Bioluminescence in Progress 9781400875689

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Bioluminescence in Progress

Cypridina hilgendorfii, a classic organism in modern research on bioluminescence.

Copious secretions

by living specimens create clouds of vivid blue light in the surrounding sea water; the active components, " luciferin " and " luciferase," which remain stable for indefinite periods of time in dried specimens like those in the photograph above, spring into lightemitting action on moistening with water.

These

tiny ostracod crustaceans, averaging 2-3 mm in length, occur in remarkable abundance in the coastal waters of Japan, where they are known as " umibotaru," the " sea-firefly." Publication of this book marks the half-century anniversary of the scholarly studies which the late Professor E. Newton Harvey began in 1916 in Japan on the Cypridina system and pursued for the rest of his life, including the three years following his retire­ ment from Princeton University in 1956.

Bioluminescence in Progress Proceedings of the Luminescence Conference sponsored by the Japan Society for the Promotion of Science and by the National Science Foundation, under the United States-Japan Cooperative Science Program, September 12-16, 1965, Hakone National Park, Kanagawa-ken, Japan

EDITED BY

Frank H. Johnson and Yata Haneda

PRINCETON, NEW JERSEY PRINCETON UNIVERSITY PRESS

1966

Copyright © 1966 by Princeton University Press Reproduction in whole or in part by or for any purpose of the governments of the United States and Japan is permitted. Library of Congress Catalog Card Number : 66-17702 Printed in the United States of America by The Colonial Press Inc., Clinton, Massachusetts Color plates printed in Japan

DEDICATED TO THE

MEMORY OF

EDMUND NEWTON HARVEY

"Some things . . . give light in the dark . . . for example, fungi, flesh, heads, scales and eyes of fish." —Aristotle, De Anima, Bk. II, Chap. 7, 419

Preface THE PAPERS in this book are the outcome of a "Luminescence Confer­ ence" which took place, September 12 to 16, 1965, amidst serene foot­ hills overtowered by majestic Mount Fuji in Hakone National Park, Japan. The Conference was held under the auspices of the recently established United States-Japan Cooperative Science Program with the joint support of the National Science Foundation and the Japan Society for the Promotion of Science. The objectives of this Conference were fundamentally the same as those of an earlier conference devoted primarily to bioluminescence, held at Asilomar, California, in 1954 and supported by the National Science Foundation through the National Research Council's Com­ mittee on Photobiology, which "recognized the mutual advantages of bringing together a group of leading investigators for a critical ap­ praisal of present knowledge, for the first-hand interchange of ex­ periences and ideas, and for the projection of likely approaches to unsolved problems".1 The present Conference had the further objec­ tives, in accordance with the purposes and policies of the U.S.-Japan Program, of promoting understanding and cooperation between Japa­ nese and American scientists, with a view toward effecting a more intimate acquaintance with each other's points of view, methods, and research materials. While the actual participants were thus limited almost wholly to Japanese and American investigators and their active collaborators, the papers of the Conference are made generally available in this book. The papers themselves are noteworthy in both scope and con­ tent, for they range from purely chemical to purely biological in their approach to the central problem—the phenomenon of light emission by living organisms—and they include some new technical reports which constitute real milestones marking the advancing frontiers of this field. It is reasonable to expect that the book will be found useful by anyone interested either in the background and status of special aspects or in a more comprehensive view of the subject as it is in progress. The editors have endeavored to allow each author maximum free­ dom and individuality in regard to style, organization, material in­ cluded, hypotheses expressed, conclusions reached, and even length of manuscript. Only minor changes have been made by the editors, 1Quoted from the preface to The Luminescence oj Biological Systems, F. H. Johnson (ed.), which was based on the 1954 Conference and published by the American Association for the Advancement of Science, Washington, D.C., 1955.

PREFACE

mostly in the interests of clarity, consistency, and conformance to cur­ rent American English usage, spelling, abbreviations, etc. Because the language of the book, except for the abstracts in Japanese, is the native language of only one of the two editors (F.H.J.), he must assume responsibility for essentially all such changes, as well as such routine matters as compilation of the indexes. These tasks were sub­ stantially lightened by the invaluable assistance of Mrs. Dorothy Hollmann of the Princeton University Press. Both editors are greatly indebted to Dr. I. Takatsuki and Mr. Y. Kishi for translating most of the English abstracts into Japanese and to Dr. 0. Shimomura for painstakingly editing all the Japanese abstracts, as well as for trans­ lating into Japanese a number of the English abstracts which were received later. Due to last-minute changes in titles, some of the titles in Japanese do not fully correspond to the titles in English, though the meanings remain essentially the same. It is a genuine pleasure to record here acknowledgement also of the assistance, cooperation, and courtesies of the many others who con­ tributed to the success of the Conference and the publication of its proceedings, with special thanks, first of all, to the Japan Society for the Promotion of Science and the National Science Foundation for the enabling support. Governor Uchiyama of Kanagawa Prefecture and Mayor Nagano of Yokosuka City provided certain facilities and memorable hospitality. Members of the staff of both the Washington and Tokyo offices of the National Science Foundation, in particular Doctors Neureiter, Ries, O'Connell, Arvey, and Mr. Miyahara, cheer­ fully helped with numerous details. Conference participants, Doctors Buck, Goto, McElroy, Y. Okada, Sugiyama, and Totter, graciously chaired the scientific sessions. The Japan Travel Bureau was efficient and pleasant in rendering their services, as were also the personnel of the Hotel Kagetsu-en where the Conference was held. Finally, the American Coordinator experienced invaluable cooperation on the part of Dr. 0. Shimomura and his colleagues in solving various problems which arose from time to time during a stay of several months at the University of Nagoya prior to the Conference. The editors, who were also the Coordinators of the Conference, express the profound appreci­ ation of all participants for the generous support, assistance, and cour­ tesies tendered. F. H. J. Y. H.

List of Contributors Robert L. Airth, The University of Texas Masamoto Akutagawa, Tokyo University of Education Jean-Marie Bassot, Oceanographic Institute, Paris Patricia Q. Behrens, The University of Texas G. Benjamin Bouck, Yale University John B. Buck, National Institutes of Health Aurin M. Chase, Princeton University Milton J. Cormier, University of Georgia Richard DeSa, University of Illinois Roger Eckert, Syracuse University James F. Ferguson, III, New England Institute for Medical Research G. Elizabeth Foerster, The University of Texas Joan Friedland, University of Illinois Taketoshi Gasha, Tokyo University of Education Quentin H. Gibson, University of Pennsylvania Toshio Goto, Nagoya University William A. Hagins, National Institutes of Health Yata Haneda, Yokosuka City Museum Frank E. Hanson, National Institutes of Health J. Woodland Hastings, University of Illinois Yoshimasa Hirata, Nagoya University Kazuo Hori, University of Georgia & Tokyo University of Education Frank H. Johnson, Princeton University Albert L. Jordon, North American Aviation, Inc. Yoshito Kishi, Nagoya University Paul Kreiss, University of Georgia Seishi Kuwabara, Agricultural College, Wageningen John Lee, New England Institute for Medical Research William D. McElroy, Johns Hopkins University Hiroshi Nakamura, Yamakatsu Pearl Co. Yo K. Okada, National Science Museum of Japan Phil M. Prichard, University of Georgia George T. Reynolds, Princeton University Yo Saiga, Tokyo University of Education Howard H. Seliger, Johns Hopkins University Osamu Shimomura, Nagoya University Edward H.-C. Sie, North American Aviation, Inc. James Spudich, University of Illinois Hans-Dietrich Stachel, University of Marburg and Princeton Uni­ versity

CONTRIBUTORS

Noboru Sugiyama, Tokyo University of Education Beatrice M. Sweeney, Yale University Edward C. Taylor, Princeton University Andrew Thanos, North American Aviation, Inc. John R. Totter, U.S. Atomic Energy Commission Frederick I. Tsuji, University of Pittsburgh Marcia Vergin, University of Illinois E. C. Wassink, Agricultural College, Wageningen Alfred S. Wesley, New England Institute for Medical Research

Contents Vll

Preface

ix

List of Contributors

3

Introduction. Frank H. Johnson Chemical Events Leading to Chemiluminescence of Lucigenine and Luminol. John R. Totter

23

The Use of Luminol as a Standard of Photon Emission. J. Lee, A. S. Wesley, J. F. Ferguson, III, and H. H. Seliger

35

The Preparation and Standardization by Different Methods of Liquid Light Sources. J. W. Hastings and G. T. Reynolds

45

Synthesis and Properties of Some Indole Derivatives Related to Cypridina Luciferin. H.-D. Stachel, E. C. Taylor, 0. Shimomura, and F. H. Johnson

51

Chemiluminescence and Fluorescence of Cypridina Luciferin and of Some New Indole Compounds in Dimethylsulfoxide. F. H. Johnson, H.-D. Stachel, E. C. Taylor, and 0. Shimomura

67

Chemilumineseence of Indole Derivatives. M. Akutagawa, T. Gasha, and Y. Saiga

83

N.

