9 10 11 12

f©

B 1

2

4

««I «Hi IMP:,

1

2

8

9

10 11 12

© ©

M Ü | « f H l i ! » HSimS !»«(?«# -KSBi^

3

4

5

6

3

4

5

6

7

8

9

10

11

12

© ©

D 1

2

7

8

9

10

11

12

©

Fig. 2. Electrophoretic separation of lactate dehydrogenase from different mouse tissues (4.5,7.5 % stepwise gradient gel, gel buffer pH 9.0). Sample volume and gel staining are the same as in Fig. 1. (A) liver, (B) muscle, (C) kidney, (D) heart. (e)(7)(B) wildtype, (1)(2)(3)(5)(9)(11) heterozygote mutant type, (4)(10)(12) homozygote mutant type.

822 Table

II. L D H s p e c i f i c a c t i v i t y e x p r e s s e d in p e r c e n t in s e v e r a l t i s s u e s of u i l d t y p e s , h e t e r o z y g o t e a n d h o m o z y g o t e m u t a n t types.

Uildtype

Mutant Heterozygote

type Homozygote

Liver

100 + 9

59 + 7

Muscle

100 + 11

41 + 10

Spleen

100 + 4

47 + 1

9.0 + 1.5

Heart

100 + 1

77 + 6

35 + 10

6 . 5 + 2.7 12.5 + 6 . 5

Kidney

100 + 7

64 + 18

32.1 + 1.1

Brain

100 + 7

69 + 14

33.6 + 5.6

Lung

100 + 5

63 + 11

11.1 + 0.6

Table

III. R e s i d u a l LDH a c t i v i t y , e x p r e s s e d in p e r c e n t , a f t e r i n c u b a t i o n at d i f f e r e n t t e m p e r a t u r e s for v a r i o u s l e n g t h s of t i m e . T h e s p e c i f i c a c t i v i t i e s at 25 °C are t a k e n as 100 % .

55 °C

65 °C 15

Tissue

Mouse

Liver

Wild Heterozygote Homozygote

77 25 1

75 17 N.D.

36 1.5 0.9

3 0.5 N.D.

Muscle

Wild Heterozygote Homozygote

99 32 8

94 21 5

29 1 0.4

2 0.2 N.D.

Heart

Wild Heterozygote Homozygote

54 58 54

37 29 27

5.7 6.2 7.3

1.2 1.7 2.5

N.D.: not

type

determinable

5

min

15

min

5

min

min

823 F r o m the t h e r m a l s t a b i l i t y e x p e r i m e n t s

(Table

III), it can be

that (1) the LDH f r o m the h e a r t was much more heat s e n s i t i v e that of the m u s c l e or the l i v e r , dominates

(liver and m u s c l e ) ,

seen than

(2) in the t i s s u e s w h e r e LDH-A

the d e c r e a s e

of a c t i v i t y was much

premore

p r o n o u n c e d in the m u t a n t s than in the w i l d t y p e s w h e r B a s in the

heart,

the d e c r e a s e of LDH a c t i v i t y was a l m o s t i d e n t i c a l in the three

types

of m o u s e . From the PAGE, LDH s p e c i f i c a c t i v i t i e s and t h e r m a l s t a b i l i t y we can c o n c l u d e that only the l o c u s coding for l a c t a t e of the m u s c l e type is a f f e c t e d by the

studies,

dehydrogenase

mutation.

Ue are not able h o w e v e r to tell w h i c h k i n d of a l t e r a t i o n we are d e a l i n g w i t h , e x c e p t that it a f f e c t s the charge of the LDH-A p e p t i d e . T h e r e f o r e , we are p l a n n i n g to p e r f o r m and 2 - d i m e n s i o n a l PAGE on the tissue

poly-

Immunoelectrophoresis

samples.

