Basic proteinases from Bothrops moojeni (caissaca) venom—I. Isolation and activity of two serine proteinases, MSP 1 and MSP 2, on synthetic substrates and on platelet aggregation

Abstract

Two serine proteinases, MSP 1 and MSP 2, were isolated from Bothrops moojeni venom by chromatographies on Sephadex G-100, DEAE-Sephacel (pH 7.5) and SP-Sephadex C-50 (pH 7.5). Both enzymes are basic glycoproteins. On sodium dodecyl sulfate-polyacrylamide electrophoresis, MSP 1 presented two close protein bands corresponding to the mol. wts of 34,000 and 32,500. MSP 2 behaved as a single-chain protein with a mol. wt of 38,000. Specific esterolytic activities of MSP 1 and MSP 2 on α- N-tosyl- l-arginine methyl ester (TAME) are 33 υmol min −1 mg −1 and 184 υmol min −1mg −1, respectively. The most sensitive substrates for the amidolytic activity of both proteinases were the thrombin substrate d-Phe-pipecolyl(Pip)-Arg-4-nitroanilide(Nan) and the glandular kallikrein substrate d-Val-Leu-Arg-Nan. MSP 1, in a concentration of 10 −8M, causes platelet aggregation in platelet-rich plasma and washed platelets. It also enhances the ADP-induced platelet aggregation. Prostaglandin E 1 (PGE 1), phenylmethylsulfonyl fluoride (PMSF) and ethylenediamine tetracetic acid (EDTA) abolished completely the aggregation induced by MSP 1. Torresea cearensis trypsin inhibitor (TCTI) inhibited both amidolytic ( K i = 1.96 × 10 −7M) and platelet-aggregating ( K i = 1.66 × 10 −7M) activities of MSP 1. The esterolytic activity of MSP 1 and MSP 2 was completely abolished by PMSF, only partially by soybean trypsin inhibitor (SBTI) and benzamidine and not affected by Trasylol. MSP 2 was also inhibited by TCTI ( K i = 0.7 × 10 −7M).