Coagulant proteinase from [formula omitted][formula omitted] venom

Abstract

The venom of Bothrops colombiensis, like other Crotalidae venoms, contains thrombin-like activity. We purified a mixture of isoenzymes by chromatography of the crude venom on DEAE-Sephacel where coagulant proteinases were separated from other proteolytic enzymes. By subsequent chromatography on Sephadex G-100 we obtained coagulant proteinase as a single band on acrylamide gel electrophoresis at pH 7.5 which showed 4 major protein bands when subjected to flat gel isoelectric focusing. This heterogeneity is presumably due to carbohydrates present in this glycoprotein. The native molecular weight of the coagulant proteinase was found to be over 90 000 by gel filtration. SDS electrophoresis showed, however, that the monomer molecular weight is around 67 000. The specific coagulant activity of the purified enzyme was increased 13 fold by purification and was 231 NIH units/mg. The optimal pH for coagulation of bovine fibrinogen was at pH 7.0. The enzyme shows maximal stability in the pH range 5–6 when incubated for 1 hr at 37 °C. The intraperitoneal LD 50 for white mice was 4.0 mg/kg. The enzyme is similar to other known coagulant proteinases from snake venoms and thus potentially useful as a therapeutic agent.