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Biochemistry of Heloderma venom
142
Abstracts of Papers
ovary, the cytoplasm was foamy with cracks at places . The ovarian atrophy was well marked in
Heteropneustes fossilis.
RBFBRBNCB3 MnTxuR, D. S. (1962a) Proc. natn . Acad. Sci. India, B) 32, 429. Me~tttR, D. S. (19626) Experientia 18, 506. MATHUR, D. S. (1965) Sci. Cult . 31, 258. MCDONAI.D, N. M. and COTTRBI.t., G. A., Wellcome Laboratories of Pharmacology, Gatty Marine Laboratory, University of St . Andrews, Fife, Scotland Pmiflcstian and mode of action of tozln 19rom Eledone clrrAiosa A toxin has been extracted and partially purified from the venom-secreting posterior salivary glands of Eledone cirrhosa (Octopoda, Cephalopods). When injected into decapod crustacea, the toxin causes a flaccid paralysis, precisely mimicking the effect in animals envenomated in vivo by Eledone. The paralysis is irreversible and is characterized by a loss of locomotory and postural function although the heart continues to beat for several hours. The toxic factor is a macromolecule, and most probably is a protein or closely associated with a protein, as it has the following properties : (a) it is destroyed by heating to 100°C for 10 min, (b) it is non-dialyzable, (c) it is precipitated by tri-chloracetic acid. Partial purification of the toxin has been achieved by : (a) saline extraction of the posterior salivary glands (crude extract, CE), ro) ammonium sulphate precipitation of the crude extract (ammonium sulphate extract, ASE), (c) gel filtration of ASE on Sephadex G-75 (Sephadex purified extract, SPE) . Starch gel electrophoresis of both CE aad ASE at pH 8~4 reveals a number of bands, a maximum of thirteen, migrating both to the anode and cathode, but electrophoresis of SPE reveals a maximum of two bands, both migrating to the cathode. This indicates that the toxin is basic in nature. The toxin is retained on Sephadex G-75 but is excluded from G-50 columns which suggests a mol. wt . in the range of 30,000 to 70,000 . Assays were done on the crab Carcinus mamas and gave an Ln~u for SPE of 8 mg/kg of crab. Preliminary experiments were done to determine the mode of action of the toxin on the nervous system in the abdomen of the Norwegian lobster Nep/rrops norvegic«.s. Dialyzed crude extracts blocked indirectly elicited contractions in both the superficial flexor muscles and the deep extensor muscles, but did not appear to affect the tonic output of the posterior branch of the third root of the ganglion which supplies the superficial flexor muscles, or the output of the dorsal muscle receptor organ. McINrosx, M. E. and WATT, D. D., University of Southern California, Laboratory of Neurological Research, Los Angeles, California, U.S .A. PmiBcation of toxins from the North American eoorpion Cestraroides sculpturatas EwL~g The venom of Centruroides sculpturatus contains four toxins (I, II, III, and m. Purification was achieved by initial separation on carboxymethylcellulose followed by gradient elution, step wise and equilibrium chromatography on Amberlite CG-50. Homogeneity was demonstrated by equilibrium chromatography on Amberlite, electrophoresis on cellulose acetate, immunoelectrophoresis using anti-venom sera and anti-toxin I and -toxin III serum, and supported by amino acid composition studies. Preliminary examination indicates a pHt value above 8~6 for the toxins . Amino acid composition studies show that methionine is absent from all four toxins . Alanine is missing from toxins I and II ; isoleucine is absent from toxins III and 1Y . Toxin I has 70 amino acids; toxin II, 64 ; and toxins III and IV, each have 78 amino acids. Minimum molecular weights based on amino acid composition studies are: toxin I, 7900 ; toxin II, 7108 ; toxin III, 9002 ; and toxin IV, 8873 . It is unknown at present if these toxins represent true molecular types, molecular variants or one or more aberrant forms. (Supported in part by Grant NB-05535 from the National Institute of Neurological Disease and Stroke, U.S.P.H .S .) MBar, D., Institut ßr gerichtliche und soziale Medizin der Universität, Franläurt a.M ., Germany HIoehemistry of He%derma venom The