Sugiyama,

The Structure of Cypridina Luciferin. Y. Kishi, T. Goto, Y. Hirata, 0. Shimomura, and F. H. Johnson

89

Activity and Inhibition of Cypridina Luciferase: Quantitative Measurement; Analysis of Inhibition by Urea; and Some Ef­ fects of Sodium and Potassium Ions. Aurin M. Chase 115 Chemistry of the Luciferases of Cypridina hilgendorjii and Apogon ellioti. F. I. Tsuji and Y. Haneda 137 Molecular Mechanisms in Bacterial Bioluminescence: On Energy Storage Intermediates and the Role of Aldehyde in the Reac­ tion. J. W. Hastings, Q. H. Gibson, Joan Friedland, and J. Spudich 151

CONTENTS

Light-Initiated Bioluminescence. Q. H. Gibson and J. W. Hast­ ings 187 Application of Luminescence in Aerospace Industry. E. H.-C. Sie, A. Thanos, and A. Jordon 195 The Luminous Fungi. R. L. Airth, G. Elizabeth Foerster, and Patricia Q. Behrens

203

The Apparent Phosphorescence of a Substance Extracted from the Mycelium of the Luminous Fungus, Omphalia jlavida. M. J. Cormier and J. R. Totter 225 Purification and Properties of the Active Substance of Fungal Luminescence. S. Kuwabara and E. C. Wassink 233 Some Notes on Semi-large-scale Cultivation of Luminous Fungi. E. C. Wassink and S. Kuwabara 247 A Note on Stimulatives of Bacterial Luminescence in Mass Cul­ ture. Hiroshi Nakamura 265 Excitation and Luminescence in Noctiluca milians. Roger Eckert 269 Scintillons: The Biochemistry of Dinoflagellate Biolumines­ cence. J. W. Hastings, Marcia Vergin, and R. DeSa 301 Crystal-like Particles in Luminous and Non-luminous Dinoflagellates. Beatrice M. Sweeney and G. B. Bouck 331 Studies on the Bioluminescence System of the Sea Pansy, Renilla reniformis. M. J. Cormier, K. Hori, and P. Kreiss

349

Bioluminescence Systems of the Peroxidase Type. M. J. Cormier, P. Kreiss, and P. M. Prichard 363 A Note on the Large Luminescent Earthworm, Oetochaetus multiporus, of New Zealand. F. H. Johnson, 0. Shimomura, and Y. Haneda 385 Isolation of the Luciferin of the New Zealand Fresh-water

xii

CONTENTS

Limpet, Latia neritoides Gray. 0. Shimomura, F. H. Johnson, and Y. Haneda 391 Quantitative Measurements of Luminescence. H. H. Seliger and W. D. McElroy 405 Firefly Bioluminescence. W. D. McElroy and H. H. Seliger

427

Unit Activity in the Firefly Lantern. John B. Buck

459

Statistics of Photon Emission and Control Mechanisms in Bio­ luminescence. W. A. Hagins, F. E. Hanson, and J. B. Buck 475 Observations on the Biochemistry of Luminescence in the New Zealand Glowworm, Arachnocampa luminosa. 0. Shimomura, F. H. Johnson, and Y. Haneda 487 Partial Purification and Properties of the Chaetopterus Lumi­ nescence System. 0. Shimomura and F. H. Johnson 495 Partial Purification of the Luminescence System of a Deep-sea Shrimp, Hoplophorus graeilorostris. F. H. Johnson, H.-D. Stachel, 0. Shimomura, and Y. Haneda 523 The Origin of Luciferin in the Luminous Ducts of Parapriaeanthus ransonneti, Pempheris klunzingeri, and Apogon ellioti. Y. Haneda, F. H. Johnson, and 0. Shimomura 533 On a Luminous Organ of the Australian Pine-cone Fish, Cleidopus gloria-maris De Vis. Yata Haneda 547 On the Comparative Morphology of Some Luminous Organs. Jean-Marie Bassot 557 Observations on Rod-like Contents in the Photogenic Tissue of PFaiasema seintillans through the Electron Microscope. Yo K. Okada 611 List of Abbreviations

627

Indexes

629

Biolurainescence in Progress

Introduction FRANK Η. JOHNSON

". . . and then I plainly faw, both with wonder and delight, that the joint of meat did in divers places fhine like rotten Wood or ftinking Fifh The Colour that accompanied the light was not in all the fame, but in thofe which fhone livelieft, it feemed to have fuch a fine Greenifh blew, as I have divers times obferved in the tails of Gloworms. But notwithftanding the vividnefs of this Light, I could not by the touch difcern the leaft degree of Heat neither I, nor any of thofe that were about me, could perceive by the fmell the leaft degree of ftink pouring on it a little pure Spirit of wine . . . I found that the light was vanifhed. But water would not fo eafily quench our feeming fires; having conveyed one of the largeft luminous pieces into a fmall Receiver [of the Pneumatical Engine (sic) ], we caufed the candles to be put out, and the pump to be plied. . . . I could perceive, upon the gradual withdrawing of the Air, a difcernible and gradual leffening of the light; which yet was never brought quite to difappear (as I long fince told you the light of Rotten Wood and Gloworms had done) " (Boyle, 1672). Unaware of the living source of the light emanating from "rotten wood" (luminous fungi), "ftinking Fifh," and "Shining Flefh" (lumi­ nous bacteria), Robert Boyle nevertheless established some of the basic properties of bioluminescence systems, e.g., that the light is generated without perceptible heat; that it is susceptible of inhibition by chemical agents (not only the alcohol in "Spirit of wine", but also various "liquors" such as "rectified Oyl of Turpentine, ftrong Spirit of Salt, and weak Spirit of Sal Armoniack"; Boyle, 1667); that the light is dependent upon air (oxygen); that only a very small pressure of air is sufficient for maximum brightness; that extinction of the light by evacuation of the air is reversible, the light immediately reappearing, even with a momentary excess intensity, on readmission of air; and other properties of less general import. For centuries, research on bioluminescence has been a prime example of pure science, for which the chief incentive has been the "wonder and delight" it proffers. The intrinsic fascination of cold light shining out of living organisms, or seemingly from the sea or decaying wood or fish, has inspired more than casual observation by renowned natural

FRANK Η. JOHNSON

philosophers and scientists (cf., Harvey, 1957), at least as far back as Gaius Plinius Secundus in the first century, and including, among others, such illustrious personages as Francis Bacon, Descartes, Hooke, Redi, Malpighi, Benjamin Franklin, Spallanzani, Priestley, Bernoulli, Humphrey Davy, Faraday, Ehrenberg, Darwin, Liebig, Pasteur, Lankester, Pfluger, and in our own century, Beijerinck, Dubois, Molisch, Kluyver, and Harvey, the last-named of whom investigated, with unparalleled dedication, virtually all aspects of luminescence. The objectives have been almost wholly uncontaminated with views toward immediate practical application, and only rarely have unanticipated discoveries been turned to significant practical use, such as the currently well-known, sensitive, and specific assay method for ATP (adenosine triphosphate) by luminescence of the firefly system (StrehIer and Totter, 1952; McElroy and Green, 1956). In principle, other luminescence systems could be turned to practical use; e.g., the bioluminescent protein, aequorin, from the jellyfish Aequorea, provides the basis of the most sensitive known specific chemical test for calcium or strontium (Shimomura, Johnson, and Saiga, 1963a); the bioluminescent protein from the marine worm Chaetopterus can be used in per­ haps the most sensitive known specific chemical test for ferrous iron (Shimomura and Johnson, this book); and bacterial luminescence offers a sensitive means of detecting contamination of the atmosphere inside space vehicles by jet fuels (Sie, Thanos, and Jordon, this book). Yet by and large the practical importance of bioluminescence is certainly not immediately obvious; it is usually of unclear, if not seriously doubtful, significance even to the organisms which possess it (cf. Harvey, 1952; Johnson, 1955). In part for this reason, perhaps, relatively few investigators at any one period of time have devoted their major research efforts to problems pertaining to luminous organisms and the light they emit. In fact, the authors of papers in this book include the majority of the world's leading investigators active in the field of bioluminescence today. Since this field is off the beaten track and the number of contem­ porary specialists is indeed small, some further introductory remarks seem apropos, all the more so because the book is made up of technical papers dealing with detailed aspects of a subject which has many facets of interest. This introduction is intended primarily to give a perspective and to help clarify, for the benefit of those who are only remotely acquainted with the field—and perhaps also of some who are rather more familiar with it—the significance and present status as well as background of the subject as a whole, and in general to