The d e t e r m i n a t i o n of s e v e r a l o t h e r e n z y m e s in d i f f e r e n t t i s s u e s already in p r o g r e s s , to see w h e t h e r they are a f f e c t e d by the

is

muta-

tion or not. The g e n e t i c a l e x p e r i m e n t s a l l o w e d us to d e t e r m i n e

the m u t a t i o n

d o m i n a n t and to c l a s s i f y the m u t a n t s as h e t e r o z y g o t e s

or

homozygotes.

The p e n e t r a n c e of the m u t a t i o n is 100 % f o l l o w i n g the r e s u l t s in Table

as

shown

I.

C r o s s e s b e t w e e n h o m o z y g o u s m u t a n t s were c o n d u c t e d and the

progeny

had a n o r m a l l i t t e r size. The fact that mice h o m o z y g o u s for the tant allele of LDH-A a p p e a r to be fully v i a b l e and f e r t i l e

raises

some q u e s t i o n as to the m e t a b o l i c f u n c t i o n and p h y s i o l o g i c a l tance of this e n z y m e " i n

mu-

impor-

vivo".

Acknowledgements

The t e c h n i c a l a s s i s t a n c e of Cliss G. B o r g w e d e l and M i s s S. K a s c h greatly

appreciated.

is

82k References

1. B a t e m a n , A . 3 . : The d o m i n a n t l e t h a l a n d A c t i o n s _3, 7 3 - 7 6 ( 1 9 7 3 )

a s s a y in the m o u s e .

Agents

2. L e g a t o r , M . S . , M a i l i n g , H . V . : T h e h o s t - m e d i a t e d a s s a y , a p r a c t i cal p r o c e d u r e f o r e v a l u a t i n g p o t e n t i o n a l m u t a g e n i c a g e n t s in m a m m a l s . In: H o l l a e n d e r , A. ( E d . ) , C h e m i c a l M u t a g e n s - P r i n c i p l e s a n d M e t h o d s for T h e i r D e t e c t i o n , V o l . 2, p. 5 6 9 - 5 8 9 , P l e n u m P r e s s , N e u Y o r k , L o n d o n 1971 3. C o h e n , M . M . , H i r s c h h o r n , K . : C y t o g e n e t i c s t u d i e s in a n i m a l s . In: H o l l a e n d e r , A. ( E d . ) , C h e m i c a l M u t a g e n s - P r i n c i p l e s a n d M e t h o d s for T h e i r D e t e c t i o n , V o l . 2, p. 5 1 5 - 5 3 4 , P l e n u m P r e s s , N e u Y o r k , L o n d o n 1971 4. E h l i n g , U . H . : S p e c i f i c - l o c u s m u t a t i o n s in m i c e . In: H o l l a e n d e r , A . , de S e r r e s , F . 3 . ( E d s . ) , C h e m i c a l M u t a g e n s - P r i n c i p l e s a n d M e t h o d s for T h e i r D e t e c t i o n , V o l . 5, p. 2 3 3 - 2 5 6 , P l e n u m P r e s s , N e u Y o r k , L o n d o n 1978 5. K l o s e , 3 . : The p r o t e i n - m a p p i n g - m e t h o d e m p l o y e d to test for c h e m i c a l l y i n d u c e d p o i n t m u t a t i o n s in m i c e . A r c h . T o x i c o l . _38, 5 3 - 6 0 (1977) 6. M a i l i n g , H . V . , V a l c o v i c , L . R . : N e u a p p r o a c h e s to d e t e c t i n g g e n e m u t a t i o n s in m a m m a l s . I n : F l a m m , W . G . , M e h l m a n , M . A . ( E d s . ) , A d v a n c e s in M o d e r n T o x i c o l o g y , V o l . 5 M u t a g e n e s i s , p. 1 4 9 - 1 7 1 , 3 o h n W i l e y & S o n s , N e u Y o r k , L o n d o n , S i d n e y , T o r o n t o 1978 7. N a r a y a n a n , K . R . , Raj, A . S . : A m e t h o d for s i m u l t a n e o u s a n a l y s i s of s e v e r a l g e n e t i c l o c i in m i c e a f t e r e l e c t r o f o c u s i n g . I n : Radola, B.3., Graesslin, D. (Eds.), Electrofocusing and Isotac h o p h o r e s i s , p. 2 2 1 - 2 3 1 , W a l t e r de G r u y t e r , B e r l i n , N e u Y o r k 1977 8. P r e t s c h , W . , N a r a y a n a n , K . R . : E r f a s s u n g von G e n m u t a t i o n e n b e i M ä u s e n d u r c h i s o e l e k t r i s c h e F o k u s s i e r u n g . H o p p e - S e y l e r 1 s Z. P h y s i o l . C h e m . 360, 345 ( 1 9 7 9 ) 9. H a r r i s , H., H o p k i n s o n , D . A . : H a n d b o o k of E n z y m e E l e c t r o p h o r e s i s in H u m a n G e n e t i c s , N o r t h - H o l l a n d P u b l i s h i n g C o m p a n y , A m s t e r d a m , O x f o r d 1976 10. B e r g m e y e r , H . U . , B e r n t , E . : U V - A s s a y u i t h p y r u v a t e and N A D H . I n : B e r g m e y e r , H . U . ( E d . ) , M e t h o d s of E n z y m a t i c A n a l y s i s , V o l . 2, p. 5 7 4 - 5 7 9 , V e r l a g C h e m i e , W e i n h e i m 1974 11. B e r g m e y e r , H . U . , B e r n t , E . , G a u e h n , K., M i c h a l , G . : H a n d l i n g of biochemical reagents and samples. In: Bergmeyer, H.U. (Ed.), M e t h o d s of E n z y m a t i c A n a l y s i s , V o l . 1, p. 1 5 8 - 1 7 9 , V e r l a g C h e m i e , W e i n h e i m 1974

