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Observations on a venom neutralizing albumin fraction isolated from serum of the northern copperhead Agkistrodon contortrix mokasen
Third Pan-American Symposium--Abstracts
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REFERENCES WAyr, D. D. and S I ~ , J. M. 0984) J. Toxicol.--Toxin Reviews 3, 181-221. SIMXRD, J. M., M~ES, H. and WATT, D. D. (1986) Pflugers Arch. 486, 620-628. Observations on a venom neutralizing albumin fraction isolated from serum of the northern copperhead Agkistrodon contortrix mokasen. SCOTTA. WmNSTE1N,PmRgE J. L~F^'O/and LEONARDA. SMITI~(Department of Toxinology, Pathology Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD 21701-5011, U.S.A.).
VENOM neutralizing capacity of serum from the northern copperhead Agkistrodon contortrix mokasen was studied. Crude venom serum was found to neutralize the hemorrhagic, proteolytic and lethal activity of A. c. mokasen venom. The serum neutralized up to 2.5 LDs0 of A. c. mokasen venom and up to 5 LDs0 of A. piscivorus venom. The serum was anti-hemorrhagic and a potent inhibitor of venom proteases and trypsin. An albumin fraction was isolated from A. c. mokasen serum by DEAE Atti-Gel-Blue chromatography and Superose-12 (molecular sieve) fast protein liquid chromatography with recycling. This fraction has a mol. wt of 64-68 kD, a pl of 4.6-4.7 and displayed anodal migration in horizontal electrophoresis. The albumin fraction exhibited the venom neutralization capacity of the crude serum, but did not inhibit venom proteases or trypsin. In concentrations of only 16.5 #g, it protected against hemorrage induced by up to 100 #g of A. c. mokasen venom. The venom neutralizing activity of serum from Lampropeltis getulus getulus is also discussed.
Some properties of a thrombin-like enzyme isolated from bushmaster Lachesis muta snake venom. ARMANDO YARLEQUI~,I N. SANcr~z,' S. CAMPOS,~ N. MARSH,2 P. BUTTERWORTH,2 R. PRICE,2 S. HYSLOP2 AND D. LANE3
(~Laboratory of Molecular Biology, ICBAR San Marcos University of Lima, Peru; 2Department of Physiology and Biochemistry, KQC, University of London; 3Department of Haematology, Charing Cross Hospital Medical School, London, U.K.). SOME STRUCTURALand functional properties of thrombin-like enzyme from L. muta venom have been investigated. Amino acid content after acid hydrolysis was determined as well as protein-associated carbohydrates in crude venom and purified enzyme. The role of diethyl pyrocarbonate and the effect of photooxidation with the dye rose Bengal have also been assayed on amidolytic activity. Using fibrinogen and human plasma as substrates have determined the release of fibrinopeptides, FPA and FPB. The results of amino acid composition were as follows: Thr 14, Ser 15, Glu 32, Pro 23, Ala 16, Val 22, lie 18, Leu 22, Phe 14, His 8, Asp 30, Lys 11, Arg 11, Met 3, Cys 18 and Tyr 2. Carbohydrate content in crude venom was 6.3% (2.5% hexosc, 2.7% hexosamine and 1.1% sialic acid) whilst in purified enzyme was 13.4% (3.4% hexose, 8.7% hexosamine and 1.3% sialic acid). On the other hand, the proteasc inhibitor diethylpyrocarbonate caused a progressive and concentrationdependant reduction in thrombin like activity with BAPNa as substrate. Enzyme activity could be regenerated overnight by the addition of hydroxylamine. Photooxidation experiments achieved a progressive reduction of enzyme activity. The results taken together suggested the involvement of histidine in the enzymatic activity and that serine is likely to be present at the active site. With respect to action-mode of this enzyme, FPA is released rapidly from purified fibrinogen in the same manner as other similar enzymes from snake venoms. However, using plasma as substrate both FPA, FPB and some other unidentified peptides are released suggesting a mechanism comparable to thrombin. Acknowledgement- This research was supported by IFS-Sweden, British Council and The Wellcome Trust, United Kingdom and CONCYTEC-Per6. Monoclonal antibodies against toxinsfiom the scorpion Centruroides noxius. F. Z. ZAMUDIO,R. S~VF~RA, G. B. GURROLA, P. HERION and L. D. POSSANI (Departamento de Bioquimica, Centro de Ingenieria Genetica y Biotecnologia--UNAM, Apartado Postal 510-3, Cuernavaca 62270, Mexico).
TOXIN II-9.2.2 from the venom of the Mexican scorpion C. noxius is a Na + channel blocker (SITGES et al., 1987). In order to correlate the structure with function of this toxin, monoclonal antibodies were prepared and characterized. Three Balb/C mice were immunized with toxin II-9.2.2 coupled to tyroglobulin. Spleen cells of the immunized mice were fused with SP2/0-Agl4 myeloma cells. Hybridoma cells producing antibodies, which recognized toxin II-9.2.2, were selected and cloned by ELISA and semi-solid agar techniques. Six independent hybridoma cell lines were obtained (BCF1 to BCF3, BCF7 to BCF9) and used to produce ascites liquid in Balb/C mice. Antibodies from the ascites fluid were purified by ammonium sulfate precipitation and ion exchange chromatography. They are immunoglobulins of the class G1 and G2a. These antibodies bind to toxin II-9.2.2 attached to the ELISA plate in a dose-dependent manner; they are displaced by addition of free toxin. Additional experiments were carried out to show that monoclonal antibodies of the hybrid BCFI and BCF8
629
REFERENCES WAyr, D. D. and S I ~ , J. M. 0984) J. Toxicol.--Toxin Reviews 3, 181-221. SIMXRD, J. M., M~ES, H. and WATT, D. D. (1986) Pflugers Arch. 486, 620-628. Observations on a venom neutralizing albumin fraction isolated from serum of the northern copperhead Agkistrodon contortrix mokasen. SCOTTA. WmNSTE1N,PmRgE J. L~F^'O/and LEONARDA. SMITI~(Department of Toxinology, Pathology Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD 21701-5011, U.S.A.).
