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Common antigens in snake sera and venoms
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
144
Abstracts of Papers
that the venom used to immunize horses for antivenin production was contaminated with snake blood or tissue fluid. The existence of common antigens in serum and venom of snakes of the same species is suggested, however. Faculté de Médecine et de Pharmacie, Laboratoire de Biochimie Médicale, Marseille, France Méthode générale de ptQl6catton des nem~otoxines animales La méthode générale que nous préconisons met en oeuvre des techniques traditionnelles aussi bien que les acquisitions récentes de la chromatographie. Les procédés classiques employés se rapportent aux phénomènes de solubilité : dissolution dans (eau pure et dialyse contre Peau, éventuellement précipitation par (acétone refroidie entre 60 et 80 pour cent de concentration en solvant . Ces procédés se sont avérés trés efficaces pour éliminer les mucoprotéines dont la présence peut compromettre la suite des opérations . Les méthodes modernes font appel à deux procédés : la filtration moléculaire et la chromatographie d'échange d'ions . La filtration moléculaire s'opère sur le gel de dextrane réticulé Sephadex G-SO dont la porosité convient aux molécules de poids moléculaire compris entre 5,000 et 10,000. Récemment, les possibilités de la méthode ont été accrues par (utilisation du `Recychrom' LKB permettant le recyclage de tout ou partie de féluat sortant de la colonne. La chromatographie d'échange d'ions est pratiquée suivant une technique `d'équilibre' avec des supports de polarité opposée (Amberlite CG-50, CM~ephadex G50 d'une part ; DEAE-Sophadex A-50 d'autre part) dans des tampons acétate d'ammonium. MIRANDA, F.,
Mrrrer .sr.~r, J. S., SHAW, S. M, and Ti~exv, L. W., Perdue University, School of Pharmacy, Depart ment of Bionucleonics, and School of Veterinary Science and Medicine, Department of Microbiology, Lafayette, Indiana, U.S .A. The detoxifying effect of cobalt-60 radiation on the venom of the hooded cobra, Ngja ngja Lyophilized Naja raja venom, reconstituted in a weak phosphate buffer, was exposed to the gamma radiation of a cobalt-60 source ; standardization being accomplished by using the per cent nitrogen of the venom as the reference point. The venom was irradiated at concentrations of 0~1, 0~5 and 1~0 mg/ml. Venom solutions were exposed to doses ranging from 6000 to 320,000 r with the exposure rate being 105 r/ sec. The effect of the irradiation on the venom's toxicity was measured by Ln,u tests in female white mice . The increase in the t.n b, value was an exponential function, varying directly with the total exposure dose and inversely with the concentration. The chromatographic patterns of the irradiated and non-irradiated venoms were compared, as were the immunodiffusion patterns of antisera produced with normal venom against irradiated and non-irradiated venoms . The possible action of radiation-produced free radicals with venom molecules and the implications were discussed. Taxai-t~sxt; T., OMORI-SATOti, T. and Muxnra, R., National Institute of Health, The 2nd Department of Bacteriology, Shinagawa-ku, Tokyo 141, Japan Pmißcation and characteriTation of the hemorrhagic priadplee ba the venom of TrJmcrcsarwJlarorirJdis The method for determining hemorrhagic activity based on the well~efined skin reaction and the parallel-line-assay showed that the venom of 7i"lmeresurus ji'avoviridis contains two hemorrhagic principles, distinct immunologically but not distinguishable in hemorrhagic action on the rabbit skin, and these principles were separable by electrophoresis or chromatography . We purified the major hemorrhagic fraction, HRI, with a 20 per cent yield and 43-fold increase in specific activity, by chromatography on DEAE~ephadex and GE-cellulose and by recycling chromatography on Sephadex G-200. The purified preparation (5~8 S) contained 7 per cent of the lethal toxicity and 0~6 per cent of the proteolytic activity originally present in the venom. We also fractionated the other hemorrhagic fraction, HR2, by chromatography on Amberlite CG-50, and succeeded is isolating the major proteinase, H,-proteinase, completely free of the hemorrhagic activity . The resulting HR2 fraction freed of H,-proteinase still contained some otherproteolytic activity. Chromatography on Bio-Rex 70 completely eliminated the proteolytic activity and resolved the hemorrhagic activity into two parts, HR2a and HR2b . From crude HR2, purification achieved was 12- and 13-fold with 25 and 12 per cent yields with respect to HR2a and HR2b, respectively. HR2a (2~4 S) was proved to be homogenous . HR2a or HR2b is indistinguishable from H,-proteinasein heat and pH stability sad in susceptibility to EDTA and cysteine . The purification of each hemorrhagic principle and of H,-proteinase and some properties of the purified preparations will be described. The results indicate that the hemorrhagic principles and the proteolytic enzymes are separate entities . OHSAKA, A.,