VERTEBRATE MITOCHONDRIAL 55S RIBOSOMES: COMPARISON OF PROTEIN PATTERNS WITH BACTERIAL AND CYTOPLASMIC R-PROTEINS BY 2-D PAGE

W. Czempiel and B. Ulbrich Institut f ü r Toxikologie und Embryopharmakologie, Freie Universität Berlin D-1000 Berlin 33, West Germany

Introduction Mammalian cells contain two types of protein synthesising systems. One, consisting of 80S ribosomes, is located in the cytoplasm and is responsible for the translation of the major portion of cell proteins including the ribosomal proteins necessary for the other system. Only a relatively small number of proteins is synthesised by the second system which is located inside the mitochondria. The mitochondrial system employs 55S ribosomes which resemble bacterial ribosomes in size and react in a similar way to inhibitors of procaryotic protein synthesis (1, 2). These observations raise the question about the origin of mitochondria and their ribosomes. To date two theories on the evolution of mitochondria have been put f o r t h . One states that these cell organelles arouse from compartmentation in a proto-eucaryotic cell. Ribosomes trapped inside the compartment then diverged from the cytoplasmic ribosomes during the evolution of the eucaryotic organism.

The second hypothesis views mitochondria as former pro-

caryotic parasites of eucaryotic cells which have become endosymbionts, transfering during this process most of the information needed for their own reproduction to the genome of the host (cf. 3). Analysing mitochondrial, cytoplasmic, and bacterial r-proteins by the same electrophoretic technique we have tried to gain information on the composition of the ribosomal particles. Comparison of the r-protein patterns may present some evidence on the relationship of mitochondrial ribosomes with the other ribosomal classes.

© 1980 Walter de Gruyter & Co., Berlin • New York Electrophoresis '79

826 Methods Isolation of Ribosomes and Ribosomal Proteins 55S mitochondrial ribosomes were isolated from digitonin-treated mitochondria prepared from r a t , pig, or chicken livers, respectively. After lysis of the mitoplasts with 1.3% Triton X-100 in KMEDT-buffer (100 mM KC1, 10 mM MgCl 2 , 0 . 1 mM EDTA, 1 mM DTE, 2 mM TES-KOH pH 7.6) crude ribosomes were prepared by centrifugation through a sucrose cushion (1.3 M sucrose, 20 mM Tris-HCl pH 7.6, 350 mM NH4C1, 20 mM Mg(CH 3 COO) 2 , 1 mM EDTA, 2 mM 2-mercaptoethanol [MSH]). 55S ribosomes were purified on 10-30% sucrose gradients in KMEDT-buffer (4). 80S poly-ribosomes were prepared from r a t or pig liver according to (5). Livers were homogenised in 2.5 vol. of b u f f e r A (0.2 M sucrose, 100 mM NH4C1, 5 mM Mg(CH 3 COO) 2 , 1 mM DTE, 20 mM Tris-HCl pH 7 . 6 ) . After clarification

