259 38 29MB
English Pages 313 [320] Year 1971
Proceedings of the International Research Conference on Proteinase Inhibitors, Munich, Nov. 1970
Proceedings of the
International Research Conference on
Proteinase Inhibitors Munich, November 4—6, 1970
edited by H . FRITZ • H . TSCHESCHE With 246 figures and 95 tables
W G DE
Walter de Gruyter - Berlin • New York 1971
Tbe quotation of registered names, trade names, trade marks, etc. in this book does not imply, even in tbe absence of a specific statement, thai tucb names are exempt from laws and regulations protecting trade markst etc. and therefore free for general use.
ISBN 3 11 003776 9 Library of Congress Catalog Card Number 70-168659 © Copyright 1971 by Walter de Gruyter & Co., Berlin All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form — by photoprint, mikrofilm, or any other means — nor transmitted nor translated into a machine language without written permission from the publisher. Printed in Germany by Walter de Gruyter & Co., Berlin. Cover designed by U. Hanisch, Berlin.
Preface
It was the aim of the First International Research Conference on Proteinase Inhibitors, held in Munich on November 4—6, 1970, to review the present status of the field, to discuss the recent advances in our knowledge on isolation, structure and mechanism of interactions between proteinases und protein proteinase inhibitors and to promote the research by mutual exchange of experience. We are beginning to understand the physiological significance of this class of proteins, which was substantiated by the primary and tertiary structures elucidated and the theory of protein-protein interaction presented. The present volume records all of the lectures and those papers and discussion remarks that contributors submitted for publication. We wish to express our gratitude to all participants for their contributions and for their open and cordial discussions. We are especially grateful to contributors which were unable to attend the Conference. We have to apologize for the limitations to the number of participants for this First Conference. The widespread and general interest and the anticipated future progress will perhaps stimulate the organization of a second conference. We would therefore appreciate any relevant suggestion submitted. On this occasion we wish to express our thanks to the following organizations we are indebted to for financial support which made possible the arrangement of this Conference: Behringwerke AG, Marburg; C. H. Boehringer & Sohn, Ingelheim; Boehringer Mannheim GmbH, Tutzing; Farbenfabriken Bayer AG, Wuppertal-Elberfeld; Farbenfabriken Hoechst AG, Frankfurt; E. Merck AG, Darmstadt; Novo Industrie, Mainz. The organization of the program was further supported by Bayer-Leverkusen, Miinchen; Beckman Instruments, Miinchen; Rohm & Haas GmbH, Darmstadt; Schering AG, Berlin. Munich, May 1971 The Organizing Committee H A N S FRITZ, K A R L HOCHSTRASSER, EUGEN W E R L E
Universität München
HARALD TSCHESCHE
Technische Universität München
Contents
List of Contributors List of Participants
x xi
Proteinase Inhibitors in Human Medicine N . HEIMBURGER, H . H A U P T , H . G . S C H W I C K
: Proteinase Inhibitors of Human Plasma
1
G. F. B. S C H U M A C H E R : Sex Hormones and Serum Proteins 21 M . H. COAN, J . T R A V I S : Interaction of Human Pancreatic Proteinases With Naturally Occurring Proteinase Inhibitors 294 E.
WERLE
: Proteinase Inhibitors in Clinical Studie
Specific Isolation
23
Methods
M. M Ü L L E R , M. G E B H A R D T : Specific Isolation and Modification Methods for Proteinase Inhibitors and Proteinases 28 G . FEINSTEIN: Isolation of Chymotrypsin Inhibitors by Affinity Chromatography through Chymotrypsin-Sepharose 38 B. KASSELL, M . B. MARCINISZYN : A Simple Method of Purification of the Basic Trypsin Inhibitor of Bovine Organs 4 3 H . FRITZ, B . BREY,
Inhibition Mechanism: Theories and Methods D . SHOTTON:
The Molecular Architecture of the Serine Proteinases
M . LASKOWSKI, J r . , R . W . D U R A N , W . R . FINKENSTADT, S . H E R B E R T , H . F . HIXSON, J r . , D . K O W A L S K I , J .
47 A.
J. A. M A T T I S , R . E. M C K E E , C. W . N I E K A M P : Kinetics and Thermodynamics of Interaction Between Soybean Trypsin Inhibitor (Kunitz) and Bovine ß Trypsin 117 H. TSCHESCHE, R. OBERMEIER : Mass Spectral Determination of Peptide Bond Hydrolysis Equilibria in Protein Proteinase-Inhibitors 135 M. LASKOWSKI, Jr.: Comments to the Modification Reaction 141 LUTHY,
R.