INTRODUCTION

insure against "missing the forest for the trees." A lengthy review would be out of place here; more or less comprehensive treatments, both technical and semi-popular or popular, have been published within the past several years (McElroy, 1960; Chase, 1960, 1964; Nicol, 1960, 1962a, b, 1963, 1964; McElroy and Glass, 1961; McElroy and Seliger, 1962, 1963; Cormier and Totter, 1964; Boden and Kampa, 1964; Klein, 1965; Schneider, 1965; Johnson and Shimomura, 1966). As a subject of fundamental research, bioluminescence has both a general and a specific significance. The former stems from the possi­ bility of contributing, through studies of luminescence systems, to an understanding of such problems as photochemical reactions, inter­ mediary metabolism, and kinetics of biological processes in nonluminous as well as luminous organisms. The specific significance is that embodied in any fundamental study of one or more aspects of a living organism or biological process as such, whether it be from the point of view of morphology, histology, cytology, biochemistry, physi­ ology, taxonomy, ecology, or whatever. With reference to the more general significance, noteworthy is the use of bioluminescence as a "tool" for investigating the kinetics of enzyme and other reactions of biological importance (cf., Johnson, 1948). For this purpose, luminescence has unique advantages, for it is perhaps the only process in the living world that has a natural, visible indicator, viz., the intensity of emitted light, of reaction velocity in the rate-limiting step; the intensity is proportional to instantaneous velocity; the light intensity can be easily and accurately measured; and under appropriate conditions the same indicator can be used for the reaction either in intact, living cells or in purified, cell-free extracts. Bacterial luminescence, in particular, has proved useful as such a tool and, in fact, has provided a key to the understanding of certain aspects of the influence of temperature, hydrostatic pressure, narcotics, and some other chemical agents on the rates of biological processes. Up to the mid 1930's, a fairly extensive literature had been accumulat­ ing along three seemingly rather separate lines of endeavor: (1) the biological effects of hydrostatic pressure, with practically no thought of a relation to temperature or narcotics; (2) the biological effects of temperature, with virtually no mention of pressure and a very un­ satisfactory understanding of the relation between temperature and narcotic action; and (3) the action of narcotics, with no known rela­ tion to the effects of pressure and a very unsatisfactory understanding of the role of temperature. In 1935, the advent of the theory of absolute reaction rates (Eyring, 1935; Glasstone, Laidler, and Eyring, 1941)

FRANK Η. JOHNSON

provided a rational basis for interpreting the effects of both tempera­ ture and hydrostatic pressure on chemical reaction rates. A few years later (Johnson, Brown, and Marsland, 1942), bacterial luminescence became the first process in living cells to which this theory was applied. The quantitative effects of increased hydrostatic pressure, at various temperatures relative to the particular temperature-activity curve for luminescence in the specific organism involved, on the rate or inten­ sity of luminescence became understandable on the basis, first, that a large volume increase of activation, probably due to a partial unfolding of the native structure of an enzyme normally limiting the light-emit­ ting process, accompanies the catalytic activity of the enzyme, and second, that an even larger volume increase of reaction characterizes a mobile equilibrium through which the enzyme undergoes a reversible, thermal denaturation from the active native state to the inactive denatured state. Various narcotics, such as alcohol, chloroform, urethane, and others, were found to promote the reversible denaturation, as indicated in an analysis of the action of increased hydrostatic pres­ sure, which caused a prompt reversal of the effects of the narcotics. While the quantitative interrelations in the influence of temperature, pressure, and narcotics on luminescence intensity were first established with living cells of luminous bacteria, some closely parallel results were eventually obtained with cell-free extracts (Strehler and Johnson, 1954) in additional support of the theory which had been advanced. Analogous results of increased pressure were subsequently obtained with respect to the alcohol narcosis of tadpoles (Johnson and Flagler, 1951a, b), as well as of single nerve fibers (Tasaki and Spyropoulos, 1957). The generality of interrelations in the biological effects of tempera­ ture, pressure, and chemical factors, elucidated in part through studies on bacterial luminescence, is attested by a wealth of more recent data (cf., Johnson, Eyring, and Polissar, 1954; Johnson, 1957). Although research on pressure-temperature relations of biological phenomena appears to be now at a low ebb, it is to be hoped that it will be resumed from the luminescence and other approaches in due course. Practically nothing is yet known concerning the influence of these factors on life processes of organisms indigenous to the deep sea, apart from some studies on barophilic bacteria (ZoBell and Johnson, 1949; ZoBell, 1952, and later papers). In regard to the more specific significance of bioluminescence re­ search, i.e., with regard to particular types of luminous organisms and their light-emitting systems, the older literature contains numerous

INTRODUCTION

studies from the viewpoints of physiology, morphology, histology, biochemistry, etc. (Harvey, 1952). In recent years Nicol (cf. his review papers referred to above) has made a number of important studies on the physiology of excitation and response of luminescence, on physical characteristics of the light, and on histology and cytology of photogenic tissues. The electrophysiology of the luminescent flash of Noctiluca is dealt with comprehensively and in detail by Eckert in this book. Morphology of photogenic organs, especially of fishes, has been investigated by Kuwabara (1955); Iwai (1958, 1959); Iwai and Asano (1958); Iwai and Okamura (1960), and by Haneda and col­ leagues (cf. Haneda and Johnson, 1962, and references in Haneda's articles in this book). With respect to the structure of photogenic cells and organs, it is noteworthy that, apart from bacteria, in which no structural feature unique to luminescent species was found by the methods available in early studies with the electron microscope (John­ son, Zworykin, and Warren, 1943) or would a prion be expected, elec­ tron microscopy has been used for the most part only within approxi­ mately the past five years. Pioneering studies of the electron micros­ copy of firefly lanterns were made by Beams and Anderson (1955) and by Kluss (1958). Some outstanding new studies with the electron microscope are now available in regard to the same (Smith, 1963) as well as other types of photogenic organs or cells, including Gonyaulax (DeSa, Hastings, and Vatter, 1963) and other dinoflagellates (Sweeney and Bouck), cephalopods (Okada), and various marine invertebrates and vertebrates (Bassot), the work of the last four of these authors being well represented by their papers in this book. The biochemistry of light-emitting systems has long been an espe­ cially fascinating subject. It may be said to have begun with the work of Dubois (1885, 1887), which is important to mention here in the interest of understanding the present status inasmuch as it led to such deeply entrenched ideas that subsequent progress was in some respects greatly furthered while in others definitely hindered. Dubois suc­ ceeded in preparing, from the West Indies elaterid firefly beetle Pyrophorus (1885) and from the boring clam Pholas (1887), crude extracts which when mixed together gave a light-emitting reaction. The procedure with each of these organisms was first to grind the photo­ genic tissue in cold water and leave the homogenate standing until the light disappeared and, second, to extract another portion of photogenic tissue from the same type of organism with hot water, and cool quickly. To the relatively heat-labile, active component in the cold-water extract he gave the name "luciferase," while to the relatively heat-

FRANK Η. JOHNSON

stable component he gave the name "luciferine." Luminescence re­ sulted on mixing at room temperature the solutions of luciferin and luciferase, and this became known as a "luciferin-luciferase reaction." The properties of diffusibility, heat stability, etc. indicated that luciferin was the substrate for the enzyme, luciferase, in biologically specific luminescence systems; luciferin of Pyrophorus would not give a light-emitting reaction with luciferase of Pholas or vice versa. Harvey carried out similar experiments, with and without certain refinements, on a great variety of luminous species and, in the period 1916 to 1931, added four more types of organisms from which extracts analogous to those of Dubois could be obtained and would give a luciferin-luciferase reaction (cf., Harvey, 1952): lampyrid fireflies (Photinus, Photuris, Luciola), ostracod crustaceans (Cypridina, Pyrocypris), a polychaete annelid (Odontosyllis), and a decapod shrimp [Systellaspis). From many, many other luminescent organisms, no extracts which would give evidence of a luciferin-luciferase reaction were obtained by the procedures used. Nevertheless, Harvey religiously tested essentially all the extracts for the possibility of light production in "cross-reaction" of the luciferin or luciferase which they contained or were intended to contain, with extracts of Cypridina, which were known to contain the components in question. Although some of his early experiments gave misleading results, it eventually became clear that (1) the majority of different types of luminous organisms would not yield by the original procedure, or various modifications thereof, crude extracts that would give a luciferin-luciferase reaction; (2) luminescent cross-reactions of the luciferin definitely or presumably contained in extracts from one type of organism with luciferase defi­ nitely or presumably contained in extracts from a different organism took place only if the organisms were biologically closely related, e.g., two genera of ostracods, or fireflies in different genera or families. Attempts by various workers to obtain extracts that give a luciferinluciferase reaction, however, have continued to the present day, in the firm belief that essentially all bioluminescence systems involve a specific enzyme-substrate system, with or without the requirement of cofactors such as ATP, which McElroy (1947) found necessary for luminescence of firefly extracts. At present, the total number (14) of luminescence systems which have been obtained in vitro from different types of luminous organisms is nearly three times the total (5) obtained up to 1931 by Dubois and Harvey. Since 1950 they include the following:* the fresh-water •Earthworms were inadvertently omitted from this list; see pp. 381 and 385.