at

10,000xg

the

supernatant was treated

with 1.3% Triton

X-100 and ribosomes were purified by sedimentation through a discontinous sucrose gradient (1.0 M; 1.8 M sucrose) in b u f f e r A. For the isolation of 70S ribosomes E.coli K12(A19) cells were disrupted by grinding with alumina. The homogenate was diluted with TMNSH-buffer (10 mM Tris-HCl pH 7.6, 10 mM Mg(CH 3 COO) 2 , 60 mM NH 4 Cl, 2 mM MSH. Ribosomes were collected by centrifugation a f t e r removal of cell debris (6). Ribosomal proteins were extracted with acetic acid, dialysed against 1% acetic acid and lyophilised. Two-dimensional electrophoresis Separation of proteins in the f i r s t dimension was performed by IEF in urea in a 4.8% T gel essentially as described in (7). However, two modifications were introduced in the separation procedure. The content of basic ampholytes was raised by addition of 0.2% 9-11 Ampholines (LKB). Isoelectric separation was performed for only 3.5 h at a constant output of 0.03 W/gel. pH-gradients were calculated a f t e r cutting the 1-D gel into 0.5 cm slices, elution of ampholytes with 0.5 ml 10 mM KCL/6 M urea and pH measurement at 25°C. Second dimension electrophoresis was performed in a 19.2% T slab gel in 6 M urea for 18 h at 60 V as described earlier (8).

827

Results The protein moiety of v e r t e b r a t e mitochondrial ribosomes consists of 70-75 p r o t e i n s . Roughly 2/3 of them belong to the large ribosomal s u b u n i t . Estimations of the molecular weight distribution based on E.coli r - p r o t e i n s as marker proteins revealed that at least 25% of the mitochondrial r - p r o t e i n s are l a r g e r than 30,000 D. Molecules smaller than 10,000 D were only o b s e r ved in the r - p r o t e i n s from chicken (Table 1).

Ribosomes

No of proteins

TP 70 E. coli

Molecular Weights

53

65,000 -

5,000 D

TP 55 chicken

70-73

50,000 -

8,000 D

TP 55 pig TP 55 r a t

70-75

51,000 - 10,000 D

70

52,000 - 10,000 D

TP 80 pig TP 80 r a t

70

28,000 -

5,000 D

70

28,000 -

4,500 D

Table 1

The mitochondrial

R-protein composition of 55S, 70S and 80S ribosomes. Molecular weights were estimated from the distance of migration in the second dimension.

ribosomes investigated

contain 3-4 acidic proteins

(pi

5 - 6 ) , of which two a r e derived from the large s u b u n i t . All other proteins are localised mainly in the n e u t r a l to basic region of the gel.

Strongly

basic proteins (pi >9.5) are missing (Fig. 1 - 3 ) . E.coli r - p r o t e i n s display a higher basicity than the mitochondrial p r o t e i n s . They contain two acidic proteins (EL7/L12, pi ~5.0) and small basic p r o teins (MW S9 S11 LI 7 s

»'o^lgpSI L250

^

OSH

S17S,800S.9

OS» L290 L 31 O

L

"0

L30 O

g, ^ Os21

L32 L33

o o

Fig. 4 Schematic drawing of the protein pattern from E.coli 70S ribosomes.

830 . ?

. 9

.