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
144
Abstracts of Papers
that the venom used to immunize horses for antivenin production was contaminated with snake blood or tissue fluid. The existence of common antigens in serum and venom of snakes of the same species is suggested, however. Faculté de Médecine et de Pharmacie, Laboratoire de Biochimie Médicale, Marseille, France Méthode générale de ptQl6catton des nem~otoxines animales La méthode générale que nous préconisons met en oeuvre des techniques traditionnelles aussi bien que les acquisitions récentes de la chromatographie. Les procédés classiques employés se rapportent aux phénomènes de solubilité : dissolution dans (eau pure et dialyse contre Peau, éventuellement précipitation par (acétone refroidie entre 60 et 80 pour cent de concentration en solvant . Ces procédés se sont avérés trés efficaces pour éliminer les mucoprotéines dont la présence peut compromettre la suite des opérations . Les méthodes modernes font appel à deux procédés : la filtration moléculaire et la chromatographie d'échange d'ions . La filtration moléculaire s'opère sur le gel de dextrane réticulé Sephadex G-SO dont la porosité convient aux molécules de poids moléculaire compris entre 5,000 et 10,000. Récemment, les possibilités de la méthode ont été accrues par (utilisation du `Recychrom' LKB permettant le recyclage de tout ou partie de féluat sortant de la colonne. La chromatographie d'échange d'ions est pratiquée suivant une technique `d'équilibre' avec des supports de polarité opposée (Amberlite CG-50, CM~ephadex G50 d'une part ; DEAE-Sophadex A-50 d'autre part) dans des tampons acétate d'ammonium. MIRANDA, F.,
Mrrrer .sr.~r, J. S., SHAW, S. M, and Ti~exv, L. W., Perdue University, School of Pharmacy, Depart ment of Bionucleonics, and School of Veterinary Science and Medicine, Department of Microbiology, Lafayette, Indiana, U.S .A. The detoxifying effect of cobalt-60 radiation on the venom of the hooded cobra, Ngja ngja Lyophilized Naja raja venom, reconstituted in a weak phosphate buffer, was exposed to the gamma radiation of a cobalt-60 source ; standardization being accomplished by using the per cent nitrogen of the venom as the reference point. The venom was irradiated at concentrations of 0~1, 0~5 and 1~0 mg/ml. Venom solutions were exposed to doses ranging from 6000 to 320,000 r with the exposure rate being 105 r/ sec. The effect of the irradiation on the venom's toxicity was measured by Ln,u tests in female white mice . The increase in the t.n b, value was an exponential function, varying directly with the total exposure dose and inversely with the concentration. The chromatographic patterns of the irradiated and non-irradiated venoms were compared, as were the immunodiffusion patterns of antisera produced with normal venom against irradiated and non-irradiated venoms . The possible action of radiation-produced free radicals with venom molecules and the implications were discussed. Taxai-t~sxt; T., OMORI-SATOti, T. and Muxnra, R., National Institute of Health, The 2nd Department of Bacteriology, Shinagawa-ku, Tokyo 141, Japan Pmißcation and characteriTation of the hemorrhagic priadplee ba the venom of TrJmcrcsarwJlarorirJdis The method for determining hemorrhagic activity based on the well~efined skin reaction and the parallel-line-assay showed that the venom of 7i"lmeresurus ji'avoviridis contains two hemorrhagic principles, distinct immunologically but not distinguishable in hemorrhagic action on the rabbit skin, and these principles were separable by electrophoresis or chromatography . We purified the major hemorrhagic fraction, HRI, with a 20 per cent yield and 43-fold increase in specific activity, by chromatography on DEAE~ephadex and GE-cellulose and by recycling chromatography on Sephadex G-200. The purified preparation (5~8 S) contained 7 per cent of the lethal toxicity and 0~6 per cent of the proteolytic activity originally present in the venom. We also fractionated the other hemorrhagic fraction, HR2, by chromatography on Amberlite CG-50, and succeeded is isolating the major proteinase, H,-proteinase, completely free of the hemorrhagic activity . The resulting HR2 fraction freed of H,-proteinase still contained some otherproteolytic activity. Chromatography on Bio-Rex 70 completely eliminated the proteolytic activity and resolved the hemorrhagic activity into two parts, HR2a and HR2b . From crude HR2, purification achieved was 12- and 13-fold with 25 and 12 per cent yields with respect to HR2a and HR2b, respectively. HR2a (2~4 S) was proved to be homogenous . HR2a or HR2b is indistinguishable from H,-proteinasein heat and pH stability sad in susceptibility to EDTA and cysteine . The purification of each hemorrhagic principle and of H,-proteinase and some properties of the purified preparations will be described. The results indicate that the hemorrhagic principles and the proteolytic enzymes are separate entities . OHSAKA, A.,