venom of the only poisonous lizards, Heloderma suspectum and Heloderma horridum, possesses besides strong toxicity, phospholipase A, hyaluronidase, kinin-releasing and arginine esterolytic activity. By gel filtration on Sephadex G-75, chromatography on DEAF-cellulose and gel filtration on Biogel P-60 the kinin-releasing enzyme (kallikrein), the arginine esterase and the toxic factor of Heloderma suspectum venom were purified . The kallikrein releases kininfrom bovine plasmaglobulin fraction, cleaves N°~-benzoylL-arginine ethyl ester (BAEE) in preference to p-toluene-sulphonyl-nL-arginine methyl ester (TAME) and is inhibited by DFP, weakly by Trasylol and not by soybean inhibitor. The arginine esterase is highly active in hydrolyzing HAEE in preference to TAME, is also inhibited by DFP and possesses marked
Abstracts of Papers
143
haemorrhagec properties. In mice massive haemorrhages are observed after subcutaneous application of this enzyme, predominantly in the eye bulb and the internal organs (lungs, kidneys, intestine) . The kininreleasing and esterolytic as well as the haemorrhagec and toxic properties of the venom are remarkably heat stable . Mwnsrssorav, H., Tel-Aviv University, Department of Zoology, Tel-Aviv, Israel Dislrlbatioo of ~enomoae soskes to lfsael in connection with agrlcaltm~al development Incidence of snake bite in human populations depends on several factors, among which the frequency of encounter between snakes and hnmanc is the most important. As h»ma_ns spend most of their time in and around their settlements, the frequency of occurrence of venomous snakes in these places may be decisive for the frequency of snake bites. Continued removal of venomous snakes from human settlements and their surrounding is reported to cause the decrease and eventual disappearance of snake populations. Observations of a few of the seven local species of venomous snakes occurring in Israel show, however, that snake populations are able to thrive even is and around human settlements, notwithstanding continued killing and collecting . In two species, Vipers palaestinae and Walterinrusia aegyptia, it was found that about 90 per cent of all the snakes delivered to the serpentarium of the Tel-Aviv University had been collected in or near agricultural settlements. Numbers of snakes delivered at the serpentaria could be used as an indication for snake populations, as there are no professional snake collectors in Israel and only snakes which are accidentally encountered are brought to the serpentaria. Numbers of snakes delivered fluctuate from year to year, but oa as average do not decrease . The factors favoring survival and reproduction of these two species of venomous snakes in agricultural settlements are their nocturnal activity and availability of food and humidity in these places . In 1965 the Plant Protection Department of the Ministry of Agriculture organized and carried out a campaign in order to exterminate jackals. This campaign resulted in a drastic reduction of the populations of mammalian and avian predators in general, in a considerable increase of vipers (Vipers palaestinae) and in an unprecedented rise in the number of snake-bite cases among the human population . The main predator of the viper seems to be the mongoose (Herpestes ichneumon), who has a high degree of immunity towards the venom of the viper. The increase in mongoose populations, beginning in 1967, was accompanied by a drop in the number of snake-bite cases. The carpet viper, F .chis crolorata, a desert snake, displayed a considerable increase in population density and an improved physical condition after irrigated agriculture had been established is the desert oasis of Eia Gedi, on the shores of the Dead Sea. It was found that these changes had been caused by an easily available source of food is the form of toads and frogs, the number of which increased with irrigated agriculture. Recently, however, the Echis population has begun to decrease, possibly as a result of the introduction of cats to the area. Mnarox, S. A., Indiana University Medical Center, Indianapolis, Indiana, U.SA. Common antigens is aoske sera and venons Sera of 14 species of