10

i

7

ooO cf

o

O

O 0 Fig. 5 Schematic drawing of the r-protein pattern from 80S cytoplasmic ribosomes. 70 spots are discernible. Beside the above mentioned acidic proteins

(MW ~22,000 D ) mitochondrial

r-protein patterns seem to contain several conserved features. A chainlike array of spots is observed between the acidic proteins and the basic region of the gel. The proteins involved (PL14, PL15, PL17, PS16 in the pig and RL15,

RL21,

RS14,

RL22,

RS13 in the r a t )

have molecular weights of

17,000-19,000 D. A similar arrangement in the E.coli pattern is formed by ES6, EL9, and EL10. On the basic side of the gel proteins of a MW of 22,000-25,000 D are grouped in a crescent-shaped formation. In pig this includes the proteins PS16, PL12, PL11, PS9, PL7/S10, PL8, PL13.

The

corresponding

group

PS11, and

in the rat pattern is formed by

RS13,

RL11, RL7, RL13, RS8, RL17, and RL25. Below this arch a horizontal line is formed by the proteins PS14, PS15, PL18, PL16, and PL19 or RL24, RL19,

RL23, and RS15, respectively.

Below this area the gel contains a

zone which is relatively free of proteins. This zone is also present in the E.coli pattern. A second curve is composed of PL27, PS20, PL25, PS21, PL26, PS24, PL33, and

PS27.

RL28,

Comparable

groups are formed by RL31, RS20, RL27,

RS21,

RS23, RS24, and RL32 in the rat and by EL15, EL16, ES8, EL13,

831 EL14, and ES9 in the E.coli p a t t e r n . A vertical linear arrangement including PL3, PL7/S10, PL18, PL20, and PS20, RL1, RL7, RL16, and RS20 or ELI, EL2, EL5, and ES8 is seen at pH ~8.4 in the mitochondrial and at ~8.6

in the bacterial p a t t e r n .

Corresponding

groups are found in

the

chicken pattern as well. No similarities were found between mitochondrial r - p r o t e i n s and the corresponding cytoplasmic r - p r o t e i n s .

As cytoplasmic r-proteins from different

species exhibit only minimal variations the pattern

of pig proteins was

omitted.

Discussion Ribosomal proteins from vertebrate mitochondria display

several

common

characteristics when separated by 2-D electrophoresis. In addition to the overall arrangement of spots apparently homologous proteins can be specified from the p a t t e r n s . (9) we observed

In agreement with the results of Matthews et al.

significant variations in the p a t t e r n s of

r-proteins from different sources. are much less conserved

mitochondrial

It seems that mitochondrial r - p r o t e i n s

than their cytoplasmic equivalents which show

only minor divergences among higher vertebrates (10, 12). So f a r the significance of different evolutionary rates of two sets of ribosomal proteins both coded by the nuclear genome is unknown. We can speculate that mitochondria are a fairly recent acquirement and therefore not yet fully adapted to their environment or that the environment is subject to constant change so instead of being eliminated in favour of the traditional system mutant proteins could multiply. Although cytoplasmic and mitochondrial r-proteins are p r e s e n t in the same organism they produce entirely different p a t t e r n s in the 2-D electrophoresis. The high basicity of the 80S proteins contrasts sharply to the more neutral 55S proteins. No similarities can be detected. Mitochondrial r-proteins on the contrary seem to have more in common with bacterial r - p r o t e i n s .

Despite the E.coli 70S proteins being slightly more

basic than 55S proteins they can be regarded to be remotely related to the

832 latter by similarities in protein distribution. Correspondence in amino acid sequence, however, cannot be deduced from similar positions in the gel so that further investigations with immunological methods or sequence analysis are necessary.

Functional homologies have to be verified by heterologous

reconstitution of single proteins with a ribosomal core particle. The electrophoretic technique employed in this study serves to separate ribosomal proteins of different provenance and is especially suited for the separation of small amounts.

Due to cathodic drift and the decay of pH

gradient basic proteins may not reach a stable position in the gel so that more importance was attached to an optimal separation of the

proteins

rather than to complete isoelectric focusing.