E. FEENEY: The Non-Bond Splitting Mechanism of Action of Inhibitors of Proteolytic Enzymes — the Conservative Interpretation 162
H . TSCHESCHE, H . KLEIN, G . REIDEL:
On the Mechanism of Temporary Inhibition
299
viii
Contents
Inhibitors of Plant Tissues T. IKENAKA, T. KOIDE, S. ODANI: Structure and Chemical Modification of Kunitz Soybean Trypsin Inhibitor . . 108 I. E. LIENER : Chemical Studies on the Site of Interaction between Trypsin and Kunitz Soybean Trypsin Inhibitor (STI)
156
Y. BIRK, A. GERTLER : Chemistry and Biology of Proteinase Inhibitors from Soybeans and Groundnuts . . . .
142
F. C. STEVENS : Lima Bean Protease Inhibitor: Amino Acid Sequence and Active Sites against Trypsin and Chymotrypsin
149
K. HOCHSTRASSER, E. WERLE: Sequential Studies on Proteinase Inhibitors Isolated by Use of Trypsin Resins. . 169 R. E. FEENEY: Comparative Biochemistry of Avian E g g White Ovomucoids and Ovoinhibitors
189
C. A. RYAN, L. K. SHUMWAY : Studies on the Structure and Function of Chymotrypsin Inhibitor I in the Solanaceae Family 175
Basic Bovine Inhibitor and Related Inhibitors R . HUBER, D . KUKLA, A . RUHLMANN, W . STEIGEMANN : T h e A t o m i c S t r u c t u r e of t h e B a s i c T r y p s i n I n h i b i t o r of
Bovine Organs. (Kallikrein Inactivator)
56
M. LASKOWSKI, J r . : Comments to the Reactive Site M . LASKOWSKI, S r . ,
S.L.SCHNEIDER,
64
K.A.WILSON, L.F.KRESS,
J . H . MOZEJKO, S . R . M A R T I N ,
U . KUCICH,
M.ANDREWS : Naturally OccurringTrypsinInhibitors: Further Studies on Purification andTemporary Inhibition
66
M. RIGBI : Studies on the Reactive Site towards Chymotrypsin and Trypsin of the Basic Trypsin Inhibitor of Bovine Pancreas
74
M. LASKOWSKI, J r . : Modification of Soybean Trypsin Inhibitor by A-Chymotrypsin
88
B. KASSELL, TSUN-WEN WANG : The Action of Thermolysin on the Basic Trypsin Inhibitor of Bovine Organs .
89
V. KEIL-DLOUHA, J.-M. IMHOFF, B. KEIL: On the Mechanism of the Interaction between Basic Pancreatic Trypsin Inhibitor and Trypsin D. CECHOVA, V. JONAKOVA, F. SORM: Amino Acid Sequence of Trypsin Inhibitor from Cow Colostrum . . . .
Pancreatic Secretory
95 105
Inhibitors
L . J . GREENE, M. H. PUBOLS: Isolation of a Secretory Trypsin Inhibitor from Human Pancreas
196
L . J . GREENE, O. GUY: Disulfide Bridges of the Bovine Pancreatic Secretory Trypsin Inhibitor — Kazal's Inhibitor 201 H . TSCHESCHE, E . W A C H T E R , S . KUPFER, R . OBERMEIER, G . REIDEL, G . HAENISCH, M . SCHNEIDER: S t u d i e s
on
Structure and Activity of Pancreatic Secretory Trypsin Inhibitors from Pig, Sheep, Dog and Cat L. J . GREENE,D. C. BARTELT: Porcine Pancreatic Secretory Trypsin Inhibitor I, Amino Acid Sequence
Seminal Inhibitors, Inhibitors and
207 . . . .
223
Fertilisation
E . FINK, G. KLEIN, F . HAMMER, G . MÜLLER-BARDORFF, H . FRITZ : P r o t e i n P r o t e i n a s e I n h i b i t o r s i n M a l e S e x G l a n d s 225 L . J . D . ZANEVELD, K . L . POLAKOSKI, R . T . ROBERTSON, W . L . W I L L I A M S : T r y p s i n I n h i b i t o r s a n d F e r t i l i z a t i o n
236
Contents G. F. B.
SCHUMACHER :
at-Antitrypsin
ix
in Seminal Fluid
245
H. INGRISCH, H. HAENDLE, E. WERLE: Inhibition by the Trypsin-Plasmin-Inhibitor from Sperm Plasma of the Dispersion of the Corona Radiata and Zona Pellucida by a Trypsin-like Enzyme from Spermatozoa . . . . 244 G. F. B. SCHUMACHER, J. R. SWARTWOUT, F. P. ZUSPAN: Fertility Experiments in Mice and Rabbits with the Trypsin-Kallikrein Inhibitor from Bovine Lung 247 G. F. B. SCHUMACHER: Alphaj-Antitrypsin in Uterine Secretions
253
H. HAENDLE, H. INGRISCH, E. WERLE: A Trypsin-Chymotrypsin Inhibitor in Human Female Cervix Secretion. 256
Inhibitors from Dog Submand. Glands, Ascaris Lumbricoides and Leeches H . FRITZ, E . JAUMANN, R . M E I S T B R , P . PASQUAY, K . HOCHSTRASSER, E . F I N K : P r o t e i n a s e I n h i b i t o r s f r o m
Dog
Submandibular Glaiîâs — Isolation, Amino Acid Composition, Inhibition Spectrum R. J. PEANASKY, G. ftf. ABU-ERREISH: Inhibitors from Ascaris Digestive Enzymes . _
Lumbricoides'.
257
Interactions. With the Host's 281
H. FRITZ, M. GEBHARD.T,: R. MEISTER, E. FINK : Trypsin-Plasmin Inhibitors from Leeches — Isolation, Amino Acid -, 271 Composition, Inhibitory Characteristics .