8

INTRODUCTION

limpet Latia (Bowden, 1950), luminous bacteria, Achromobacter and Photobaetenum (Strehler, 1953; McElroy, Hastings, Sonnenfeld, and Coulombre, 1953), dinoflagellate protozoa, Gonyaulax (Hastings and Sweeney, 1957), the teleost fishes Parapriaeanthus and Apogon (Haneda and Johnson, 1958; Haneda, Johnson, and Sie, 1958), lumi­ nous fungi, Collybia and Armillaria (Airth and McElroy, 1959), the sea pansy Renilla (Cormier, 1959), the acorn worm Balanoglossus (Dure and Cormier, 1961, 1963), the hydromedusan jellyfish Aequorea and Halistaura (Shimomura, Johnson, and Saiga, 1962a; 1963b, c), and the polychaete annelid Chaetopterus (Shimomura and Johnson, this book). According to present evidence, which is convincing in some instances but uncertain in others, no special cofactors are required for luminescence of nearly half of these systems, viz., those of Cypridina, Latia, Pholas, decapod shrimp, protozoa, and fish. The specific cofactors or activators of the other systems are various; they include ATP and Mg + + (firefly), FMNH 2 (fiavine mononucleotide, reduced form) and long-chain aliphatic aldehyde (bacteria), DPNH (diphosphopyridine nucleotide, reduced form) (fungi), DPA (3',5', diphospho-adenosine) (sea pansy), H 2 O 2 (acorn worm), CN - (Odontosyllis, cf., Shimomura, Beers, and Johnson, 1964), Ca++ or Sr+ + (jellyfish), and Fe++ and a peroxide (Chaetopterus). For any one system, complete purification of the essential compo­ nents is prerequisite to investigating the reaction mechanism, determin­ ing the quantum efficiency, analyzing the structure of the light emitting molecule and achieving its synthesis, and learning the biochemical relationship, if any, to other systems. A critical appraisal of the present outlook all but justifies the view that this is practically a virgin field of endeavor. Some of the reasons are as follows. First of all, the substrates in only three different systems have been chemically identified and the enzymes of these systems essentially fully purified. Firefly luciferase was crystallized in 1956 by Green and McElroy, and firefly luciferin in 1957 by Bitler and McElroy. The structure of firefly luciferin was established and proved by its total synthesis in 1961 by White, McCapra, Field, and McElroy. Cypridina luciferin was crystallized in 1957 by Shimomura, Goto, and Hirata, and its structure established in 1965 by Kishi, Goto, Hirata, Shimo­ mura, and Johnson (reported in this book; total synthesis has since been achieved). Cypridina luciferase was brought to essentially com­ plete purification in 1961, independently by Shimomura, Johnson, and Saiga and by Tsuji and Sowinski. Purified bacterial luciferase was crystallized in 1965 by Kuwabara, Cormier, Dure, Kreiss, and Pfuderer.

FRANK Η. JOHNSON

Bacterial luciferin has become a matter of definition. To the extent that FMNH 2 may be considered the "luciferin" of this system, which presumably also requires any one of a number of possible long-chain aliphatic aldehydes-—no activating aldehyde has yet been isolated from luminous bacteria-—it may be considered that bacterial luciferin has been synthesized. Recent studies of this system have given evidence of a precursor compound (Terpstra, 1963) and of several intermediary reactants (Hastings and Gibson, 1963) in the aerobic oxidation of FMNH 2 leading to the production of light. The designation of any particular one in the series as "luciferin" involves some departure from the original meaning of the term, as foreseen in discussions by Harvey and Tsuji (1954). An added semasiological complication re­ sults from the discovery (Gibson, Hastings, and Greenwood, 1965) that luminescence of purified bacterial luciferase, in the absence of FMN but in the presence of aldehyde, can be initiated by irradiation with ultraviolet or visible light, and the further discovery (Hastings, private communication) that the emission spectrum is the same as that of the complete system. This discovery has implications concerning the func­ tioning of other types of biological systems. In any event, it is to be noted that, while the diffusible, oxidizable organic substrate, or "luciferin," is chemically far from the same in these three systems, the analogous component of no other luciferin-luciferase system has yet been chemically identified. Thus, although there has been a tendency to conclude, on various indirect lines of evidence, that the luciferins of essentially all, not just the few known or partially known, different types of luminous or­ ganisms are different from each other, and somewhat to disdain the idea that there may be a thread of comparative biochemistry among luminescence systems (Glass, 1961), the conclusion is possibly a bit premature. Moreover, as Cormier and Totter (1964) point out, indole derivatives or compounds "that mimic an indole" are represented in the luciferins extracted from several different systems—Cypridina, fish, the acorn worm; also, the sea pansy (Hori and Cormier, 1965). It may become necessary to omit fish from this list if it is ultimately proved that the luciferin extracted from it originates in Cypridina it has eaten, as discussed by Haneda et al. in this book. The luciferin and luciferase components of the fish and Cypridina systems crossreact to produce light; i.e., the luciferin and luciferase from either the fish or Cypridina are functionally interchangeable (Haneda, Johnson, and Sie, 1958; Johnson, Haneda, and Sie, 1960; Sie, McElroy, Johnson, and Haneda, 1961), and crystalline luciferin from the fish Parapria-

INTRODUCTION

canthus is chemically the same, in major respects, as that from Cypri­ dina (Johnson, Sugiyama, Shimomura, Saiga, and Haneda, 1961). This is, thus far, a unique exception to the rule that luminescent cross-reactions take place only between components extracted from biologically closely related species, a circumstance favoring the view that the fish luciferin does indeed derive from ingested Cypridina. On the other hand, evidence presented by Tsuji and Haneda in this book favors the view that each of these two types of distantly related organisms produces its own functional luciferase. It is worth noting that the quantum efficiency of light emission has been determined with the three systems that- have been sufficiently purified to permit reliable data, and with some startling results. Thus, a seemingly fantastic quantum efficiency of unity was found for the luminescent oxidation of firefly luciferin (Seliger and McElroy, 1959, 1960). Under best conditions, the luminescent oxidation of Cypridina luciferin is characterized by a quantum efficiency of 0.28 ± 15% (Johnson, Shimomura, Saiga, Gershman, Reynolds, and Waters, 1962). The quantum efficiency in the luminescent oxidation of FMNH2 by bacterial luciferase depends on the chain length of the aliphatic alde­ hyde supplied (Hastings, Spudich, and Malnic, 1963). With dodecyl aldehyde and partially purified luciferase, quantum efficiencies of 0.28 and 0.34, depending on the enzyme preparation used, were found by Cormier and Totter (1957), and an efficiency of 0.3, with decanal and enzyme purified by the method of Hastings, Riley, and Massa (1965), was found by Hastings and Gibson (1963). A quantum yield of about 0.27 per bacterial luciferase molecule was calculated by Hastings, Riley and Massa (1965). For one other bioluminescence system a reasonable value of the quantum efficiency of the practically pure system is available, subject to correction when a definitive value for the molecular weight of the photogenic substance is obtained, viz., that of the reaction of the bioluminescent protein, aequorin, with calcium. On the basis of an indirectly estimated molecular weight of 35,000 for aequorin, the quantum efficiency amounts to 0.14 (Johnson et al., 1962). All these values are much higher than the usual quantum yields of non-biological substances in aqueous solution. For instance, the quantum yield of chemiluminescence of dimethylbiacridylium nitrate in alkaline solution has an average value of 0.016, whether the chemi­ luminescence is induced by H2O2 or by the enzyme xanthine oxidase plus hypoxanthine (Totter, 1964). Among the above-mentioned biological systems, the one extracted and purified from the jellyfish Aequorea is of more than passing in-