Acknowledgements This work was supported by grants from the Deutsche Forschungsgemeinschaft awarded to Sonderforschungsbereich 29 (Embryopharmakologie). The expert technical assistance of Mrs. Andrea Schmiedel is appreciated.

References 1.

O'Brien, T.W.: Handbook of Genetics, Vol. King e d . ) Plenum Press, New York 1976.

5 pp.

535-580,

(R.C.

2.

Denslow, N.D., O'Brien, T.W.: Eur. J . Biochem. 91, 441-448 (1978).

3.

Reijnders, L . : J . Mol. Evol. 5, 167-176 (1975).

4.

Czempiel, W.,

5.

Falvey, A . K . , Staehelin, T . : J . Mol. Biol. 53, 1-19 (1970).

6.

Schreiner, G., Nierhaus, K . H . : J . Mol. Biol. 81, 71-82 (1973).

7.

Czempiel, W. : Electrofocusing and Isotachophoresis, Walter de Gruyter, Berlin-New York 1977.

Klose, J . ,

Bass,

R.:

FEBS Lett.

62, 259-262 (1976).

pp.

405-412,

8.

Kaltschmidt,

9.

Matthews, D . E . , Hessler, R . A . , O'Brien, T.W.: FEBS Lett. 86, 76-80 (1978).

10. Delauney, J . , 312 (1973).

E . , Wittmann, H.G.: Anal. Biochem. 36, 401-412 (1970).

Creusot, F . ,

11. Ramjoué, H . - P . ,

Schapira, G. : Eur. J . Biochem. 39, 305-

Gordon, J . :

J.

Biol. Chem. 252,

9065-9070 (1977).

ISOELECTRIC PEROXIDASE PATTERNS.AS INDICATORS FOR GROWTH AND FOR THE EFFECT OF GROWTH REGULATORS IN TOBACCO TISSUE CULTURES

W. Rücker and J. Markotai Pharmakognostisches Institut der Universität Wien A-1090 Vienna, Austria

Introduction Tobacco tissue cultures show a high sensitivity to the influence of growth substances. This sensitivity, expressing itself in general by changes of growth and differentiation (1, 2), can be demonstrated especially by changes in the activity of several zones in the peroxidase patterns (3-5)• The objective of this investigation is to assess some possible connections between alterations of growth and peroxidase isoenzyme patterns under the influence of different growth hormones. The tests were conducted on two stocks of tobacco tissue cultures which were kept under different growth conditions. These moot sensitive stocks, stabilized by long lasting cultivation, provide us with especially suitable experimental material for testing the effects of growth substances.

Material and Methods The influence of the following growth substances on callus differentiation and isoelectric peroxidase patterns has been tested: indoleacetic acid (IAA), gibberellic acid A3 (GA3) and benzylaminopurine (BAP). These are representatives of three important groups of growth regulators: the Auxins, the Gibberellins -9 from 10

and the Cytokinins.

g/ml to 10

g/ml. These substances were tested either individually

or in different combinations.

© 1980 Walter de Gruyter & Co., Berlin • New York Electrophoresis '79

The concentration range extended

&3h Biological material: The stock cultures used stemmed from the pith tissue of Nicotiana tabacum var. Wisconsin and were maintained in Murashige and Skoog medium (6). They were kept in the dark and under the continual influences of either I M & BAP 10 g/ml -stock b- or 2,4-dichlorphenoxyacetic acid (2,4 D) 10~6g/ml -stock c- (Fig.l). Experimental conditions: During the experiments for testing these different phytohormones, the tissues were exposed to a 12 hour day-night illumination rhythm. For each of the experiments 12 or 24 replicates were used. The duration of the experiments was six weeks for the tissue of the b-stock and four weeks for the tissue of the c-stock. The callus growth was estimated by measuring the terminal weights of the fresh and the dry tissue and substracting these values from the initial tissue values. Gonditions of isoelectric focusing: For the enzyme analyses, the cultures were lyophilized and 200 mg of powdered tissue was dissolved in 1.0 ml E^O, pressed through miracloth and centrifuged. Ten or twenty ul of the sap had been used for the enzyme separation. The amounts of the tissue to be analysed corresponded to two or four mg of dry tissue respectively. The peroxidase systems were separated by isoelectric focusing (IEF) in layers of granulated gels. This was performed with a carrier arrpholite: Ampholine pH 3.5 - pH 10 and with Sephadex G 75 superfine on 20 x 20 cm glass plates. The peroxidase patterns were detected by taking a print with buffered paper impregnated with a methanol solution containing 1% peroxide and 1% o-dianisidine (7~9).