List of Contributors
G . M . A B U - E R R E I S H , D . C . B A R T E L T , Y . B I R K , B . B R E Y , M . H . COAN, D . C E C H O V A , R . W . D U R A N , V . D L O U I I Ä - K E I L , R . E . FEENEY, O . GUY,
G . FEINSTEIN,
M . HAENDLE,
K . HOCHSTRASSER,
E.FINK,
G . HAENISCH,
R . HUBER,
E . R . FINKENSTADT, F . HAMMER,
T . IKENAKA,
H.FRITZ,
H . HAUPT,
M.GEBHARDT,
N . HEIMBURGER,
A . GERTLER,
S . HERBERT,
J . M . I M H O P F , H . I N G R I S C H , V . JONÄKOVÄ,
L.J.GREENE,
H . F . HIXSON,
E . JAUMANN,
Jr.,
B . KASSELL,
R . E . M C K E E , B . K E I L , U . K U C I C H , G . KLEIN, H . KLEIN, T . KOIDE, D . KOWALSKI, L . F . KRESS, D . KUKLA, S . KUPFER, M . LASKOWSKI, S r . , M . LASKOWSKI, J r . , I . E . LIENER, J . A . LUTHY, M . B . MARCINISZYN, S . R . M A R T I N , J . A . MATTIS, R . MEISTER, J . H . MOZEJKO, M . M Ü L L E R , G . MÜLLER-BARDORFF, C . W . NIEKAMP, R . OBERMEIER, S . O D A N I , P . PASQUAY, R . J . PEANASKY, M . H . PUBOLS, G . R E I D E L , M . R I G B I , A . R Ü H L M A N N , C . A . R Y A N , M . S C H N E I D E R , S . L . S C H N E I DER, G . F . B . S C H U M A C H E R , H . G . S C H W I C K , D . SHOTTON, L . K . S H U M W A Y , F . S O R M , W . STEIGEMANN, F . C . STEVENS, J . R . SWARTWOUT, J . A . T R A V I S , H . T S C H E S C H E , E . W Ä C H T E R , T . - W . W A N G , VELD, F . P . ZUSPAN
E.WERLE, K.A.WILSON, L. J.D.ZANE-
List of Participants
ACHER, R., Laboratoire de Chimie Biologique, 96, Boulevard Raspail, Paris VI e , France
LASKOWSKI, M., Jr., Purdue University, Dept. of Chemistry, Lafayette, Indiana 47907, U. S. A.
BIRK, Y., The Hebrew University of Jerusalem, Rehovot Campus, P. O. B. 12, Israel
LASKOWSKI, M., Sr., Roswell Park Memorial Institute, 666 Elm Street, Buffalo, New York 14203, U. S. A.
CECHOVÀ, D., Czechoslovak Academy of Science, Institut of Organic Chemistry and Biochemistry, Flamingovo naméstl 2, Praha 6, CSSR
LIENER, I. E., University of Minnesota, Dept. of Biochemistry, 140 Gortner Laboratory, St. Paul, Minnesota 55101, U. S. A.
KEIL-DLOUHÀ, V., Institut de Chimie des Substances Naturelles, 91 Gif-Sur-Yvette, France
PEANASKY, R. J., The University of South Dakota, Dept. of Biochemistry, Vermillion, South Dakota 57069, U. S. A.
FEENEY, R. E., University of California, 209 Roadhouse Hall, Davis, California 95616, U. S. A. FEINSTEIN, G., Tel-Aviv University, Dept. of chemistry, Tel-Aviv, Israel
Bio-
FINK, E., Institut für Klinische Chemie und Klinische Biochemie, 8 München 15, Nußbaumstr. 20, W.Germany FRITZ, H., Institut für Klinische Chemie und Klinische Biochemie, 8 München 15, Nußbaumstr. 20, W.-Germany GREENE, L. J., Brookhaven National Laboratory, Upton, L. I., N. Y . 11973, U. S. A. HEIMBURGER, N., Behringwerke AG, 355 W.-Germany
Marburg,
HUBER, R., Max Planck-Institut für Eiweiß- und Lederforschung, 8 München 15, Schiller Straße 46, W.Germany HOCHSTRASSER, K., Institut für Klinische Chemie und Klinische Biochemie, 8 München 15, Nußbaumstr. 20, W.-Germany IKENAKA, T., Osaka College of Science, Toyonaka, Japan KASSELL, B., The Medical College of Wisconsin, Dept. of Biochemistry, 561 North Fifteenth Street, Milwaukee, Wisconsin
RIGBI, M., The Hebrew University of Jerusalem, Dept. of Biological Chemistry, Jerusalem, Israel RYAN, C. A., Washington State University, Dept. of Agricultural Chemistry, Pullman, Washington 99163, U. S. A. SCHUMACHER, G. F. B., The University of Chicago, Dept. of Obstetrics and Gynecology, 5841 Maryland Avenue, Chicago, Illinois 60637, U. S. A. SHOTTON, D., The Molecular Enzymology Laboratory, Department of Biochemistry, University of Bristol, Bristol BS 9 1 AE, England STEVENS, F. C., The University of Manitoba, Dept. of Biochemistry, Winnipeg 3, Manitoba, Canada TRAVIS, J., The University of Georgia, Dept. of Biochemistry, Athens, Georgia 30601, U. S. A. TSCHESCHE, H., Organisch-Chemisches Laboratorium der Technischen Universität München, Lehrstuhl für Organische Chemie und Biochemie, 8 München 2, Arcisstr. 21, W.-Germany WERLE, E., Institut für Klinische Chemie und Klinische Biochemie, 8 München 15, Nußbaumstr. 20, W.-Germany ZANEVELD, L. J . D., The University of Georgia, Dept. of Biochemistry, Athens, Georgia 30601, U. S. A.