FRANK Η. JOHNSON

terest, for at least two reasons. First, it appears to be comprised of a single organic component, the protein aequorin, which specifically requires only Ca + + or Sr++ for luminescence in aqueous solution (Shimomura, Johnson, and Saiga, 1962a). Moreover, efforts to find evidence that this protein possesses the properties of an enzyme have given only negative results. Thus the terms "luciferin" and "luciferase" cannot be construed to apply to this system, at least in their usual, albeit not altogether precise, meaning. In the opinion of the present author, there is no real justification for referring in recent reviews (Nicol, 1963; Boden and Kampa, 1964) to aequorin as a type of luciferin. A more appropriate term would seem to be a type of "photoprotein," suggested by Shimomura and Johnson in the paper on Chaetopterus in this book. The actual number of systems analogous to that of Aequorea, i.e. systems which consist of a single organic component, remains to be determined. Isolation of this system was undoubtedly delayed because of the profoundly embedded idea that probably all bioluminescence systems involve distinct and separable luciferin and luciferase components. A second, unusual point of interest about the luminescence of purified aequorin is that quantitatively the same amount of light is emitted whether the aequorin is added to solutions equilibrated with pure hydrogen, pure oxygen, or air. Only three types of luminous organisms had been known in which the luminescence system could, under appropriate conditions, emit significant, visible light in virtually the complete absence of oxygen, viz., certain radiolarians, jellyfish, and ctenophores (Harvey, 1952). A fourth type of luminescence system, that of Balanoglossus requires H2O2 but not oxygen directly (Cormier and Dure, 1963). It has been suggested (Hastings and Gibson, 1963; McElroy and Seliger, 1963; Cormier and Totter, 1964) that aequorin is analogous to the kinetically evidenced, relatively long-lived, reduced enzyme-oxygen adduct, "intermediate II," of the bacterial system, which luminesces on the addition of aldehyde in absence of oxygen (Hastings and Gibson, 1963). It would be interesting to have definite evidence concerning this conjecture, for example through the biochemical isolation and the determination of the chemi­ cal properties of intermediate II. Of the luminescence systems which have been successfully extracted from 14 different types of organisms, 10 have been significantly purified in the sense that at least a meaningful absorption spectrum has been obtained of at least one of the essential components. These 10 include, along with the fully purified systems of the firefly, Cypn-

INTRODUCTION

dina, and bacteria, those of certain fish, fungi, jellyfish, Odontosyllis, sea pansy, the fresh water limpet Latia, and Chaetopterus. It is interesting to note, apropos of the sponsorship of this book and the Conference upon which it is based, that purification of four-fifths of these systems, i.e., all except those of the firefly and fungi, has resulted in part from active cooperation between Japanese and American scientists. Furthermore, purification of two of these systems (Latia and Chaetopterus), reported here for the first time, as well as elucida­ tion of the structure of Cypridina luciferin, was accomplished in a project directly supported in part by the U.S.-Japan Cooperative Science Program. Progress in research on bioluminescence during the past several years, especially from a biochemical point of view, has been impressive, despite the relatively few investigators involved. Some of the puzzling features of systems discussed in this book, for example the seemingly particulate nature of fungal luciferase (Airth and Foerster, 1962) and the precise role of scintillons (DeSa, Hastings, and Yatter, 1963) in protozoan luminescence, as well as other such problems which are disconcerting to the specialist and easily recognized by the thoughtful reader, will no doubt be resolved in due course. In the biochemistry of luminescence the problems which remain are manifold, challenging, and involve certain special difficulties, not the least of which is the difficulty of getting an adequate amount of the raw material. The era is past when a significant biochemical discovery could be made with a few individual specimens, as when Harvey demonstrated the Odontosyllis luciferin-luciferase reaction with ex­ tracts of only two of these worms, each perhaps a centimeter long and one or two millimeters in diameter. About 25,000 worms, collected with the aid of hand-nets by many friends and members of the Bermuda Biological Station, were required for obtaining microgram quantities of very highly purified luciferin and determining some of its properties (Shimomura, Johnson, and Saiga, 1963d), including the fact that it is a colorless and non-fluorescent substance, rather than a fluorescent one as had been supposed earlier from observations with partially purified preparations (McElroy, 1960; McElroy and Seliger, 1963). Such differences between results obtained with highly purified preparations and with partially purified preparations emphasize the real importance of the former. Misleading conclusions often arise from observations and data pertaining to the latter type of preparation; e.g., solutions of Cypridina luciferin, which for years were considered highly purified (ca., 2000 fold, Anderson, 1935) gave no evidence of

FRANK Η. JOHNSON

fluorescence under ultraviolet light, whereas solutions of crystalline luciferin (purified ca. 40,000 fold) are brilliantly fluorescent under ultraviolet. Misleading results may easily be obtained also with regard to the action of cofactors, activators, inhibitors, etc.; e.g., with the above-mentioned purified (ca. 2000X) Cypridina preparations, it appeared that low concentrations of cyanide combined irreversibly with the luciferin, preventing its luminescent reaction with luciferase (Giese and Chase, 1940), whereas with pure luciferin the same and higher concentrations of cyanide had no effect, except at extremely low luciferin concentrations at which the luciferin is particularly unstable and its destruction is catalyzed by traces of heavy metals and cyanide (Johnson, Shimomura, and Saiga, 1962b). As already pointed out, the great majority of bioluminescence sys­ tems are chemically unidentified. In most instances one or more of the active components are probably present in only infinitesimal amounts, amidst numerous other substances having more or less closely similar chemical and physical properties. Moreover, an essential component may be unstable in the presence of small concentrations of oxygen. Further, an essential component is frequently very unstable in crude or partially purified extracts, as compared to the finally isolated product. For obtaining the large quantities of raw material needed, only bacteria, fungi, and protozoa can be feasibly supplied through cultivation in the laboratory. Elsewhere, with the exception of fireflies, which can be procured by enlisting an army of juveniles at a nominal fee, Nature seems to have contrived to keep luminous organisms out of the hands and homogenizers of the biochemist. Some of the most in­ teresting types of organisms, for example many luminescent fishes, can only be had as an occasional specimen brought up by means of complicated gear from the depths of the sea; others, such as the firefly squid Watasenia, though seasonally collectable in vast numbers with the aid of fishermen using surface nets, generally fail to survive a brief trip to a nearby laboratory, and no way has yet been found to restore the property of luminescence after these organisms have suc­ cumbed. The tiny fireworm Odontosyllis also occurs in large numbers, but only for a period of half an hour or so, beginning about an hour after sunset, and only for 3 or 4 days after a full moon; no better method of collecting them has been found than by small nets in the hands of the maximum possible number of individuals who can be induced to go out for this purpose, preferably in a grand flotilla of small boats. The reasonably large (up to 10 cm in diameter) jellyfish Aequorea is also best collected by hand net when it appears at erratic

INTRODUCTION

times during summer months. The worm Chaetopterus occurs in parch­ ment-like tubes, buried in the sub-tidal ocean floor, or on the floor but overgrown with various types of marine life, and it generally requires the skills of a scuba diver to dislodge it from its habitat. The large, brightly luminous earthworm Octochaetus lives in thick clay sub-soil from which it can be recovered only by blind, back-straining search with spade or fork, unless a steam shovel is available or a bulldozer happens to strike a bonanza of them. The New Zealand glowworm Arachnocampa occurs most abundantly on virtually un­ reachable ceilings and walls in deep recesses of dark, slippery caves. These examples could easily be multiplied. They add to the challenge. Although progress in research on bioluminescence throughout the past several years has been gratifying, we would perhaps do well to temper any rejoicing over recent successes with the sobering reflection that even in the most thoroughly studied system, namely the firefly, no satisfying answer can yet be given to the seemingly simple, innocent question, "how does it flash?" May bioluminescence continue in progress! REFEREN CES

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FRANK Η. JOHNSON

addressed to the Publisher and presented to the R. Society. Phil. Trans. Roy. Soc., London 7:5108-5116. Chase, A. M. 1960. The measurement of luciferin and luciferase. In D. Glick (ed.) Methods of Biochemical Analysis, Vol. 8, pp. 61-117. Wiley (Interscience), New York. . 1964. Bioluminescence—Production of light by organisms. In A. C. Giese (ed.), Photophysiology, Vol. 2, pp. 389-421. Academic Press, Inc., New York. Cormier, M. J. 1959. Adenosine-5'-triphosphate requirement for lumi­ nescence in cell-free extracts of ReniUa reniformis. J. Am. Chem. Soc. 81:2592. Cormier, M. J., and L. S. Dure. 1963. Studies on the bioluminescence of Balanoglossus biminiensis extracts. I. Requirement for hydrogen peroxide and characteristics of the system. J. Biol. Chem. 238:785789. Cormier, M. J., and J. Totter. 1957. Quantum efficiency determinations on components of the bacterial luminescence system. Biochim. Biophys Acta. 25:229-237. . 1964. Bioluminescence. Ann. Rev. Biochem. 33:431-458. DeSa, R., J. W. Hastings, and A. E. Vatter. 1963. Luminescent "crystalline" particles: an organized subcellular bioluminescent system. Science 141:1269-1270. Dubois, R. 1885. Fonction photogenique des pyrophores. Compt. Rend. Soc. Biol. 37:559-562. . Note sur la fonction photogenique chez Ies Pholades. Compt. Rend. Soc. Biol. 39:564-566. Dure, L. S., and M. J. Cormier. 1961. Requirements for luminescence in extracts of a balanoglossid species. J. Biol. Chem. 236:PC48-PC49. . 1963. Studies on the bioluminescence of Balanoglossus bi­ miniensis extracts. II. Evidence for the peroxidase nature of bal­ anoglossid luciferase. J. Biol. Chem. 238:790-793. Eyring, H. 1935. The activated complex in chemical reactions. J. Chem. Phys. 3:107-115. Gibson, Q. H., J. W. Hastings, and C. Greenwood. 1965. On the molec­ ular mechanism of bioluminescence. II. Light-induced luminescence. Proc. Natl. Acad. Sci. U.S. 53:187-195. Giese, A. C., and A. M. Chase. 1940. The effects of cyanide on Cypridina luciferin. J. Cellular Comp. Physiol. 16:237-246. Glass, B. 1961. Summary. In W. D. McElroy and B. Glass (eds.), A Symposium on Light and Life, pp. 817-910 see under, "Bio-

