A. Yarleque, S. Campos, E. Escobar, F. Lazo, N. Sanchez, S. Hyslop, N. A. Marsh, P. J. Butterworth and R. G. Price. Isolation and characterization of a fibrinogen-clotting enzyme from venom of the snake Lachesis muta muta(Peruvian bushmaster). Toxicon27, 1189-1197, 1989.-A fibrinogen-clotting enzyme from the venom of the Peruvian bushmaster snake was purified to homogeneity by gel filtration on Sephadex G-100 followed by DEAE-cellulose ion-exchange chromatography using a linear ionic strength gradient with NaCl. The specific activity of the enzyme was 866 NIH U/mg, representing a 55-fold purification, with a recovery of 45%. The amino acid composition was Asx30, Thr14, Ser15, Glx33, Pro23, Gly22, Ala15, Val22, Cys18, Met3, Ile18, Leu23, Tyr2, Phe13, His8, Lys11, Arg11. The total carbohydrate content was 13.4%, comprised of 3.4% hexose, 8.7% hexosamine and 1.3% sialic acid. The enzyme was active against the synthetic amide substrate α-N-benzoyl-dl-arginine-p-nitroanilide (BAPNA) and against the ester substrates α-N-benzoyl-l-arginine ethyl ester (BAEE) and tosyl-l-arginine methyl ester (TAME). Kinetic parameters for TAME esterolysis were: Vmax, 135 μmoles/min/mg and Km, 2.5 × 10-4M. The pH optimum was 8.0. Vmax for BAPNA amidolysis was 0.363 μmoles/min/mg and Km, 7.5 × 10-5M. Enzyme activity was reduced by diethylpyrocarbonate and by photo-oxidation, suggesting that the enzyme is a serine protease with a histidine residue involved in the active site. The enzyme released fibrinopeptide A rapidly from purified human fibrinogen and fibrinopeptide B more slowly. Factor XIII was not activated and the clotting activity was not inhibited by heparin. A dose of 50 μg/kg brought about defibrinogenation in anaesthetized rats but rabbits were unaffected. A dose of 80 μg/kg defibrinogenated conscious rats after 5 hr. There were no hypotensive or haemorrhagic effects.
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Acknowledgements-We acknowledge financial support from the International Foundation for Science, Sweden, Concejo Nacional de Ciencia y Tecnologia CONCYTEC, Peru, and the Wellcome Trust, U.K.
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