snakes (four wlubrids, four crotalids, two boids, one elapid, one hydrophid, one viperid and one atractaspid) showed precipitin bands when reacted in gel diffusion tests against 10 antivenins (three elapid, three viperid, three crotalid and one wlubrid). Serum of Agkistrodon corrtortrix reacted with nine of the 10 antivenins ; sera of Crotales horridus, Pituophis melanoleucus and Thamnophis sirtalis reacted with eight each . Fewest reactions were given by the sera of Python molurus, Constrictor constrictor and Atractaspis microkpidota . One component common to the sera of all snakes tested reacted with Nqja ngja antivenin. Another component common to all except the boids reacted with Trimeresurus antivenin. A component found only is the sera of Pituophis and Thamnophis reacted with Dispholidus antivenin. Precipitin lines produced by the sera of Naja ngja, Hydrophls cyanocinctus, Vipers russeUl, Agkistrodon coruortrix and Crotales horrfdus did not show fusion with lines produced by the venoms of these species, hence they cannot be presumed identical with major venom antigens. Similarly, no reactions of identity were observed between four electrophoretically separated fractions of Ngja ngja venom and the components of Ngja serum that react with Ngja antivenin. These venom fractions gave no reaction with a precipitin serum produced in rabbits against the serum of Naja ngja . This serum showed one band with whole Ngja ngja venom but no bands with the venoms of Agkistrodon contortrix, A, rhodostoma, Crotales horridrrs and Vipers russeUi. A precipitin serum similarly produced against the serum of Crotales horridus gave a band with C. horridus venom but none with the other venoms tested. A precipitin serum against the serum of the non-venomous colubrid Natrtx sipedon did not react with any of the vesoms tested . Snake serum components reacting with antivenin were of slow or intermediate electrophoretic motility with the exception of one component in the fast moving fraction of Ngja serum that reacted with Ngja antivenin. Most of the reactions observed between antivenins and snake sera can be explained by assuming
Abstracts of Papers
ovary, the cytoplasm was foamy with cracks at places . The ovarian atrophy was well marked in
Heteropneustes fossilis.
RBFBRBNCB3 MnTxuR, D. S. (1962a) Proc. natn . Acad. Sci. India, B) 32, 429. Me~tttR, D. S. (19626) Experientia 18, 506. MATHUR, D. S. (1965) Sci. Cult . 31, 258. MCDONAI.D, N. M. and COTTRBI.t., G. A., Wellcome Laboratories of Pharmacology, Gatty Marine Laboratory, University of St . Andrews, Fife, Scotland Pmiflcstian and mode of action of tozln 19rom Eledone clrrAiosa A toxin has been extracted and partially purified from the venom-secreting posterior salivary glands of Eledone cirrhosa (Octopoda, Cephalopods). When injected into decapod crustacea, the toxin causes a flaccid paralysis, precisely mimicking the effect in animals envenomated in vivo by Eledone. The paralysis is irreversible and is characterized by a loss of locomotory and postural function although the heart continues to beat for several hours. The toxic factor is a macromolecule, and most probably is a protein or closely associated with a protein, as it has the following properties : (a) it is destroyed by heating to 100°C for 10 min, (b) it is non-dialyzable, (c) it is precipitated by tri-chloracetic acid. Partial purification of the toxin has been achieved by : (a) saline extraction of the posterior salivary glands (crude extract, CE), ro) ammonium sulphate precipitation of the crude extract (ammonium sulphate extract, ASE), (c) gel filtration of ASE on Sephadex G-75 (Sephadex purified extract, SPE) . Starch gel electrophoresis of both CE aad ASE at pH 8~4 reveals a number of bands, a maximum of thirteen, migrating both to the anode and cathode, but electrophoresis of SPE reveals a maximum of two bands, both migrating to the cathode. This indicates that the toxin is basic in nature. The toxin is retained on Sephadex G-75 but is excluded from G-50 columns which suggests a mol. wt . in the range of 30,000 to 70,000 . Assays were done on the crab Carcinus mamas and gave an Ln~u for SPE of 8 mg/kg of crab. Preliminary