VENOM neutralizing capacity of serum from the northern copperhead Agkistrodon contortrix mokasen was studied. Crude venom serum was found to neutralize the hemorrhagic, proteolytic and lethal activity of A. c. mokasen venom. The serum neutralized up to 2.5 LDs0 of A. c. mokasen venom and up to 5 LDs0 of A. piscivorus venom. The serum was anti-hemorrhagic and a potent inhibitor of venom proteases and trypsin. An albumin fraction was isolated from A. c. mokasen serum by DEAE Atti-Gel-Blue chromatography and Superose-12 (molecular sieve) fast protein liquid chromatography with recycling. This fraction has a mol. wt of 64-68 kD, a pl of 4.6-4.7 and displayed anodal migration in horizontal electrophoresis. The albumin fraction exhibited the venom neutralization capacity of the crude serum, but did not inhibit venom proteases or trypsin. In concentrations of only 16.5 #g, it protected against hemorrage induced by up to 100 #g of A. c. mokasen venom. The venom neutralizing activity of serum from Lampropeltis getulus getulus is also discussed.
Some properties of a thrombin-like enzyme isolated from bushmaster Lachesis muta snake venom. ARMANDO YARLEQUI~,I N. SANcr~z,' S. CAMPOS,~ N. MARSH,2 P. BUTTERWORTH,2 R. PRICE,2 S. HYSLOP2 AND D. LANE3
(~Laboratory of Molecular Biology, ICBAR San Marcos University of Lima, Peru; 2Department of Physiology and Biochemistry, KQC, University of London; 3Department of Haematology, Charing Cross Hospital Medical School, London, U.K.). SOME STRUCTURALand functional properties of thrombin-like enzyme from L. muta venom have been investigated. Amino acid content after acid hydrolysis was determined as well as protein-associated carbohydrates in crude venom and purified enzyme. The role of diethyl pyrocarbonate and the effect of photooxidation with the dye rose Bengal have also been assayed on amidolytic activity. Using fibrinogen and human plasma as substrates have determined the release of fibrinopeptides, FPA and FPB. The results of amino acid composition were as follows: Thr 14, Ser 15, Glu 32, Pro 23, Ala 16, Val 22, lie 18, Leu 22, Phe 14, His 8, Asp 30, Lys 11, Arg 11, Met 3, Cys 18 and Tyr 2. Carbohydrate content in crude venom was 6.3% (2.5% hexosc, 2.7% hexosamine and 1.1% sialic acid) whilst in purified enzyme was 13.4% (3.4% hexose, 8.7% hexosamine and 1.3% sialic acid). On the other hand, the proteasc inhibitor diethylpyrocarbonate caused a progressive and concentrationdependant reduction in thrombin like activity with BAPNa as substrate. Enzyme activity could be regenerated overnight by the addition of hydroxylamine. Photooxidation experiments achieved a progressive reduction of enzyme activity. The results taken together suggested the involvement of histidine in the enzymatic activity and that serine is likely to be present at the active site. With respect to action-mode of this enzyme, FPA is released rapidly from purified fibrinogen in the same manner as other similar enzymes from snake venoms. However, using plasma as substrate both FPA, FPB and some other unidentified peptides are released suggesting a mechanism comparable to thrombin. Acknowledgement- This research was supported by IFS-Sweden, British Council and The Wellcome Trust, United Kingdom and CONCYTEC-Per6. Monoclonal antibodies against toxinsfiom the scorpion Centruroides noxius. F. Z. ZAMUDIO,R. S~VF~RA, G. B. GURROLA, P. HERION and L. D. POSSANI (Departamento de Bioquimica, Centro de Ingenieria Genetica y Biotecnologia--UNAM, Apartado Postal 510-3, Cuernavaca 62270, Mexico).
TOXIN II-9.2.2 from the venom of the Mexican scorpion C. noxius is a Na + channel blocker (SITGES et al., 1987). In order to correlate the structure with function of this toxin, monoclonal antibodies were prepared and characterized. Three Balb/C mice were immunized with toxin II-9.2.2 coupled to tyroglobulin. Spleen cells of the immunized mice were fused with SP2/0-Agl4 myeloma cells. Hybridoma cells producing antibodies, which recognized toxin II-9.2.2, were selected and cloned by ELISA and semi-solid agar techniques. Six independent hybridoma cell lines were obtained (BCF1 to BCF3, BCF7 to BCF9) and used to produce ascites liquid in Balb/C mice. Antibodies from the ascites fluid were purified by ammonium sulfate precipitation and ion exchange chromatography. They are immunoglobulins of the class G1 and G2a. These antibodies bind to toxin II-9.2.2 attached to the ELISA plate in a dose-dependent manner; they are displaced by addition of free toxin. Additional experiments were carried out to show that monoclonal antibodies of the hybrid BCFI and BCF8