INTRODUCTION luminescence," pp. 847-858. The Johns Hopkins Press, Baltimore, Md. Glasstone, S., K. J. Laidler, and H. Eyring. 1941. The Theory of Rate Processes. McGraw-Hill, New York. Green, A. A., and W. D. McElroy. 1956. Crystalline firefly luciferase. Biochim. Biophys. Acta 20:170-176. Haneda, Y., and F. H. Johnson. 1958. The luciferin-luciferase reaction in a fish, Parapriacanthus beryciformes, of newly discovered lu­ minescence. Proc. Natl. Acad. Sci. U.S. 44:127-129. . 1962. The photogenic organs of Parapriacanthus beryciformes Franz and other fish with the indirect type of luminescent system. J. Morphol 110:187-198. Haneda, Y., F. H. Johnson, and H.-C. Sie. 1958. Luciferin and lucif­ erase extracts of a fish, Arogon marginatus, and their luminescent cross-reactions with those of a crustacean, Cypridina hilgendorfii. Biol. Bull. (WoodsHole) 115:336. Harvey, E. N. 1952. Bioluminescence. Academic Press, New York. . 1957. A History of Luminescence. American Philosophical Society, Philadelphia. Harvey, E. N., and F. I. Tsuji. 1954. Luminescence of Cypridina luciferin without luciferase together with an appraisal of the term luciferin. J. Cellular Comp. Physiol. 44:63-76. Hastings, J. W., and Q. H. Gibson. 1963. Intermediates in the bioluminescent oxidation of reduced flavine mononucleotide. J. Biol. Chem. 238:2537-2554. Hastings, J. W., W. H. Riley, and J. Massa. 1965. The purification, properties, and chemiluminescent quantum yield of bacterial lucif­ erase. J. Biol. Chem. 240:1473-1481. Hastings, J. W., J. Spudich, and G. Malnic. 1963. The influence of aldehyde chain length upon the relative quantum yield of the bioluminescent reaction of Achromobacter fischeri. J. Biol. Chem. 238: 3100-3105. Hastings, J. W., and Β. M. Sweeney. 1957. The luminescent reaction in extracts of the marine dinoflagellate, Gonyaulax polyedra. J. Cellular Comp. Physiol. 49:209-225. Hori, K., and M. J. Cormier. 1965. Studies on the bioluminescence of Renilla reniformis. Y. Absorption and fluorescence characteristics of chromatographically pure luciferin. Biochim. Biophys. Acta 102:386396. Iwai, T. 1958. A study of the luminous organ of the apogonid fish,

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INTRODUCTION

Klein, Η. Α. 1965. Bioluminescence. J. Β. Lippincott Co., Philadel­ phia/New York. Kluss, B. C. 1958. Light and electron microscope observations on the photogenic organ of the firefly, Photurus pennsylvanica, with special reference to the innervation. J. Morphol., 103:159-186. Kuwabara, S. 1955. Some observations on the luminous organ of the fish, Paratraehichthys prosthemius Jordon and Fowler. J. Shimonoseki Col. Fisheries 4:81-85. Kuwabara, S., M. J. Cormier, L. S. Dure, P. Kreiss, and P. Pfuderer. 1965. Crystalline bacterial luciferase from Photobaeterium fischeri. Proc. Natl. Acad. Sci. U.S. 53:822-828. McElroy, W. D. 1947. The energy source for bioluminescence in an isolated system. Proe. Natl. Acad. Sci. U.S. 33:342-345. . 1960. Bioluminescence. Federation Proc. 19:941-947. McElroy, W. D., and B. Glass (eds.). 1961. Light and Life. Johns Hopkins Press, Baltimore, Md. McElroy, W. D., and A. Green. 1956. Function of adenosine triphos­ phate in the activation of luciferin. Arch. Biochem. Biophys., 64:257271. McElroy, W. D., J. W. Hastings, V. Sonnenfeld, and J. Coulombre. 1953. The requirement of riboflavine phosphate for bacterial lu­ minescence. Science 118:385-386. McElroy, W. D., and H. H. Seliger. 1962. Biological luminescence. Sci. Am. 207:76-89. . 1963. The chemistry of light emission. Adv. Enzymol. 25:119166.

Nicol, J. A. C. 1960. The regulation of light emission in animals. Biol. Rev. Cambridge Phil. Soc. 35:1-42. . 1962a. Bioluminescence. Proc. Roy. Soc. London, Ser. A, 265:355-359. . 1962b. Animal luminescence. In 0. Lowenstein (ed.), Adv. Comp. Physiol. Biochem. 1:217-273. Academic Press, New York. . 1963. Luminescence in animals. Endeavour 22:37-41. . 1964. Luminous creatures of the sea. Sea Frontiers 10:143-154. Schneider, F. 1965. Pleins feux dans la nature. Sciences et Avenir, No. 222, 538-543; 573. Seliger, H. H., and W. D. McElroy. 1959. Quantum yield in the oxida­ tion of firefly luciferin. Biochem. Biophys. Res. Commun. 1:21-24. . 1960. Spectral emission and quantum yield of firefly biolu­ minescence. Arch. Biochem. Biophys. 88:136-141.

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Shimomura, 0., J. R. Beers, and F. H. Johnson. 1964. The cyanide ac­ tivation of Odontosyllis luminescence. J. Cellular Comp. Physiol. 64:15-22. Shimomura, 0., T. Goto, and Y. Hirata. 1957. Crystalline Cypridina luciferin. Bull. Chem. Soc. Japan 30:929-933. Shimomura, 0., F. H. Johnson, and Y. Saiga. 1961. Purification and properties of Cypridina luciferase. J. Cellular Comp. Physiol. 58: 113-124. Shimomura, 0., F. H. Johnson, and Y. Saiga. 1962a. Extraction, puri­ fication and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J. Cellular Comp. Physiol. 59:223-240. . 1962b. Action of cyanide on Cypridina luciferin. J. Cellular Comp. Physiol. 59:265-272. . 1963a. Microdetermination of calcium by aequorin lumines­ cence. Science 140:1339-1340. . 1963b. Further data on the bioluminescent protein, aequorin. J. Cellular Comp. Physiol. 62:1-8. . 1963c. Extraction and properties of halistaurin, a biolumines­ cent protein from the hydromedusan Halistaura. J. Cellular Comp. Physiol. 62:9-16. . 1963d. Partial purification and properties of the Odontosyllis luminescence system. J. Cellular Comp. Physiol. 61:275-292. Sie, H.-C., W. D. McElroy, F. H. Johnson, and Y. Haneda. 1961. Spectroscopy of the Apogon luminescent system and of its cross reaction with the Cypridina system. Arch. Biochem. Biophys. 93:286291. Smith, D. S. 1963. The organization and innervation of the luminescent organ in a firefly, Photuris pennsylvanica (Coeloptera). J. Cell. Biol. 16:323-359. Strehler, B. L. 1953. Luminescence in cell-free extracts of luminous bacteria and its activation by DPN. J. Am. Chem. Soc. 75:1264. Strehler, B. L., and F. H. Johnson. 1954. The temperature-pressure-in­ hibitor relations of bacterial luminescence in vitro. Proc. Natl. Acad. ScL U.S. 40:606-617. Strehler, B. L., and J. R. Totter. 1952. Firefly luminescence in the study of energy transfer mechanisms. I. Substrate and enzyme determina­ tion. Arch. Biochem. Biophys. 40:28-41. Tasaki, I., and C. S. Spyropoulos. 1957. Influence of changes in tem­ perature and pressure on the nerve fiber. In F. H. Johnson (ed.),

INTRODUCTION

The Influence of Temperature on Biological Systems, pp. 201-220. American Physiological Society, Inc., Washington, D.C. Terpstra, W. 1963. Investigations on the identity of the light-emitting molecule in Photobacterium phosphoreum. Biochim. Biophys. Acta 75:355-364. Totter, J. R. 1964. The quantum yield of the chemiluminescence of dimethylbiacridylium nitrate and the mechanism of its enzymically induced chemiluminescence. Photochem. Photobiol. 3:231-241. Tsuji, F. I., and R. Sowinski. 1961. Purification and molecular weight of Cypridina luciferase. J. Cellular Comp. Physiol. 58:125-129. White, E. H., F. McCapra, G. Γ. Field, and W. D. McElroy. 1961. The structure and synthesis of firefly luciferin. J. Am. Chem. Soc. 83: 2402-2403. ZoBell, C. E. 1952. Bacterial life at the bottom of the Philippine trench. Science 115:507-508. ZoBell, C. E., and F. H. Johnson. 1949. The influence of hydrostatic pressure on the growth and viability of terrestrial and marine bac­ teria. J. Bacteriol. 57:179-189.