experiments were done to determine the mode of action of the toxin on the nervous system in the abdomen of the Norwegian lobster Nep/rrops norvegic«.s. Dialyzed crude extracts blocked indirectly elicited contractions in both the superficial flexor muscles and the deep extensor muscles, but did not appear to affect the tonic output of the posterior branch of the third root of the ganglion which supplies the superficial flexor muscles, or the output of the dorsal muscle receptor organ. McINrosx, M. E. and WATT, D. D., University of Southern California, Laboratory of Neurological Research, Los Angeles, California, U.S .A. PmiBcation of toxins from the North American eoorpion Cestraroides sculpturatas EwL~g The venom of Centruroides sculpturatus contains four toxins (I, II, III, and m. Purification was achieved by initial separation on carboxymethylcellulose followed by gradient elution, step wise and equilibrium chromatography on Amberlite CG-50. Homogeneity was demonstrated by equilibrium chromatography on Amberlite, electrophoresis on cellulose acetate, immunoelectrophoresis using anti-venom sera and anti-toxin I and -toxin III serum, and supported by amino acid composition studies. Preliminary examination indicates a pHt value above 8~6 for the toxins . Amino acid composition studies show that methionine is absent from all four toxins . Alanine is missing from toxins I and II ; isoleucine is absent from toxins III and 1Y . Toxin I has 70 amino acids; toxin II, 64 ; and toxins III and IV, each have 78 amino acids. Minimum molecular weights based on amino acid composition studies are: toxin I, 7900 ; toxin II, 7108 ; toxin III, 9002 ; and toxin IV, 8873 . It is unknown at present if these toxins represent true molecular types, molecular variants or one or more aberrant forms. (Supported in part by Grant NB-05535 from the National Institute of Neurological Disease and Stroke, U.S.P.H .S .) MBar, D., Institut ßr gerichtliche und soziale Medizin der Universität, Franläurt a.M ., Germany HIoehemistry of He%derma venom The venom of the only poisonous lizards, Heloderma suspectum and Heloderma horridum, possesses besides strong toxicity, phospholipase A, hyaluronidase, kinin-releasing and arginine esterolytic activity. By gel filtration on Sephadex G-75, chromatography on DEAF-cellulose and gel filtration on Biogel P-60 the kinin-releasing enzyme (kallikrein), the arginine esterase and the toxic factor of Heloderma suspectum venom were purified . The kallikrein releases kininfrom bovine plasmaglobulin fraction, cleaves N°~-benzoylL-arginine ethyl ester (BAEE) in preference to p-toluene-sulphonyl-nL-arginine methyl ester (TAME) and is inhibited by DFP, weakly by Trasylol and not by soybean inhibitor. The arginine esterase is highly active in hydrolyzing HAEE in preference to TAME, is also inhibited by DFP and possesses marked
Abstracts of Papers
143
haemorrhagec properties. In mice massive haemorrhages are observed after subcutaneous application of this enzyme, predominantly in the eye bulb and the internal organs (lungs, kidneys, intestine) . The kininreleasing and esterolytic as well as the haemorrhagec and toxic properties of the venom are remarkably heat stable . Mwnsrssorav, H., Tel-Aviv University, Department of Zoology, Tel-Aviv, Israel Dislrlbatioo of ~enomoae soskes to lfsael in connection with agrlcaltm~al development Incidence of snake bite in human populations depends on several factors, among which the frequency of encounter between snakes and hnmanc is the most important. As h»ma_ns spend most of their time in and around their settlements, the frequency of occurrence of venomous snakes in these places may be decisive for the frequency of snake bites. Continued removal of venomous snakes from human settlements and their surrounding is reported to cause the decrease and eventual disappearance of snake populations. Observations of a few of the seven local species of venomous snakes occurring in Israel show, however, that snake populations are able to thrive even is and around human settlements, notwithstanding continued killing and collecting . In two species, Vipers palaestinae and Walterinrusia aegyptia, it was