Purification and characterization of a coagulant enzyme from Trimeresurus flavoviridis venom.
An enzyme bearing thrombin-like specificity has been purified to homogeneity from the venom of Trimeresurus flavoviridis (the Habu snake). The enzyme is a monomer with a molecular weight of 23,500 as determined by analytical gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein contains approximately 210 amino acid residues and has a relatively high content of aspartic acid and glutamic acid. The isoelectric point was 4.8 and the extinction coefficient at 280 nm for a 1% solution was 11.5. The enzyme acted directly on fibrinogen to form a fibrin clot with 2.0 NIH units. Analysis by high performance liquid chromatography of enzyme-treated fibrinogen revealed the release of a peptide identical in composition to thrombin-induced fibrinopeptide A, but no peptide corresponding to fibrinopeptide B was detected. The enzyme showed esterase and amidase activities on synthetic substrates containing arginine. The enzyme exhibited higher activity toward tosyl-L-arginine methyl ester (TAME) but 6-times lower activity toward benzoyl-L-arginine p-nitroanilide when compared with bovin thrombin. The esterase activity was inhibited by diisopropylfluorophosphate and at a slower rate by phenylmethanesulfonyl fluoride, but was least affected by tosyl-L-lysine chloromethyl ketone, showing that the enzyme is a serine protease like thrombin. The enzyme showed a bell-shaped pH dependence of kcat/Km for hydrolysis of TAME, with a maximum around pH 8.5.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D003435:Crotalid Venoms / Q000302:isolation & purification*; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D012911:Snakes; D013379:Substrate Specificity; D014361:Trypsin Inhibitors / Q000494:pharmacology
D000596:Amino Acids; D003435:Crotalid Venoms; D014361:Trypsin Inhibitors; D005340:Fibrinogen; D010450:Endopeptidases