title: Isolation and characterization of a fibrinogen-clotting enzyme from venom of the snake, Lachesis muta muta (Peruvian bushmaster).
abstract: 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 alpha-N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) and against the ester substrates alpha-N-benzoyl-L-arginine ethyl ester (BAEE) and tosyl-L-arginine methyl ester (TAME). Kinetic parameters for TAME esterolysis were: Vmax, 135 mumoles/min/mg and Km, 2.5 x 10(-4) M. The pH optimum was 8.0. Vmax for BAPNA amidolysis was 0.363 mumoles/min/mg and Km, 7.5 x 10(-5) M. 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 micrograms/kg brought about defibrinogenation in anaesthetized rats but rabbits were unaffected. A dose of 80 micrograms/kg defibrinogenated conscious rats after 5 hr. There were no hypotensive or haemorrhagic effects.
authors.affiliation: Faculdad de Ciencias Biologicas, Universidad Nacional Mayor de San Marcos, Lima, Peru.
authors.forename: A
authors.initials: A
authors.lastname: Yarleque
authors.forename: S
authors.initials: S
authors.lastname: Campos
authors.forename: E
authors.initials: E
authors.lastname: Escobar
authors.forename: F
authors.initials: F
authors.lastname: Lazo
authors.forename: N
authors.initials: N
authors.lastname: Sanchez
authors.forename: S
authors.initials: S
authors.lastname: Hyslop
authors.forename: N A
authors.initials: NA
authors.lastname: Marsh
authors.forename: P J
authors.initials: PJ
authors.lastname: Butterworth
authors.forename: R G
authors.initials: RG
authors.lastname: Price
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001777:Blood Coagulation / Q000187:drug effects; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003435:Crotalid Venoms / Q000032:analysis* / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D012697:Serine Endopeptidases / Q000032:analysis / Q000302:isolation & purification*
chemical_list: D000596:Amino Acids; D003435:Crotalid Venoms; C062785:fibrinogen clotting enzyme, Lachesis muta muta; D012697:Serine Endopeptidases