Table Of ContentHandbook of
Experimental Pharmacology
Continuation of Handbuch der experimentellen Pharmakologie
Vol. 52
Editorial Board
G.V. R. Born, Cambridge . A. Farah, Rensselear, NY
H. Herken, Berlin· A.D. Welch, Memphis, TN
Advisory Board
S. Ebashi . E. G. Erdos· V. Erspamer . U. S. von Euler· W. S. Feldberg
G. B. Koelle· O. Krayer . M. Rocha e Silva· 1. R. Vane· P. G. Waser
W. Wilbrandt
Snake Venoms
Contributors
E. X. Albuquerque· C. A. Alper· A. Bdolah . A. L. Bieber
G. M. Bohm . I. L. Bonta· V. Boonpucknavig . P. Boquet
R. A. Bradshaw· C. H. Campbell· C. C. Chang· P. A. Christensen
E. Condrea . P. Efrati . A. T. Eldefrawi . M. E. Eldefrawi
R. A. Hogue-Angeletti . S. I wanaga . E. Karlsson . C. Y. Lee
S. Y. Lee· B. W. Low· S. A. Minton· A. Ohsaka . C. Ouyang
H. A. Reid· P. Rosenberg· A. M. Rothschild· Z. Rothschild
F. E. Russell· Y. Sawai . W. H. Seegers· V. Sitprija . D. J. Strydom
T. Suzuki· G. L. Underwood· B. B. Vargaftig
Editor
Chen-Yuan Lee
With 208 Figures
Springer-Verlag Berlin Heidelberg New York 1979
CHEN-YUAN LEE, MD, Professor
National Taiwan University, College of Medicine,
No.1. Jen Ai Road, 1st Section, Taipei, Taiwan, Republic of China
1SBN-13: 978-3-642-66915-6 e-ISBN-I3: 978-3-642-66913-2
DOT: 10.1007/978-3-642-66913-2
Library of Congress Cataloging in Publication Data. Main entry under title: Snake venoms. (Handbook of experimental
pharmacology: New Series; v. 52). Includes bibliographies and index. 1. Snake venom. 2. Snake venom - Physiological
effect. 3. Snake venom - Toxicology. I. Albuquerque, E. X. II. Lee, Chen Yuan, 1915-. III. Series: Handbuch der
experimentellen Pharmakologie: New series; v. 52. QP905.H3 vol. 52 615'.1'08s [615'.36] 78-14789.
This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned
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© by Springer-Verlag Berlin Heidelberg 1979.
Softcover reprint of lbe hardcover I st edition 1979
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statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general
use.
2122/3130-543210
Preface
The past decade has been a period of explosion of knowledge on the chemistry and
pharmacology of snake toxins. Thanks to the development of protein chemistry,
nearly a hundred snake toxins have been purified and sequenced, representing one of
the largest families of sequenced proteins. Moreover, the mode of action of these
toxins has been largely elucidated by the concerted efforts of pharmacologists,
electro physiologists, and biochemists. As a result of these studies, some of the snake
toxins, e.g., a-bungarotoxin and cobra neurotoxins, have been extensively used as
specific markers in the study of the acetylcholine receptors. Indeed, without the
discovery of these snake toxins, our knowledge of the structure and function of
nicotinic acetylcholine receptors would not have advanced so rapidly.
The contribution of snake venom research to the biomedical sciences is not
limited to the study of cholinergic receptors. Being one of the most concentrated
enzyme sources in nature, snake venoms are also valuable tools in biochemical
research. Venom phosphodiesterase, for example, has been widely used for structural
studies of nucleic acids; proteinase, for the sequence studies of proteins and pep tides ;
phospholipase Az, for lipid research; and L-amino acid oxidase for identifying optical
isomers of amino acids. Furthermore, snake venoms have proven to be useful agents
for clarifying some basic concepts on blood coagulation and some venom enzymes,
e.g., thrombin-like enzymes and pro coagulants have been used as therapeutic agents.
The discovery of bradykinin as a hypotensive peptide released by the venom of
Bothrops jararaca is another important contribution derived from venom research.
Other components of certain snake venoms, e.g., presynaptic neurotoxins, crotamine,
cardiotoxins, etc., are of current interest as potential tools for studies of transmitter
release, ion channel, and biomembrane structure.