Results and Discussion Each series of the growth hormone experiments will be reported separately depending whether they are individually applied or applied in combinations with each other. The influence of a single application of the following growth substances: -7 I M , GA3 and BAP, in a concentration range of 10 'g/ml to 10 ^g/ml. The phytohormones euployed elicit different morphological and biochemical influences. The two tissue stocks respond to the hormone influence with sim-

835 ilar growth and peroxidase patterns. However, there were some quantitative differences, probably resulting from different storage of growth substances and from different cytokinin dependency (10). 1)Callus growth Under the influence of I M , the growth of both tissue strains is increasingly stimulated. The optimal concentration in the c-stock is -S

-6

10 g/ml I M (Pig. 2) and in the b-stock 10 g/ml I M . Under the influence of GA3, there is a tendency towards tissue growth inhibition (Fig.2). Despite great differences in the growth results achieved in both tissue strains, a similar inhibition is able to be recognized. This similarity can be identified by the course of the growth curves and from the growth optima which in both cases are achieved by -7

10 g/ml GA3- An optimal GA3 concentration exerts strong stimulatory effects in the c-stock while the b-stock develops only weakly. This is possibly due to a growth stimulating after effect of 2,4 D (11, 12). Under the influence of BAP, the dose-effect-relations resemble those after the GA3 treatment. This resemblance in the course of growth inhibition is striking in the c-stock tissue (Fig.2). In the b-stock tissues, by means of budding, to which only this tissue strain is able to do, there occurs an additional tissue production such that the inhibition of callus growth cannot be detected appropriately by the determination of dry weight. 2)Isoelectric peroxidase patterns The isoelectric enzyme separation of tobacco tissue, with a pH gradient of 3-5 to 10, reveals several zones of peroxidase activity. According to their response to the influence of growth regulators, they can be separated into functional groups (13, 14). The zones of group A and B are closely connected in the acidic pH range while group C is located in the basic range. The zones of group C are rather unstable, therefore, in the following we will confine our observations to the enzyme groups A and B only.

836

^ O

A

A

(

.... "

»

m

h

^

> Wi

Fig.l: Tobacco tissue cultures and their isoelectric peroxidase patterns. The tissues of stock b were rather compact. They undergo a slower growth rate and require cytokinin for their growth. The tissues of stock c are cytokinin independant and only consist of a semi friable callus and grow relatively fast. In stock b the enzyme group A and B exhibit several intensively marked zones. In contrast, stock c shows only a few more or less observable zones in the peroxidase pattern.

Fig.1 represents both tissue culture stocks used. In dependence upon the influence of different growth substances during the cultivation period, not only growth and morphology, but also the peroxidase patterns of the two stocks are different. However, in both cases the isoenzyme pattern characteristic for tobacco callus tissue is maintained. Stc IAA 7

BAP

GA 3 5

10 .10 ?10" 10"

7

10

6

io"

5

7

10'

10"6 10~5 1_A ± B

i* H m ©

1

^mm

CONTROL

Fig.2: Growth curves and isoelectric peroxidase patterns of the tobacco tissue culture stock c after increasing the concentration of IAA, GA3 or BAP. In comparing the growth curves with the peroxidase patterns it is shown that the occurance and the intensity of the first two zones in the acid pH range (enzyme group A) are in strong correlation with tissue growth. In tissues that are growth inhibited, these zones are less intensively expressed or do not appear at all. The cytokinins are solely responsible for the increase of the intensity in the zones of the Bgroup in the isoelectric peroxidase patterns.

