Purification And Biochemical Characterization Of Thrombin-like Enzymes From The Venom Of Agkistrodon Halys Ussuriensis Emelianov

Snake venom thrombin-like enzymes, a member of serine proteases, can convert fibrinogen into fibrin. The resulting fibrin forms a non-cross-linking fibrin microclot which is susceptible to degration by plasmin and thus can be used to decrease the blood viscosity because of comsuption of fibrin. Thrombin-like enzymes have been used extensively in clinical medicine to cure and prevent thrombotic and myocardial infraction diease. In this study, two fibrinogenolytic proteases, termed as enzyme I and enzyme II, were isolated and purified from the venom of Agkistrodon halys ussuriensis by means of liquid chromatographies involving cation-exchange, gel-filtration followed by anion-exchange chromatography. Biochemical characterization of these enzymes showed they also exhibited both in vitro fibrinogen-clotting and arginine-esterase activities. Serine protease inhibitors including phenyl methyl Sulfonyl Fluoride (PMSF), Benzamidine as well as N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK) were found to affect their activities, suggesting they belonged to serine protease family. Ethylene diamine tetraacetic acid (EDTA), the generally applied ion chelator, did not show inhibitory activity to these enzymes, indicating they were non-metal proteases. Incubation with fibrinogen, SDS-PAGE analysis revealed that Enzyme I could only cleave fibrinogen to release a small amount of fibrinopeptide A, while enzyme II could release both fibrinopeptide A and a small amount of fibrinopeptide B.Both Enzyme I and Enzyme II can preferentially degrade the Aa-chain of fibrinogen .When the incubation prolonged, the Enzume II was found to degrade γ-chain of fibrinogen by SDS-PAGE assay. Both of them were consisted of a single polypeptide chain on SDS-PAGE. Analysis proved the molecular weights for enzyme I, 34kDa, and enzyme II, 38kDa, respectively. Using (N a-benzoyl-L-arginine ethyl ester) BAEE as sustrate, the optimum pH of both Enzyme I and Enzume II is 8.0, and the optimum temperature of them is 60 ℃ and 50 ℃. Sequencing of N-terminus amino acid residues of Enzyme II showed higher identity of other source thrombin-like enzymes...