Chemical Events Leading to Ghemiluminescence of Lucigenine and Luminol JOHN

R.

TOTTER

1

ABSTRACT

It has been shown that enzymatic reduction of lucigenine in the presence of oxygen leads to chemiluminescence. The redox potential of lucigenine is —0.093 and is independent of pH. This is sufficiently high that it is capable of reduction by H 0 2 ~ or N H 4 O H at pH's above 10 rapidly enough to account for the production of light seen with these reagents. The nature of the oxidation steps following reduction are not yet fully elucidated. Determination of the effect of maintaining a constant oxygen supply on the intensity of chemiluminescence by luminol in K2S2O8 solution leads to the suggestion that one of the steps leading to light production in this case is also a reduction by H 0 2 ~ . This leads to a tentative sequence of reactions involving a radical attack and formation of H 2 0 2 (through a series of reactions with 0 2 ) and of

23

JOHN R. TOTTER dehydro 5-amino phthalazinedione which exchanges its nitrogen for oxygen. The peranhydride (?) formed is reduced to activated aminopthalate ion by Η (>2~. Stoichiometry also requires a reduction in the uncatalyzed H2O2 chemiluminescence if aminophthalate is the end product. 1

United States Atomic Energy Commission, Washington 25, D.C.

LUCIGENINE AND LUMINOL

LUCIGENINE

In a series of researches covering the past seven years the lumines­ cent response of dimethylbiacridylium nitrate (Iucigenine) to enzymic reactions, particularly xanthine oxidase and its substrates, have been studied (Totter, Scoseria, and Medina, 1959; Totter, Medina, and Scoseria, 1960; Totter, de Dugros, and Riveiro, 1960; DeAngelis and Totter, 1964; and Totter, 1964). These investigations have fully estab­ lished that lucigenine behaves as a redox dye in a manner closely resembling methylene blue and other hydrogen transfer agents long used in biochemistry. The difference lies in the fact that upon autooxidation a small fraction of the reduced lucigenine gives rise to methylacridone in an excited state and the latter emits light or trans­ fers its excitation energy to other intermediates which then emit at their characteristic fluorescent wavelengths. On the basis of kinetic evidence (Totter, 1964) it was postulated that the small fraction of reduced lucigenine that gives rise to methyl­ acridone first undergoes a second reduction to produce N,N-dimethylbiacridan, and is then attacked by a radical—probably arising from the auto-oxidation of the first reduction product—which abstracts a hydrogen atom. Oxygen is presumed to react with the biacridan radi­ cal, and through rearrangement and a reverse pinacol-type reaction the latter splits into a molecule of methylacridone and one of a some­ what more reduced form. In later work Totter and Philbrook (1965) have determined the redox potential of lucigenine (—0.093 volts). Calculations were made showing that either hydrogen peroxide or ammonium hydroxide at elevated pH's is thermodynamically capable of reducing this com­ pound to produce a "steady-state" level of the reduced intermediate of sufficient concentration to account for the light intensity seen in uncatalyzed reactions with these reagents. It was found that the reac­ tion with NH 4 OH is slow in contrast with the auto-oxidation or the hydrogen peroxide reaction, and consequently in the presence of even the small amount of peroxide formed by auto-oxidation of the reduced dye the H2O2/O2 redox couple could easily dominate the (presumed) NH4OH /N2H4 couple. Thus, it is possible to inhibit light production in NH 4 OH by passing air or oxygen through the solution, a finding

JOHN

R.

TOTTER

which seems strange in the light of the supposed almost universal re­ quirement of oxygen for chemiluminescence. The equation for reduction of lucigenine by NH4OH was written by Totter and Philbrook (1965) as follows: D2+ + 2 NH4OH + OH- — DH+ + N2H4 + 3 H2O No chemical or quantitative kinetic evidence verify this postulate. The compound DH+ ridanyl-9'-acridylium ion) was suggested by (1964) as a probable intermediate because

was then available to (10,10'-dimethyl-9-acDeAngelis and Totter of the insolubility of

S 18

(NH 4 OH) 2 (OH") 2 χ IO 2

Figure 1. The relationship between maximum light intensity and "initial velocity" and the NH4OH and OH" concentrations of mixtures of NHiOH 0.005 M in lucigenine. Closed circles, maximum intensity; some uncertainty exists over the precise maximum at the lower concentrations and they are expressed as a range. Open circles, "initial velocity"; these were taken from the second minute of light evolution to avoid interference by the "blank" value.

LUCIGENINE AND LUMINOL

dimethyl 9,9' Δ biacridan (D0). However, it was found that the re­ duction potential of lucigenine determined with a dropping mercury electrode is independent of pH (Totter and Philbrook, 1965), therefore, it seems possible that the equation should be written: D2+ + 2 NH4OH + 2 OH" — D0 + N2H4 + 4 H2O The D0 in its nascent state (a biradical?) must normally be removed too rapidly by subsequent reactions to accumulate in concentrations sufficiently high to precipitate. It can, of course, be readily prepared by anaerobic reduction or in bulk where there is insufficient air to prevent its accumulation. Results presented in Figure 1 indicate that the "equilibrium" con­ centration of the intermediate which is limiting in light production may be related to the square of both the NH4OH and OH- concen­ trations. In these experiments 0.005 M lucigenine was mixed with NH4OH to the final molarities indicated in Figure 2 (14.3, 11.3, 7.5, 5.3) and the time course of light intensity followed by means of a photomultiplier tube attached to an amplifier and recorder. It may be seen that with a 2.7-fold excursion in NH4OH concentration there is a corresponding 27-fold excursion in maximum light intensity. In an earlier publication (Totter and Philbrook, 1965) it was assumed that the light intensity at "equilibrium" would be related to the first power of the NH4OH concentration. This seemed logical since the first reduction (if there are in fact two successive ones) might be expected to be limiting and the equation for the "equilibrium" could be written [D0] =

ki[D2+][NH4OH]2[0H~]2 fc_i[N2H4]

(J)

In the absence of extraneous N2H4 (and appreciable O2) the initial concentrations of N2H4 and D0 at steady state would be equal and therefore

(

fr, \ 1/2 γ-) ([D2+][NH4OH]2[OH-]2)1/2

(2)

The occurrence of a higher power dependence, then, must be either because there is a small but significant contamination of the NH4OH with hydrazine, because the reaction with oxygen in solution is suffi­ cient to influence the results, or because the equations are otherwise incorrect. Evidence for the essential correctness of equation (1), however, is

JOHN

R.

TOTTER

obtainable from the curves of Figure 2. The equation for time rate of change of would be: (3) 1 formed is extremely small (Totter and PhilSince the amount of brook, 1965) when compared with i.e.:

the relationship may be treated as the familiar equation

in which the assumption is made that the light intensity I is propor-

T i m e In Minutes

Figure 2. The time course of light emission of mixtures of NH ( OH and Iucigenine. The molarities of NH.OH (14.3, 11.3, 7.5, 5.3) are given on the figure. The final concentration of Iucigenine in all cases was 0.005 M.

28

LUCIGENINE

AND

LUMINOL

T i m e In Minutes

Figure 3. A plot of In against time as explained in the text. The light intensity at time for each of the lower three curves of Figure 1 was multiplied by the inverse ratio of its maximum intensity (/,„»*) to that of the highest curve. The points are averages of the four values thus obtained.

tional to [ D ° ] . This has the general solution:

and may be "linearized" by rearrangement and converting into the logarithmic form:

where 7 max is the maximum intensity, the intensity at time t, and C is a constant. A plot of the data from Figure 2 is given in this form in Figure 3. All four curves have been converted into a single curve by multiplying the data from the lowest curve by 27, the second by 10, and the third

29

JOHN

R.