837 After the transferal of tissue fragments onto the new media, changes occur in conformity to the new growth and hormone conditions. The alterations in the peroxidase patterns partly reveal a strong correlation with growth (enzyme group A). This effects is independent from growth horax>nes which cause growth stimulation or inhibition. Partly they also express the amount of applied cytokinin (enzyme group B). Independent of growth, the intensity of the zones in group B increase together with the BAP-concentration. Following the application of I M and GA3, however,the appearance of these latter zones is inhibited (Fig.2). On the basis of morphological and biochemical results, certain zones of the isoelectric pattern can be considered as functional groups. In addition, it is shown that the intensity of certain zones allows us to decide whether or not the tissues are optimally stimulated or inhibited by means of growth hormones. Experiments with hormones used in the following combinations:IAA & BAP, IAA & GA3, GA3 & BAP, in the concentration range of 10~9g/ml to 10 _5 g/ml. The purpose of these experiments is to show what will occur when two hormones are applied at the same time. This simultaneous effect will express itself in possible changes in tissue growth or peroxidase patterns. BAP takes a dominating role in the combination of IAA & BAP. Growth and peroxidase patterns are similar to the results that are characteristic of cytokinin action -striking growth inhibition in higher concentrations and in correlation with the observed growth inhibition, a decrease in the intensity of the zones of the A-group and an additional underscoring the zones of the B-group- (Fig.2 and 3)- In any case, one cannot exclude the influence of exogenously applied IAA on BAP action. This, however, is not shown in the growth and peroxidase patterns, since the bud differentiation in the b-stock under the BAP treatment is decreased through IAA and inhibited totally in high IAA concentration. In the combination of GA3 & BAP, GA3 influences the BAP action on growth and peroxidase. A comparison of the results from experiments with BAP alone and in combination with GA3 shows that GA3 weakens the growth inhibiting action of BAP (Fig.4). This effect produces a rather uniform growth and

838 makes a well pronounced appearance in the zones of the A-group in the isoelectric peroxidase patterns. Exceptions are found only in the combination -9 of 10

g/ml of both growth hormones. This tends to have only a weak stimu-

lation on growth and is evident in all combinations with higher BAP concentration that inhibit growth. In these instances the zones mentioned are either less pronounced or not visible at all. The occurence of the characteristic BAP zones (enzyme group B) is either strongly attenuated or totally inhibited. In the combination experiments of IAA & GA3 there is a strong uniform growth pattern over the entire concentration range. This indicates mutual stimulatory actions of both growth hormones. With the simultaneous presence of GA3, the cells are able to exhibit optimal growth at the already low IAA concentration. The growth stimulating action of higher IAA concentrations is not additionally effected by GA3- However, the observed GA3 growth inhibition appears to be compensated by the influence of IAA. In the peroxidase patterns, the uniform growth is acconpanied by a strong uniform intensity in the zones of the A-group. Results of the growth and enzyme determinations allow us to infer a regulatory mode of action of GA3 (15)- When singly applied, the GA3 effect becomes visible through the accentuation of the first zones in the A-group. In the combination experiments, GA3 has a definite stimulating effect on growth action. The stimulating action of IAA is pronounced through the concomitant presence of GA3. However in combination with BAP, the inhibitory action of BAP is decreased by GA3In contrast to these results, BAP in combination with IAA impedes the action of exogenously applied IAA. The reason is probably due to an increased rate of auxin breakdown by cytokinin, whereby the exogenously applied IAA is trapped and not able to participate in the physiological growth processes. The results of our experiments demonstrate a close correlation between the intensity of special zones in the peroxidase patterns (group A) with growth. By just glancing at the peroxidase patterns, it is possible to see if the added growth substances produced stimulation or inhibition in the growth

839 BAP g / m l

FIG. 3

TO"' nr8 10"' 10"' io

10

© I- •

«SM «Hi

*

10'

Î.A 18

© ©!

BAP g/ml

O

• DIMINISHED

GROWTH (