Proteinase Inhibitors of Human Plasma N . HEIMBURGER, H . HAUPT a n d H . G . SCHWICK
Behringwerke AG, Marburg (Lahn)
Human plasma may be assumed to contain far more than a hundred single proteins. It is generally agreed that each protein has a specific function. The most simple function might be the transport of inorganic ions and organic substances. Transport functions involve binding, and plasma contains such proteins which bind generally or specifically, e. g. transferrin acts as carrier of iron only, whereas albumin combines with such different substances as fatty acids, heme, bilirubin, copper and hormones. In special cases, not only a single, but several proteins are engaged in the same function: Thyroxin is bound by three proteins, prealbumin, albumin but only specifically by the so-called Thyroxin binding globulin. Finally there are proteins in plasma which do not reveal any biological activity as single proteins until after interaction with one or more plasma factors. The interaction may have the character of a cascade reaction, as typified not only in the activation of coagulation and fibrinolysis but also in the kallikrein and complement system. The final products of the activation chains are enzymes. It is not surprising therefore, that human plasma contains proteins which can bind and neutralize these enzymes. These proteins are generally termed proteinase inhibitors (PI). It is thought that their function is to limit the action of the proteolytic enzymes to the biological requirements. First observations concerning the antitryptic activity of serum were obtained in 1894 by 1 Fritz-Tschesche, Proceedings
and PERNOSSI [ 1 ] , In 1 8 9 7 three papers appeared concerning the proteinase inhibitors of serum; namely from C A M U S and G L E Y [ 2 ] , FERMI
PUGLIESE a n d C O G G I [ 3 ] a n d H A H N [ 4 ] , I n
1900
[5] reported the concentration of the antitryptic activity by salt fractionation procedures in the albumin fraction. In the following years many workers tried to find out the chemical character of the P I ' s : D E L E Z E N N E LANDSTEINER
a n d POZERSKY [ 6 ] , S C H W A R T Z [ 7 ] , B A U E R [ 8 ] a n d J O B L I N G [ 9 ] assumed the antitryptic factors to be lipids because they were destroyed by treatment with chloroform. Their hypothesis, however, was not generally accepted [10, 11, 12], At the same time H E D I N [ 1 3 ] , W I E N S [ 1 4 ] , M E Y E R [ 1 5 ] and E I S N E R [ 1 6 ] occupied themselves with the biological nature of the PI and their mechanism of action. They came to the conclusion that the "antifermente" had the character of antibodies. As a result of the discovery of the antitryptic activity of serum, its concentration and partition was studied during various diseases, e. g. in Pneumonia, an increase in antitryptic activity in the beginning of sickness, an elevated level in the acute phase and a reduction in the third phase was found ( A S C O L I and BEZZOLA, 1 9 0 3 [ 1 7 ] ) . Deviations of normal values were observed in various infectious diseases [ 1 8 , 1 9 , 2 0 ] . Also in cancer the trypsin inhibitors were found to be increased, but did not prove to be specific [21],
Independently from the antitryptic activity the antithrombin efficiency of human serum was observed in 1 9 0 5 by M O R A W I T Z [ 2 2 ] ,
2
N . HEIMBURGER, H . HAUPT a n d H . G . SCHWICK
Separation of the inhibitors begun by L A N D in 1 9 0 0 was continued by S C H M I T Z [ 2 3 ] in 1 9 3 8 ; he used the methods of N O R T H R O P and K U N I T Z [ 2 4 ] which already had been proved in the isolation of the peptide inhibitor of the pancreas. By this technic a TCA-soluble inhibitor which inactivated trypsin in molar ratios, but not chymotrypsin, was isolated. His results were confirmed by G R O B [ 2 5 ] and D U T H I E and L O R E N Z [ 2 6 ] who used methods according to S C H M I T Z [ 2 3 ] . But the procedures used resulted in partially denaturated inhibitors which could not be exactly characterized. This is the reason why the Pi's isolated by the above mentioned scientists cannot be attributed to one of the now known inhibitors. In 1939, S M I T H and L I N D S L E Y [27], using the recently introduced electrophoresis, localized the main antitryptic activity of serum in the albumin zone. More than ten years later two inhibitors were demonstrated in serum by S H U L M A N [28], J A C O B S S O N [29] in 1953 reported their electrophoretic separation. Some time later the same author [30] published that 90% of the antitryptic activity is localized in the ^-globulin zone and 10% in the oe2-globulin zone and antiplasmin activity in the slower a2-zone only. A 12-fold concentration of the «j-trypsin-inhibitor was reported by M O L L and co-workers [31] in 1958 before B U N D Y and M E H L succeeded in its isolation in a native state [32] in 1959. Human plasma is now known to contain at least six Pi's. Starch gel-electrophoresis and fibrincontaining agar plates, as used in the sandwich technic [33, 34], have proved to be very helpful in the discovery, identification and isolation of these Pi's. This technic is demonstrated in Fig. 1. To prove the presence of PI, plasma is separated electrophoretically in starch gel. The gel is then cut into two discs; the upper one is colored with amido black and the lower one is used to cover agar-plates containing heat-inactivated fibrin. As soon as the proteins have entered the fibrinagar film, troughs parallel to the migration direction are cut and filled with enzyme solutions The substrate plates are then incubated at 37° C for about 20 hours. During this time the proSTEINER [5]
teases enter the gel. Diffusion is evidenced by lysis of the fibrin. Only in the electrophoretic positions of the inhibitors fibrin remains free
!