found that about 90 per cent of all the snakes delivered to the serpentarium of the Tel-Aviv University had been collected in or near agricultural settlements. Numbers of snakes delivered at the serpentaria could be used as an indication for snake populations, as there are no professional snake collectors in Israel and only snakes which are accidentally encountered are brought to the serpentaria. Numbers of snakes delivered fluctuate from year to year, but oa as average do not decrease . The factors favoring survival and reproduction of these two species of venomous snakes in agricultural settlements are their nocturnal activity and availability of food and humidity in these places . In 1965 the Plant Protection Department of the Ministry of Agriculture organized and carried out a campaign in order to exterminate jackals. This campaign resulted in a drastic reduction of the populations of mammalian and avian predators in general, in a considerable increase of vipers (Vipers palaestinae) and in an unprecedented rise in the number of snake-bite cases among the human population . The main predator of the viper seems to be the mongoose (Herpestes ichneumon), who has a high degree of immunity towards the venom of the viper. The increase in mongoose populations, beginning in 1967, was accompanied by a drop in the number of snake-bite cases. The carpet viper, F .chis crolorata, a desert snake, displayed a considerable increase in population density and an improved physical condition after irrigated agriculture had been established is the desert oasis of Eia Gedi, on the shores of the Dead Sea. It was found that these changes had been caused by an easily available source of food is the form of toads and frogs, the number of which increased with irrigated agriculture. Recently, however, the Echis population has begun to decrease, possibly as a result of the introduction of cats to the area. Mnarox, S. A., Indiana University Medical Center, Indianapolis, Indiana, U.SA. Common antigens is aoske sera and venons Sera of 14 species of snakes (four wlubrids, four crotalids, two boids, one elapid, one hydrophid, one viperid and one atractaspid) showed precipitin bands when reacted in gel diffusion tests against 10 antivenins (three elapid, three viperid, three crotalid and one wlubrid). Serum of Agkistrodon corrtortrix reacted with nine of the 10 antivenins ; sera of Crotales horridus, Pituophis melanoleucus and Thamnophis sirtalis reacted with eight each . Fewest reactions were given by the sera of Python molurus, Constrictor constrictor and Atractaspis microkpidota . One component common to the sera of all snakes tested reacted with Nqja ngja antivenin. Another component common to all except the boids reacted with Trimeresurus antivenin. A component found only is the sera of Pituophis and Thamnophis reacted with Dispholidus antivenin. Precipitin lines produced by the sera of Naja ngja, Hydrophls cyanocinctus, Vipers russeUl, Agkistrodon coruortrix and Crotales horrfdus did not show fusion with lines produced by the venoms of these species, hence they cannot be presumed identical with major venom antigens. Similarly, no reactions of identity were observed between four electrophoretically separated fractions of Ngja ngja venom and the components of Ngja serum that react with Ngja antivenin. These venom fractions gave no reaction with a precipitin serum produced in rabbits against the serum of Naja ngja . This serum showed one band with whole Ngja ngja venom but no bands with the venoms of Agkistrodon contortrix, A, rhodostoma, Crotales horridrrs and Vipers russeUi. A precipitin serum similarly produced against the serum of Crotales horridus gave a band with C. horridus venom but none with the other venoms tested. A precipitin serum against the serum of the non-venomous colubrid Natrtx sipedon did not react with any of the vesoms tested . Snake serum components reacting with antivenin were of slow or intermediate electrophoretic motility with the exception of one component in the fast moving fraction of Ngja serum that reacted with Ngja antivenin. Most of the reactions observed between antivenins and snake sera can be explained by assuming