The present treatise is an attempt to offer a comprehensive review of the entire
field of snake venom research. The volume is devided into four parts. Part I starts
with the history of venom research, followed by a chapter on the distribution of
venomous snakes and another on their venom glands. Part II deals with chemistry
and biochemistry of snake venoms, including enzymes, snake toxins, nerve growth
factors, and metal and nonprotein constituents in snake venoms. One special chapter
is devoted to the three-dimensional structure of postsynaptic toxins and another to
the evolution of snake toxins. Part III describes the pharmacologic effects of snake
toxins and enzymes, especially phospholipase Az, on nerve, muscle, circulatory
system, blood vessels, blood cells, blood coagulation, etc. One chapter specially deals
with the use of snake toxins for the study of the acetylcholine receptor, and another
for the study of microvessel damage. Part IV describes the immunologic and clinical
aspects, including antigenic properties of snake venoms, production of antivenin,
VI Preface
common antigens, vaccination, snake venoms and compliment system, and the
symptomatology, pathology, and treatment of snake bites. One chapter is devoted to
nephrotoxicity of snake venoms.
A variety of descriptive terms, such as neurotoxins ((X-neurotoxins, postsynaptic or
curaremimetic neurotoxins, and presynaptic neurotoxins), cardiotoxins (cytotoxins,
direct lytic factors, membrane active toxins, etc.), myotoxins, myonecrotic toxins,
vasculotoxins, etc., are used by different authors in this volume. The editor is aware of
the increasing criticism among toxinologists concerning use of such phenom
enological nomenclature. At the fifth International Symposium on Animal, Plant and
Microbial Toxins, Costa Rica, 1976, a round table discussion on "Nomenclature of
Naturally Occurring Peptides" was held and consequently an International
Committee on this problem was formed. Since, so far, no agreement on the
nomenclature system has been reached, the editor believes that there is no alternative
but to use such phenomenological nomenclature, provided that the name used is well
defined and not confused with another.
The contributors of this volume are highly qualified specialists in their specific
areas of research. The editor wishes to thank his collaborators who have not only
contributed important chapters but have also cooperated fully in the final prepara
tion of this volume. It is hoped that this volume will be a most comprehensive
reference book on snake venoms and serve as a stimulating guide for future research
not only to toxinologists but also to investigators of other disciplines.
The editor cannot conclude this preface without expressing his deepest gratitude
to Dr. BERNHARD WITKOP, Chief of the Laboratory of Chemistry, NIADD, and Dr.
M. D. LEAVITT, Jr., Director of Fogarty International Center, National Institutes of
Health, Bethesda, Maryland, U.S.A., who invited him to participate in the Scholors
in-Residence Program from July 1976 to February 1977, during which period the
manuscripts of most chapters of this volume were edited. Had the editor not been
given this opportunity of sabbatical leave, it would not have been possible to
accomplish such a heavy task for a busy administrator as a dean of a medical school.
The editor is grateful also to Mrs. M. SWENSON for her secretarial help during his stay
at the Stone House, Fogarty International Center.
September 1978, Taipei CHEN-YUAN LEE
Table of Contents
Part I: History, Ecological and Zoological Aspects
CHAPTER 1
History of Snake Venom Research. P. BOQUET . 3
References . . . . . . . . . . . . . . . . 11
CHAPTER 2
Classification and Distribution of Venomous Snakes in the World.
G. UNDERWOOD. With 4 Figures. 15
References . . . . . . . . . . . . . . . . . . . . . . . 36
CHAPTER 3
The Venom Glands of Snakes and Venom Secretion. A. BDOLAH. With 13 Figures
A. Introduction. . . . . . . . . . . 41
B. General Morphology and Histology. 41
I. Venom Glands of Elapidae. . . 41
II. Venom Glands of Viperidae . . 43