a,M
Ml
C1IN Alal AT1II axX
a,A
Fig. 1. Identification of six proteinase inhibitors in human plasma. Starch gel electrophoresis used in combination with fibrin agar plates according to the sandwich technic. A. Electrophoregram of plasma stained with amido-black; fibrin plates after being covered with and lysed by elastase (B), plasmin (C), trypsin (D) and chymotrypsin (E).
from attack. By this technic plasma can be demonstrated to contain inhibitors against elastase, plasmin, trypsin, chymotrypsin, in total six, from which five proved to be polyvalent. The Pi's were determined according to their electrophoretic mobility and to their, first recognized or most important, physiological function, as far as known, at the time of isolation. Many scientists have been involved in the isolation and identification of the Pi's, and in most cases, these occurred independent of each other. In some cases proteinase inhibitors were well characterized chemically, but their inhibitor function was at that time still unknown, e. g. aj-antitrypsin (ax A) was isolated in 1955 in our
Proteinase Inhibitors of Human Plasma
laboratories by SCHULTZE and co-workers [35] as (^-3.5-glycoprotein. Its identity as the main trypsin inhibitor of plasma was not recognized until 1962 [36] when fibrin-agar electrophoresis [33] was used to control our processing of ccj3.5-glycoprotein. Then, we became aware too, that the inhibitor is irreversibly denaturated when the pH of the solution drops below pH 5. We designated the inhibitor as «^antitrypsin [36] and recognized it to be identical with the inhibitor isolated three years previously by BUNDY a n d MEHL [32],
a2-Macroglobulin (oc2 M) also was not identified as an inhibitor until 9 years after its isolation [37] and chemical characterization in our laboratories [35, 38, 39]. ai-Antichymotrypsin (ocjX) was also isolated and characterized by our laboratories [40] and identified three years later as the most specific inactivator present in serum [34]. Antithrombin III (AT III) was purified by ABILDGAARD [41] in 1967 and at the same time also in our laboratories [42], However, before this, a concentrated, but impure preparation of A T I I I was a c h i e v e d b y LOEB [43], HENSEN and LOELIGER
[44]
and MARKWARDT and
WALS-
MAN [45].
The inter a-trypsininhibitor (Ial) was first described as n protein by STEINBUCH [46]. Four years later in 1965 a protein with the chemical properties of the n protein was isolated and characterized as an inhibitor [47]. The inactivator efficiency of human serum for Cl-esterase was observed by RATNOFF and LEPOW [48] in 1957. Partial purification and some properties were later reported in 1961 by the same scientists [49]. About this time a2-neuraminoglycoprotein was isolated by SCHULTZE and co-workers [50]. Its function was unknown. However, in 1969 [51] it was shown to be identical with Cl-inactivator.
3
Isolation Fractionation charts for all the six Pi's from human serum are shown in tables 1—4. Concerning the methods used, it can be stated generally, that crude purification of the PI was obtained by their different solubilities in ammonium sulfate, fractionation with Rivanol, ethanol precipitation and specific adsorption steps. A high degree of purification was obtained with zone electrophoresis and sieve gel-filtration. a a -M isolation was started with plasma. To prevent kallikrein contact-activation, plasma should be drawn in plastic tubes and/or with peptide inhibitors. Otherwise a2-M partially saturated with kallikrein will be obtained [52]. For the isolation of CI INA and Ial freshly recalcified serum is used. Processing is started with adsorption on DE-32; during the buffer gradient elution Ial is obtained in a fraction separate from CI INA and can then be highly purified by ammonium sulfate precipitation and Sephadex G-150 gel-filtration. ajA, a j X and AT III are enriched in the mother liquid of albumin after crystallization with ammonium sulfate [53]. All three inhibitors are characterized by their solubility in a 50% saturated ammonium sulfate solution. oc1A and otjX were separated from each other by their different precipitability with Rivanol. Further purification of a x X was achieved by zone electrophoresis at pH 5.2, and of xxA by ethanolfractionation and electrophoresis too. The most important steps in the isolation of AT III are the adsorption on Ca 3 (P0 4 ) 2 and the zone electrophoresis. The last fractionation step of all procedures required gel-filtration in order to separate the monomer forms of the inhibitors from the polymers generated during the isolation procedure.
