- Email: [email protected]
Biochemical studies on the toxic nature of snake venom
Abstracts of Papers
161
Y.~x~, C. C., I{aohsiung Medical College, Department of Biochemistry, I{aohsiung, Taiwan, China Slochemical atadiea an the tozk nature of s~èe venom A toxic protein devoid of enzyme activities has been highly purified from Formosan cobra venom. The crystalline toxin, cobrotoxin, was found to be homogeneous and to be the main toxin protein in the venom. It consists of a single peptide chain cross-linked intramolecularly by 4 disulfide bonds with amino terminal leucine and carboxy terminal asparagine. The minimal molecular weight calculated from amino acid composition (62 residues) is 6949 . Since the complete sequence of amino acids in cobrotoxin has been determined and the positions of disulfide bonds clarified, a two-dimensional schematic diagram for the structure of cobrotoxin was proposed. On reduction with ß-mercaptoethanol, cobrotoxin displays 8 SH groups with a concurrent loss is toxicity. The reduced toxin yields oa reoxidation, a biologically active product with complete restoration of toxicity and antigeaicity, specific rotation close to the native value and an infrared spectrum identical with that of cobrotoxin . The results indicate that the integrity of the disulfide bonds in cobrotoxia is essential for lethality. The optical rotatory dispersion (ORD) of cobrotoxin is quite different from those of the usual proteins in having a right-handed a-helical structure and circular dichromism (CD) spectrum suggests that the cobrotoxin molecule contains a ß-structure. On reductive cleavage of the disulfide bonds, cobrotoxin becomes a random conformation and the ORD and CD curves change greatly. However, the reoxidized toxin gave essentially the same ORD and CD curves as cobrotoxin which suggests that cornet reformation on helical structure occurred by air-0xidation of the reduced protein. Antisera have bcen prepared by injecting increasing doses of cobrotoxin with Freund's adjuvaat into rabbits. Immunoglobulia G isolated was allowed to react with cobrotoxin at equivalence. The antigenantibody precipitates were dissolved is 053 M formic acid and the antibody was separated from antigen through a column of Sephadex G-100. The purified antibody was proved to be free from antigen and 100 precipitable with oobrotoxin, and the neutralizing capacity increased 17 5-fold over that of the Antsera. The purified antibody was digested into univalent fragments with mercuri-pepsin . The fragments do not form insoluble aggregates with antigen but form a soluble antigen-antibody complex. The univalent fragments together with the purified antibody may provide valuable tools for an immunochemical approach to the elucidation of the toxic nature of snake venom.
Zr ozxuv, E., FRSprncF~r G. S., Mnurron, F., Liea ~~e Y, S. and Sxur ov, A., Hebrew University, Department of Entomology and Venomous Animals, Jerusalem, Israel ; University of Illinois, Department of Ento urology, Urbane, Illinois, U.S.A., and School of Medicine, Department of Medical Biochemistry, Marseille, France The effect of scorpion venom on lame Injection of scorpion venom into intact or ligated (thus nullifying the central nervous system) larvae of blowflies causes an immediate paralysis accompanied by strong contraction of the test animals, demonstrating the peripheral nerve-muscle stimulating effect of the venom (Any and WEIaa, 1959 ; P~utv~s and RussaLt, 1967). The duration of this contrastive paralysis may range from several seconds to several hours, depending on the amount of venom injected. This effect of scorpion venom on fly larvae formed the basis of a very rapid and sensitive test for the quantitative estimation of scorpion venom. The venom of eighteen different species of scorpions was tested for its contrastive-paralyzing activity in fly larvae and its lethal effect on mice (r-n estimations) . No correlation was found between the two biological activities. These data suggested the possibility that there are different components of scorpion venom responsible for each of the above two biological activities . This hypothesis was verified by the following experimental results: Purified proteins from Androctonus australtr venom (Mue~n~ et al., 1964, 1966) which were highly toxic to mice were completely ineffective against fly larvae when tested for their contrastive-paralyzing activity. Furthermore these proteins were not lethal to the larvae . A clear separation between larvae and mice toxic components was achieved both by starch gel electrophoresis and Sephadex G-50 column chromatography of the crude venom of A. oustralis. It has been shown that the coatractive-paralyzing fraction was the sole fraction lethal to fly larvae . An electrophoretical analysis of the Sephadex larval-active fraction revealed the presence of several protein bands, only one of which contained the larval toxic activity. Ia its mobility characteristics the above component closely resembled those of the larvae-active component previously separated by ea identical procedure from the crude venom. The above mentioned larvae-active component was ineffective on mice. It is concluded that scorpion venoms may contain different wmponents with specific toxic effects against different groups of animals.