C. The Fine Structure of the Secretory Cell During the Venom Regeneration
Cycle. . . . . . . . . . . . . . . . . 45
D. Intracellular Transport of Venom Proteins. 49
E. Venom Synthesis and Secretion. . . . . . 51
I. The Venom Regeneration Cycle. . . . 51
II. Synthesis and Secretion of Different Venom Components . 53
III. Total Venom Yield and the Amount of Venom Expelled During the
Bite . . . . . 54
F. Concluding Remarks 54
References . . . . . . 55
VIII Table of Contents
Part II: Chemistry and Biochemistry of Snake Venoms
CHAPTER 4
Enzymes in Snake Venom. S. IWANAGA and T. SUZUKI. With 25 Figures
A. Introduction. . . . . . . . . . . . . . 61
B. Distribution of Enzymes in Snake Venoms. 61
C. Methods for Purification, Isolation, and Crystallization of Snake Venom
Enzymes . . . . . . . . . . . . . . 64
I. Polyacrylamide Gel Electrophoresis. 66
II. Isoe1ectric Focusing. . . . . . . 66
III. Molecular-Sieve Chromatography. . 68
IV. Ion-Exchange Chromatography. . . 70
D. Biochemical Properties of Snake Venom Enzymes. 75
I. Oxidoreductases . . . . . 75
1. L-Amino Acid Oxidase . . . . . 75
2. Lactate Dehydrogenase. . . . . 84
II. Enzymes Acting on Phosphate Esters. 84
1. Endonuclease . . 87
2. Phosphodiesterase . . . . . . . 87
3. 5'-Nucleotidase . . . . . . . . 93
4. Nonspecific Phosphomonoesterase 96
5. "Paraoxonase" (O,O-Diethyl O-p-Nitrophenyl Phosphate,
O-p-Nitrophenyl Hydrolase) .... 98
III. Enzymes Acting on Glycosyl Compounds. 99
1. Hyaluronidase .... 99
2. Heparinase-like Enzyme .... 100
3. NAD Nucleosidase. . . . . . . 101
IV. Enzymes Acting on Peptide Bonds. 103
1. Endopeptidases . . . . . 104
2. Peptidases . . . . . . . 113
3. Arginine Ester Hydrolases . 114
4. Kininogenase . . . . . . 118
V. Enzymes Acting on Carboxylic Ester Bonds 121
1. Phospholipase A2 . . . . . . . . . 121
2. Phospholipase B and Phospholipase C . 138
3. Acetylcholinesterase . . . . 140
VI. Enzyme Acting on Arylamides . 143
E. Summary 144
References . 144
Table of Contents IX
CHAPTER 5
Chemistry of Protein Toxins in Snake Venoms. E. KARLSSON. With 11 Figures
A. Introduction. . . . . . . . . . . . . . . . . . . . . . . . 159
B. Toxins with Postsynaptic Neurotoxin-Membrane Toxin Structure 161
I. Curaremimetic Toxins. . . . . . . 161
1. Introduction. . . . . . . . . . . . . 161
2. Isolation of Curaremimetic Toxins. . . . 163
3. Characteristics of Curaremimetic Toxins. 164
4. Interaction with the Acetylcholine Receptor. 168
5. Structural Information. 168
6. Chemical Modifications 175
a) Amino Groups 175
b) Arginine Residues 178
c) Carboxyl Groups 179
d) Tryptophan. . . 179
e) Tyrosine . . . . 180
f) Disulfide Bridges. 181
g) Histidine . . 181
h) Modifications Involving a Large Increase in Size 181
7. Discussion 182
II. Membrane Toxins. 183
1. Introduction 183
2. Mode of Action. 183
3. Structural Information 185
4. Chemical Modifications 188
C. Toxins with Phospholipase Structure. 188
I. Notexin and its Homologues 188
II. Taipoxin. . . . 189
III. Crotoxin. . . . . . . . . 192
IV. f3-Bungarotoxin. . . . . . 194
V. Enhydrina schistosa Myonecrotic Toxins 194
VI. Some Other Toxic Phospholipases A . 195
VII. Pharmacologic and Biochemical Effects . 196
1. Presynaptic Neurotoxicity . . . . . 197
2. Inhibition of High Affinity Choline Uptake. 197
3. Postsynaptic Effects and Myotoxicity. . . . 197
4. Antagonism by High Mg2+, Ca2+, and Low Ca2+ 198
VIII. Phospholipase A Activity and Presynaptic Neurotoxicity 198
IX. Concluding Remarks 199
D. Other Toxins . . . . . . 200
I. Crotamine. . . . . . 200
II. Convulxin and Gyroxin 201
III. Mojave Toxin . . . . 201
IV. Other Crotalid Toxins. 201
x Table of Contents
V. Viperotoxin . . . . . .. . . . . . 202
VI. Toxins from Bungarus caeruleus Venom 202
1. Ceruleotoxin. . . . . . . . . . 202
2. Post-and Presynaptic Neurotoxins. 202
E. Conclusion 203
References . . . . . . . . . . . . . . . . 204
CHAPTER 6
The Three-Dimensional Structure of Postsynaptic Snake Neurotoxins:
Consideration of Structure and Function. BARBARA W. Low. With 11 Figures
A. Introduction . . . . . . . . . . . . . . . . . . . . . . . .. 213
B. The Postsynaptic Neurotoxins: Survey of Three-Dimensional Prototype