The following abbreviations are used: otjA, ^-Antitrypsin; a 2 M, a 2 -Macroglobulin; o^X, aj-Antichymotrypsin; A T III, Antithrombin III; Ial, Inter a-Trypsininhibitor; C l I N A , Cl-Inactivator.
1*
4
N . HEIMBURGER, H . HAUPT a n d H . G .
Table 1. Fractionation Flow chart for
SCHWICK
Antitrypsin and aj-Antichymotrypsin
ppt, precipitation/spt, separation
Proteinase Inhibitors of Human Plasma
Table 2. Fractionation Flow Chart for Cl-Inactivator and Inter-oc-Trypsininhibitor
ppt, precipitation/spt, separation
5
6
N . HEIMBURGER, H . HAUPT a n d H . G .
SCHWICK
Table 3. Fractionation Flow Chart for Antithrombin III
ppt, precipitation/spt, separation
Proteinase Inhibitors of Human Plasma
Table 4. Fractionation Flow Chart for a2-Macroglobuün
ppt, precipitation/spt. separation
7
8
N . HEIMBURGER, H . HAUPT a n d H . G . SCHWICK
Special Sensitivity The PI proved to be extremely sensitive, during fractionation, to fractionation methods generally used in protein chemistry; e. g. as illustrated by the following examples: a,A tends to form high molecular partially-inactivated aggregates when precipitated by ethanol. Despite this we use this fractionation step because good elimination of all other «-globulins is achieved therewith.
Fraction number
NS
1
¡¡¡¡¡jT . I B f 1
~
Fig. 2. Denaturation of the proteinase inhibitors by normal fractionation methods as demonstrated by starch gel
8
c 2
£ j f f l J »•»•• f
In Fig. 2 A separation can be seen of the polymers and dimers from the monomer form on Sephadex G-100 and their electrophoretic characterization. Aggregation of AT III occurs already during dialysis against water and is accompanied by inactivation (Fig. 2B). It becomes more evident also when the ion strength of plasma or serum is reduced. a 2 M is inactivated when it has been precipitated by ammonium sulfate about or above the neutral point as evidenced by its increased mobility in polyacrylamide gels (Fig. 2C). Ial can be aggregated by heating [54] to 56° C for 30 minutes. Although the aggregates formed are of too high a molecular weight for polyacrylamide gel sieving, they remain still active as trypsin inhibitors (Fig. 3). By treatment of plasma with sulfhydryl reagents, a 2 M is preferentially inactivated and probably CI INA as well. Addition of ammonia, in a concentration so that a pH of 8.5 is not exceeded, causes a relative specific, and approximately quantitative inactivation of a 2 M in plasma. This inactivation is irreversible in contrast to that produced by aliphatic amines [55, 56]. Finally it should be mentioned that a progressive denaturation can be frequently observed when the pH is below 5. The preceding examples show the difficulties encountered in the isolation of the inhibitors of serum in a native state, particularly because of their high sensitivity. Proved
electrophoresis. ^
A ) Aggregation of (^-antitrypsin following ethanol separation of the aggregates on Sephadex G-100 and characterization of the fractions (below). ^ -Aggregation of antithrombin III in an ion-poor
C) Inactivation of a 2 -macroglobulin as evidenced by its increased mobility after (NH 4 ) 2 S 0 4 pption. in alkaline medium: 1 . 5 0 % ppt at pH 8.0 and 2. at pH 5.0 (NS = normal serum)
Proteinase Inhibitors of Human Plasma
fractionation methods cause an alteration in structure, which is of basic importance for the interaction with the specific enzymes. oc2M CÏ INA loci ATIII o^A
(CI INA, a 2 M) remain near the start point. In between the inter-a-globulins, Ial and AT III are found, the latter nearer to the front due to its lower molecular weight. Tab. 5 shows the most relevant data of the Pi's Plasma is rich in a t A and a 2 M. Ten weight per cent of the plasma proteins are inhibitors,
Fig. 3. Stability of the inhibitors under various conditions. Lysis of fibrin agar plates being covered with starch gel electrophoretograms of A) B) C) D)
normal plasma, plasma heated at 56°C for 30 min., plasma treated with thioglycollate (final conc. 0,1M), plasma adjusted to pH 8.5 using NH 4 OH, (troughs contain trypsin).
Chemical Characterization All six Pi's are characterized by their electrophoretic mobility in Polyacrylamide gels in which migration is a function of the net charge, the form and the molecular seize (Fig. 4): the low molecular aj-globulms (a x A, a x X) migrate in front of all, the high molecular a 2 -globulins
«ja
NS
NS
Fig. 4. Polyacrylamide gel-electrophoresis. Characterization of the isolated inhibitors. NS = normal serum; 1. a r a n t i t r y p s i n ; 2. oci-antichymotrypsin; 3, inter a-trypsininhibitor; 4. antithrombin III; 5. CI inactivator; 6. a 2 -macroglobulin.
considering the molar concentration, a t A is the predominant. The molar concentration of the others gives a total of 40% of o^A. It should be noted, that Pi's are all glycoproteins. Especially
Table 5. Concentration and characterization of the Inhibitors in Human Scrum Inhibitors
1. 2. 3. 4. 5. 6.