161
Y.~x~, C. C., I{aohsiung Medical College, Department of Biochemistry, I{aohsiung, Taiwan, China Slochemical atadiea an the tozk nature of s~èe venom A toxic protein devoid of enzyme activities has been highly purified from Formosan cobra venom. The crystalline toxin, cobrotoxin, was found to be homogeneous and to be the main toxin protein in the venom. It consists of a single peptide chain cross-linked intramolecularly by 4 disulfide bonds with amino terminal leucine and carboxy terminal asparagine. The minimal molecular weight calculated from amino acid composition (62 residues) is 6949 . Since the complete sequence of amino acids in cobrotoxin has been determined and the positions of disulfide bonds clarified, a two-dimensional schematic diagram for the structure of cobrotoxin was proposed. On reduction with ß-mercaptoethanol, cobrotoxin displays 8 SH groups with a concurrent loss is toxicity. The reduced toxin yields oa reoxidation, a biologically active product with complete restoration of toxicity and antigeaicity, specific rotation close to the native value and an infrared spectrum identical with that of cobrotoxin . The results indicate that the integrity of the disulfide bonds in cobrotoxia is essential for lethality. The optical rotatory dispersion (ORD) of cobrotoxin is quite different from those of the usual proteins in having a right-handed a-helical structure and circular dichromism (CD) spectrum suggests that the cobrotoxin molecule contains a ß-structure. On reductive cleavage of the disulfide bonds, cobrotoxin becomes a random conformation and the ORD and CD curves change greatly. However, the reoxidized toxin gave essentially the same ORD and CD curves as cobrotoxin which suggests that cornet reformation on helical structure occurred by air-0xidation of the reduced protein. Antisera have bcen prepared by injecting increasing doses of cobrotoxin with Freund's adjuvaat into rabbits. Immunoglobulia G isolated was allowed to react with cobrotoxin at equivalence. The antigenantibody precipitates were dissolved is 053 M formic acid and the antibody was separated from antigen through a column of Sephadex G-100. The purified antibody was proved to be free from antigen and 100 precipitable with oobrotoxin, and the neutralizing capacity increased 17 5-fold over that of the Antsera. The purified antibody was digested into univalent fragments with mercuri-pepsin . The fragments do not form insoluble aggregates with antigen but form a soluble antigen-antibody complex. The univalent fragments together with the purified antibody may provide valuable tools for an immunochemical approach to the elucidation of the toxic nature of snake venom.
Zr ozxuv, E., FRSprncF~r G. S., Mnurron, F., Liea ~~e Y, S. and Sxur ov, A., Hebrew University, Department of Entomology and Venomous Animals, Jerusalem, Israel ; University of Illinois, Department of Ento urology, Urbane, Illinois, U.S.A., and School of Medicine, Department of Medical Biochemistry, Marseille, France The effect of scorpion venom on lame Injection of scorpion venom into intact or ligated (thus nullifying the central nervous system) larvae of blowflies causes an immediate paralysis accompanied by strong contraction of the test animals, demonstrating the peripheral nerve-muscle stimulating effect of the venom (Any and WEIaa, 1959 ; P~utv~s and RussaLt, 1967). The duration of this contrastive paralysis may range from several seconds to several hours, depending on the amount of venom injected. This effect of scorpion venom on fly larvae formed the basis of a very rapid and sensitive test for the quantitative estimation of scorpion venom. The venom of eighteen different species of scorpions was tested for its contrastive-paralyzing activity in fly larvae and its lethal effect on mice (r-n estimations) . No correlation was found between the two biological activities. These data suggested the possibility that there are different components of scorpion venom responsible for each of the above two biological activities . This hypothesis was verified by the following experimental results: Purified proteins from Androctonus australtr venom (Mue~n~ et al., 1964, 1966) which were highly toxic to mice were completely ineffective against fly larvae when tested for their contrastive-paralyzing activity. Furthermore these proteins were not lethal to the larvae . A clear separation between larvae and mice toxic components was achieved both by starch gel electrophoresis and Sephadex G-50 column chromatography of the crude venom of A. oustralis. It has been shown that the coatractive-paralyzing fraction was the sole fraction lethal to fly larvae . An electrophoretical analysis of the Sephadex larval-active fraction revealed the presence of several protein bands, only one of which contained the larval toxic activity. Ia its mobility characteristics the above component closely resembled those of the larvae-active component previously separated by ea identical procedure from the crude venom. The above mentioned larvae-active component was ineffective on mice. It is concluded that scorpion venoms may contain different wmponents with specific toxic effects against different groups of animals.