Isolation and characterization of a fibrinogen-clotting enzyme from venom of the snake, Lachesis muta muta(Peruvian bushmaster)

A. Yarleque, S. Campos, E. Escobar, F. Lazo, N. Sanchez, S. Hyslop, N. A. Marsh, P. J. Butterworth, R. G. Price

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27 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)1189-1197
Number of pages9
JournalToxicon
Volume27
Issue number11
DOIs
StatePublished - 1989

Bibliographical note

Funding Information:
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.

Copyright:
Copyright 2014 Elsevier B.V., All rights reserved.

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