Purification and partial characterization of a thrombin-like enzyme from the venom of the bushmaster snake, Lachesis muta noctiv aga

Abstract

A thrombin-like enzyme has been purified from the venom of Lachesis muta noctivaga (41-fold purification and 43% yield). The steps included: gel filtration with Sephadex G-100, hydroxylapatite and DEAE-cellulose chromatography, and finally Sephadex G-150 filtration (twice). The material was homogeneous in polyacrylamide electrophoresis and gel filtration on Sephadex G-150. The enzyme is a glycoprotein of molecular weight 36,300 as determined by gel filtration. the thrombin-like enzyme hydrolyses tosyl- l-arginine methyl ester ( Km = 1·45 × 10 −4 M, Vm = 353 μmole/min·mg, Kcat = 212sec −1) with an optimum pH of 8·30. The enzyme also hydrolyses α- N-benzoyl- dl-arginine p-nitroanilide and is competitively inhibited by benzamidine, β-naphtamidine and phenylguanidine. Clotting and amidase activities are inhibited by diisopropylfluorophosphate. The enzyme molarity was determined by active site titration with p-nitrophenyl- p′-guanidino benzoate (92% pure). Injected in dogs 2 μg of the purified enzyme reduce, in 30 min, the plasma fibrinogen concentration to values less than 15% of the original level. In vitro the activity for human fibrinogen-fibrin conversion was equivalent to 1650 ± 12 NIH thrombin units/mg protein enzyme.