TOTTER

b y 2.1, the inverse ratios of of the highest concentration of NH4OH. The values thus obtained show remarkably close concordance except at readings near the maxima reached. This is shown b y the drift to too high values at 8 to 10 min on the curve. These errors are probably owing to slight errors in determining the experimental maxima, which are magnified by the mathematical procedure. The ordinate intercept (lnC) represents a small blank value, probably because of p H alone since some light is given off when N a O H replaces The final determination of the correct kinetic equation awaits further study. T h e present indications are that equation (1) is nearly correct under conditions of long-lived light emission and with very low 0 2 concentrations but may not be fully descriptive of the actual mechanism. T h e presence of traces of impurities strongly affects the reactions, and fully reproducible data are not easy to obtain. When oxygen is present the equation for formation of and oxidation of the reduced intermediate should be added (Totter and Philbrook, 1965).

or

(4) The final expression becomes, for the "equilibrium" steady-state:

(5) where is the ionization constant of water. The (usually) low concentration of makes it necessary to use the conservation equation for 0 2 expressed in the denominator. This, together with the assumption that and makes it possible to explain readily the behavior of the light emission from the _ -lucigenine system when air or oxygen is conducted through the solutions (Totter and Philbrook, 1965) as well as to account for the relationship of the maximum intensity and the N H 4 O H concentration. LUMINOL

White, Zafiriou, Kagi, and Hill (1964) have isolated 3-aminophthalic acid as a major end-product of the reaction of luminol (5-amino-2,3phthalazine-l,4-dione) with oxygen in dimethylsulfoxide. The chemiluminescence of luminol and the fluorescence of aminophthalate ion

30

LUCIGENINE AND LUMINOL

occur at concordant wavelengths. It is therefore highly likely that aminophthalate ion is the emitting molecule. It is also likely that this compound results from the oxidation of luminol in aqueous alkaline solutions by potassium peroxydisulfate. The evidence is based on the transient appearance of a fluorescent compound with wavelength and molar fluorescence intensity identical with those of aminophthalate ion at the same rate that luminol disappears (Totter, Stevenson, and Philbrook, unpublished data). Totter, Stevenson, and Philbrook (1964) have indicated that the phthalazinediones are probably oxidized by S208= through a chain re­ action in which a hydrogen atom and an electron are removed. The pH dependence of those reactions has been studied (Totter and Phil­ brook, 1965) and it was found that with a constant oxygen supply the maximum light intensity increases linearly with OH- ion concentra­ tion up to about IO-2 M. Above this concentration the maximum in­ tensity is nearly constant or declines slightly. On the other hand, with unaerated solutions there is a hyperbolic relationship up to pH 12. The pK of this apparent dissociation curve is very close to 11.74, the same as that for hydrogen peroxide. These data have been interpreted as indicating that hydrogen peroxide is formed in the complex chain re­ action and reduces an oxidation product of luminol to produce the activated intermediate which emits. The change in relationship with O2 supply is entirely analogous to equation (4) above for the reduc­ tion of lucigenine by hydrogen peroxide and involves the necessity or lack of necessity for the conservation equation for O2. A reduction is required by the stoichiometry of the reaction. Ap­ parently some workers have assumed that this reduction is one involv­ ing only H2O2. It is not possible to account for the light intensity, however, on this basis, since the oxygen concentration in solution can scarcely rise above 1.25 X IO-3M under atmospheric pressure. The light intensity reached is manyfold that expected from this O2 con­ centration. The promotion of chemiluminescence in both the phthalazinediones and alkylbiacridylium compounds by alkali is thus seen to be simply the result of lowering the value of the redox potential of the O2/H2O2 couple sufficiently to reduce the proper intermediates (Totter and Philbrook, 1965). At a pH of 12 the potential of this couple is near O volts, quite low enough to reduce appreciably lucigenine with a poten­ tial of —0.093 volts. The operation of the unfavorable equilibria in­ volved gives the long "steady-state" emission (really a very slow first-order decay) of light seen with the uncatalyzed reactions. The

JOHN

R.

TOTTER

"LUCIGENINE" ION

METHYL ACRIDONE

AMINOPHTHALATE ION Figure 4. Summary of the author's suggestions for Iucigenine and for luminol.

32

LUCIGENINE AND LUMINOL

reduction potentials of the "oxidized" phthalazinedione compounds are not known but must be slightly below zero to react as observed. Since only the starting compounds and the final compounds in the reactions are known with certainty it is hazardous to speculate about the intermediates in both chemiluminescences. However, the clear recognition of reduction steps permits a somewhat more meaningful series of reactions to be written than could be done formerly. The author's suggestions for lucigenine and for luminol are sum­ marized in Figure 4. REFERENCES

DeAngelis, W. J., and J. R. Totter. 1964. J. Biol. Chem. 239:1012. Totter, J. R. 1964. Photochem. Photobiol. 3:231. Totter, J. R., E. Castro de Dugros, and C. Riveiro. 1960. J. Biol. Chem. 235:1839. Totter, J. R., V. J. Medina, and J. L. Scoseria. 1960. J. Biol. Chem. 235:238. Totter, J. R., and G. E. Philbrook. 1965. Photochem. Photobiol. 5:177. Totter, J. R., J. L. Scoseria, and V. J. Medina. 1959. Anales Fac. Med., Montevideo 44:463. Totter, J. R., W. Stevenson, and G. E. Philbrook. 1964. J. Phys. Chem. 68:752. White, E. H., 0. Zafiriou, H. H. Kagi, and J. H. M. Hill. 1964. J. Am. Chem. Soc. 86:940.

The Use of Luminol as a Standard of Photon Emission J. L E E , 1 J. F.

FERGUSON,

A.

S.

III,1

WESLEY, AND H .

H.

1

SELIGER

2

ABSTRACT

Experimental procedures are described for the use of the chemiluminescence of luminol (5-amino-2,3-dihydro-l,4-phthalazinedione) as a quantitative source of photons, permitting the absolute calibration of phototube detectors. A method is described for the measurement of the quantum yields of chemiluminescent and bioluminescent reactions which does not require the absolute calibration of phototube spectral efficiency. New England Institute for Medical Research, Ridgefield, Connecticut. McCollum-Pratt Institute and Department of Biology, Johns Hopkins University, Baltimore, Maryland. Contribution No. 467 of the McCollum-Pratt Institute. 1

2

35

LEE, WESLEY, FERGUSON, SELIGER

Most measurements of light intensities of bioluminescent and chemiluminescent reactions are made in arbitrary units. The initial light in­ tensity is assumed to be proportional to the rate of the reaction, and in those cases where substrate is added in excess the "flash height" observed is directly proportional to the enzyme concentration in the rate-limiting step. For more detailed studies of the mechanism of enzyme action and for consistency among different laboratories in the reporting of data such as the specific activity of purified luciferase preparations it is desirable to be able to reference the relative readings to absolute intensity in numbers of quanta per second. In the course of a study of methods for accurate calibration of the spectral sensitivity of photomultipliers (Lee and Seliger, 1965a) and their use in measuring the quantum yields of some chemiluminescent and bioluminescent reactions (Lee and Seliger, 1965b), we have de­ termined the experimental conditions for the use of the luminol chemi­ luminescent reaction as a convenient and reproducible standard of total light emission. This paper reports experimental methods for obtaining accurately known numbers of photons of visible light from the luminol oxidation in both aqueous and dimethylsulfoxide solutions. Such a photon stand­ ard is valuable not only in the field of chemiluminescence but also in astrophysics and spectroscopy in general. This photon standard com­ plements a radioactive scintillation standard now available (Hastings and Weber, 1963). Apparatus and Procedure If the product of both the detector efficiency and the physical geometry are to be calibrated, then the particular shape of the reaction cell is of minor importance so long as the relative light intensity measurements of bioluminescence are carried out in the same shape of cell. However for calibration of the phototube photocathode separately the emission from the reaction container will generally be non-isotropic and will require a refraction correction (Lee and Seliger, 1965a). We shall discuss the calibration of a phototube photocathode sep­ arately from the combined detector-geometry calibration in order to make the treatment more general. In our experiments a fluorescence

LUMINOL AS PHOTON EMISSION STANDARD cell with a transparent flat bottom was used. In this case the refrac­ tion correction can easily be calculated. In principle, however, a spherical container should be used, in which case no refraction correc­ tion is needed. The cell used was a fluorescence cuvette with a transparent bottom surface of 1 cm2 area. The cell was supported inside a cylindrical phototube housing, with the photomultiplier tube at one end. The cell was masked with black tape to cover the overlapping, fused, bottom edges. This precaution was taken to avoid an edge effect previously encountered (Lee and Seliger, 1964). The top of the cylinder was cov­ ered with several layers of black cloth to ensure light-tightness. A Keithley 240 Regulated High Voltage Supply was used to supply 1,000 volts to an EMI 6097-S photomultiplier tube. The signal was monitored with a calibrated Keithley 416 High Speed Picoammeter. This equipment was stabilized with a Sola 500 VA CVS-I line voltage stabilizer. The recorders used were a Foxboro 9356 EV recorder with integrator and an Esterline Angus "Speed Servo" recorder. The absorp­ tion spectra of the solutions used were obtained with a Cary 14 re­ cording spectrophotometer. One milliliter of an aqueous or DMSO solution of luminol was de­ livered to the cuvette inside the phototube housing. The other reactants and catalysts were squirted into the cuvette in the dark with syringes. The output of the phototube in amperes was recorded as a function of time, and after approximately 99% of the total light had been emitted there was little error involved in extrapolating the curve to zero light intensity or zero current. The area under the curve, in coulombs, could then be related directly to the total number of photons emitted by the luminol solution. If, from previous measurements of the quantum yield of luminol we know that the 1.0 ml of solution will emit L photons, the average photon spectral efficiency of the phototube at a fixed gain G determined by the cathode high voltage is given by