ocj-Antitrypsin aj-Antichymotrypsin Inter «-Trypsininhibitor Antithrombin III CT-Inactivator a 2 -Macroglobulin
mg/100 ml C. Mean ± SD 290.0 48.7 50.0 29.0 23.5 260.0 701.2
± 45.0 ± 6.5 ± 2.9 ± 3.0 ± 70.0
MW
,uMol
Peptide Content (%)
Carbohydrate Content ( % )
54.000 69.000 160.000 65.000 104.000 820.000
54.0 7.0 3.1 4.5 2.3 3.3
86 73 90 85 65 92
12.2 24.6 8.4 13.4 34.7 7.7
10
N . HEIMBURGER, H . HAUPT a n d H . G . SCHWICK
Table 6. % of Carbohydrate Residues in Proteinase Inhibitors
Hexoses Fucose Acetylhexosamines Sialic acid
Oij-Antitrypsin
o^-Antichymotrypsin
Inter a-trypsin inhibitor
Antithrombin III
ClInactivator
10
22000
4.5
8.5 4-6
32
H . FRITZ, B . BREY, M . MÜLLER a n d M . GEBHARDT
ducible. The amount of active trypsin molecules present was titrated with p-nitrophenyl-p'guanidinobenzoate according to CHASE and S H A W [ 1 8 ] . It is remarkable that the amount of trypsin molecules able to complex with some inhibitors is near the proportion found from their titration curves. The high amount of basic inhibitors bound by the trypsin-cellulose resin is most likely due to additional non-specific adsorption, as this support contains a considerable amount of negatively charged groups [16]. Some loss of pig pancreatic trypsin inhibitor by enzymatic degradation is also possible [8, 19], From the similar amounts of bdellins and soybean trypsin inhibitor bound to the resin it can be concluded that the net charge of the inhibitor has a much stronger influence on the binding capacity than the molecular volume. Isolated Inhibitors In Table 2 the yields and purities of some trypsin inhibitors are shown isolated in our laboratory. Noticeable are the higher yields with permanent inhibitors which do not show the phenomenon of temporary inhibition. Obviously these inhibitors are also resistant to enzymatic degradation during contact with the enzyme resins. — The inhibitors from lung and liver of sheep and cattle are eluted from the resin column in 2 fractions; the first contains approximately 75% of the bound inhibitor and the second, which appears much later in the effluent, the residual
amount up to 100%. We have no explanation of this separation. — Enzymatic degradation mainly causes the loss of the other, temporary inhibitors shown in the Table. In these cases rapid working and extensive cooling during the binding, washing, and elution steps increase the recoveries. Some loss may also be due to solubilization of the enzyme resin when a high amount of inhibitor is bound and thus the protein content of the resin is greatly increased. Summarizing the observations we can say that the purity of the inhibitors after dissociation (of the complex), elution and gel filtration is influenced by the support used, the ionic strength of the washing buffers, and to some extent the degree of contamination (concentration of the basic inhibitor from sheep lung in the extracts is very low just as the degree of purity, see Table 2) in the crude extracts. Further purification of the inhibitors is relatively easily achieved by ion exchange chromatography or gel filtration. Enzymatic Cleavage Most of the inhibitors isolated by the resin method are not homogeneous. The permanent inhibitors isolated from plant seeds by HOCHSTRASSER et al. [6, l i b ] consisted mainly of two different forms, separable by ion exchange chromatography, but with the same biochemical characteristics and identical amino acid compositions. The temporary inhibitors from
Table 2. Inhibitors Isolated in Larger Amounts For experimental details see text and references Inhibitor f r o m Pancreas (pig, sheep) Lung (sheep) Lung, liver (cattle) Seminal vesicles (guinea pigs) Boar seminal plasma Leeches (Bdellins) a
A f t e r gel filtration,
b
Ref.
Trypsin-resin support
Yield [ % ]
10 21 22
polyamphoteric E M A " polyamphoteric E M A polyanionic E M A
90-- 1 0 0 80-- 1 0 0
70-- 9 0 3 5 -- 5 0 85-- 9 5
10, 13, 14
polyamphoteric E M A
80-- 9 5
80
13 12
Polyacrylamide polyamphoteric E M A
63-- 8 5 65-- 7 4
Ethylene-maleic acid copolymer.