Structure; Deviant Toxins, Chemical Modification Studies. Preliminary
Review. . . . . . . . . . . . . . . . . . . . 215
I. Primary Sequence of Erabutoxin b. . . . . . 215
II. Three-Dimensional Structure of Erabutoxin b . 216
1. Molecular Size and Shape. . . . . . . . . 216
2. Backbone Chain Conformation in the Erabutoxin Molecule. 219
III. Residue Sequences: Invariant and Conservative Substitutions in the
Long and Short Chain Series of Neurotoxins. . . . . . . 221
IV. Chemistry and Chemical Modification Studies. . . . . . . 227
1. Residues not Subject to Study by Group-Specific Reagents. 228
a) Serine-Threonine 228
b) Glycine . . 228
c) Proline . . . . 228
d) Asparagine 229
e) Alanine, Leucine, Isoleucine, and Valine. 229
2. Invariant and Conservatively Substituted Residues as Studied
by Chemical Modification. 229
a) Disulfide Linkages 230
b) Tyrosine . . . . . . 230
c) Tryptophan . . . . . 230
d) Aspartic Acid, Glutamic Acid. 230
e) Arginine . . . . . . . . . 231
f) Amino Groups. . . . . . . 231
3. Toxin Modification by Deletion or by Size Increase. 231
a) Carboxyl-Terminal Deletion of a Long Chain Toxin. 231
b) Polymerization. . . . . . . . . . . . . . . . . . 231
4. Summary . . . . . . . . . . . . . . . . . . . . . 231
V. Physicochemical Studies of Neurotoxins and Their Derivatives. 233
1. Spectroscopic Studies of Native Toxin Conformation in Aqueous
and Aqueous-Organic Solvent. . . . . . . . . . . . . . . . 233
Table of Contents XI
a) Optical Rotatory Dispersion (ORD) and Circular Dichroism
(CD) Spectra. . . . . . . . . . . . . . . . . . . . . . 233
b) Laser-Raman Spectra . . . . . . . . . . . . . . . . . . 233
2. Spectroscopic Studies of Chemically Modified and/or Denatured
Neurotoxins . . . 234
a) Disulfide Linkages 234
b) Tyrosine . . . . 234
c) Tryptophan . . . 234
VI. Theoretical and Model Studies of Neurotoxin Conformation and
Three-Dimensional Structure . . . . . . . . 234
1. Prediction of Conformation in the Neurotoxins . 234
a) Helix . . . . 234
C(
b) PP leated Sheet. . . . . . . . . . . . 235
c) PT urns . . . . . . . . . . . . . . . 235
2. Predictions of Three-Dimensional Structure in the Neurotoxin
Series . . . . . . . . . . . . 235
a) Model Structure Studies. . . 235
b) Energy Minimization Studies. 235
c) Theoretical Chemical Models. 235
VII. X-ray Crystal Structure Studies of Other Neurotoxins 236
1. Erabutoxin a (Asn 26 Erabutoxin b). . . . . . . . 236
2. Neurotoxin b from Laticauda semifasciata (Philippines) . 236
3. Erabutoxin c . . . . . . . . . . . . . . . . . . . . 237
4. Laticotoxin a and Cobrotoxin. . . . . . . . . . . . . 237
C. Structure-Function Relationship in the Postsynaptic Neurotoxins. The
Three-Dimensional Structure of Erabutoxin b as Prototype in both Long
and Short Toxins. . . . . . . . . . . . . . . . . . . . . . . 237
I. Erabutoxin b and the Short Toxins: The Reactive Site. . . 237
II. Erabutoxin b and the Short Toxins: Nonreactive Site Regions. 240
III. Erabutoxin and the Long Toxins: Reactive Site. . . . . .. 241
IV. Erabutoxin and the Long Toxins: Nonreactive Site Regions. 241
V. Long and Short Toxins: Biochemical and Biological Differences. 242
VI. Erabutoxin b and the Short Toxin Series: Detailed Intramolecular
Packing . . . . . . . . . . . . . . . . . . . . . . . . 243
1. PP leated Sheet and PT urns. . . . . . . . . . . . . .. 243
2. Intramolecular Interactions: Particularly Those Involving In-
variant and Type-Conserved Residues. . . . . . . . . . . 243
a) Main Chain-Main Chain and Main Chain-Side Chain Hydro-
gen Bonds. . . . . . . . . . . 243
b) Side Chain-Side Chain Interactions 244
D. Conclusions. . . . . . . 245
I. The Present View. . . . . . . . . . . 245
1. Structure: General . . . . . . . . 247
2. Comparison of Short and Long Toxins. 247
3. Structural and Functional Residues. 250
a) Structural Residues . . . . . . . . 251
Description:The past decade has been a period of explosion of knowledge on the chemistry and pharmacology of snake toxins. Thanks to the development of protein chemistry, nearly a hundred snake toxins have been purified and sequenced, representing one of the largest families of sequenced proteins. Moreover, the