60-- 7 5
Purity 3 [ % ]
> 90 60-- 8 0
Specific Isolation and Modification M e t h o d s
pancreas glands [20], leeches [12] and seminal vesicles [13,14] showed three and more different forms with identical or similar amino acid compositions. The occurence of these different inhibitor forms is at least partly due to the trypsin resin method used. Dr. HOCHSTRASSER in nearly each case obtained a mixture of the native inhibitor and a modified form in which an arg-X or lys-X bond had been broken. He could prove that this Cterminal lysine or arginine residue is part of the reactive site of the inhibitor. Temporary inhibitors from animal organs contained forms with broken bonds at the reactive center, but also with other split bonds. In some cases we isolated active inhibitor forms With missing Nor C-terminal peptides [8, 13, 14, 23, 24]; these peptides are apparently not necessary for the inhibitory reaction. An interesting example is the trypsin-specific inhibitor from guinea pig seminal vesicles [13,14]. By the resin method we produced this inhibitor in fully modified form (arg-X bond broken) •— this form is inactivated by carboxypeptidase B; however, when isolated by ion exchange chromatography, only the native inhibitor (arg-X bond intact) was obtained. When Dr. F I N K treated the native form with the trypsin resin, he obtained the modified one, too. The trypsin-plasmin inhibitors from leeches [12] are also modified to some extent at the reactive center during this isolation procedure. These results do not contradict the elegant modification theories of LASKOWSKI Jr. and Co-workers [25], because the dissociation and the elution of the inhibitors from the trypsin resin complexes take place slowly, so that a thermodynamic equilibrium can be approximated. By kinetically controlled dissociation (sudden drop of pH) of the complexes only non-modified inhibitor forms are to be expected. The modification reactions during the isolation procedure with trypsin resins are not always disadvantageous; in many cases the groups of HOCHSTRASSER, TSCHESCHE and our laboratory were able to elucidate in this way the reactive sites of inhibitors. 3 Fritz-Tschesche, Proceedings
33
Preparation of Modified Inhibitors Water-insoluble enzyme resins are also suitable for the preparation of chemically modified inhibitors. The trypsin-kallikrein inhibitor from bovine organs accumulates in the kidneys shortly after i. v. injection. By contrast the tetramaleyl derivative of this inhibitor is excreted in the urine [26], In this latter derivative all amino groups, except that of the lysine residue in position 15, are substituted [27]. We synthesized the tetramaleyl inhibitor, which is nearly as active against trypsin, chymotrypsin, plasmin, (and kallikrein) proteinases as the native one, at first according to the method published by CHAUVET and A C H E R [28]: We treated the inhibitortrypsin complex with maleic anhydride and separated the substituted components by gel filtration [27]. Our new method is as follows [17]: The waterinsoluble trypsin resin (support: polyacrylamide) is saturated with the inhibitor; unbound inhibitor is washed off. The suspension of the complex is reacted with an excess of maleic anhydride, and afterwards the reaction products are washed out with buffer solutions from the resulting insoluble maleylated complex. The complex is dissociated by the action of weakly acidic salt-buffer solutions. After desalting and lyophilisation, the analytically pure inhibitor is obtained, its structure is shown in Fig. 3. It may be mentioned that the degree of purity of the native stock inhibitor (starting material) was only about 70%. We tried to synthesize for the first time another inhibitor derivative in a similar manner: The insoluble inhibitor-trypsin complex was treated with pyridoxal 5'-phosphate, and reduced with NaBH4 to link covalently phosphopyridoxyl groups to the inhibitor (and bound enzyme). After dissociation of the complex a mixture of mono-, di- and trisubstituted inhibitor derivatives was obtained [17]. As important as the simplicity of this one-step method is the fact that the modified trypsin resin retains full binding capacity. It can be used re-
34
H . FRITZ, B . BREY, M . MÜLLER and M . GEBHARDT
peatedly for the same procedure. The method allows the synthesis of many modified and substituted inhibitors with full inhibitory activity as
Insolubilized Inhibitors In future probably more importance will be given to insolubilized inhibitors than to enzyme resins. Affinity chromatography using inhibitor resins allows the isolation of enzymes more simply and more rapidly than by present methods. T o obtain further basic data we prepared both polyanionic and polyamphoteric EMA-resins of the basic trypsin-kallikrein inhibitor (BPTI) from bovine organs [29]. The specific binding capacity of these resins (Table 3) is sufficiently high for trypsin, chymotrypsin and plasmin, but it is very low for kallikreins from organs in which we are especially interested. All the other problems and results in using these inhibitor EMA-resins are similar to those already mentioned in connection with the trypsin EMAresins. Dissociation of the insoluble complexes in slightly acidic salt-buffer solutions yields satisfying results for trypsin, chymotrypsin and plasmin; the kallikrein complex dissociates in weakly acidic guanidine-HCl solutions. Investigations aimed to obtain inhibitor resins with higher binding capacities for kallikreins
Table 3 Polyanionic
(A) and Polyamphoteric EMA-Resins
(N)
Inhibitor
For experimental details see references [29, 5] The trypsin-kallikrein inhibitor from bovine organs was used Weight ratio by preparing the inhibitor resin Fig. 3. Tertiary Structure of the Trypsin-Kallikrein Inhibitor from Bovine Organs According to HUBER et al. [32]. R . : Site of substitution by maleyl or 5'-phosphopyridoxyl residues.
the reactive center is protected during the modification reaction. The search for the reactive center of "lysine"-inhibitors is facilitated for the same reasons.
A-resin E M A : inhibitor
1:5
N-resin 3:5
1:5
3:5
Inhibitor amount in [%] a still able to complex with Trypsin Chymotrypsin Plasmin Kallikrein a
50
28
18
6
48
48
30
37
>
30
>
42
ch-< - n
\
NH
Hr
CÍÚ
Substrate
CH,
\
)cH-
CH,
Fig. 5 Tyrosine
\NH
^CH-
OH
216
THR- 226 SER-189
SER-195
I
A
HC CH
Elastase Substrate
I II
HC CH
VI
OH