Isolation from Gloydius blomhoffii siniticus Venom of a Fibrin(ogen)olytic Enzyme Consisting of Two Heterogenous Polypeptides

Objective: This study was undertaken to isolate a fibrin(ogen)olytic enzyme from the snake venom of Gloydius blomhoffii siniticus and to investigate the enzymatic characteristics and hemorrhagic activity of the isolated enzyme as a potential pharmacopuncture agent. Methods: The fibrinolytic enzyme was isolated by using chromatography, sodium dodecyl sulfatepolyacrylamide gel electrophoresis, and fibrin plate assay. The characteristics of the enzyme were determined by using fibrin plate assay, protein hydrolysis analysis, and hemorrhage assay. Its amino acid composition was determined. Results: The fibrin(ogen)olytic enzyme with the molecular weight of 27 kDa (FE-27kDa) isolated from G. b. siniticus venom consisted of two heterogenous disulfide bond-linked polypeptides with the molecular weights of 15 kDa and 18 kDa. When more than 20 μg of FE-27kDa was applied on the fibrin plate, fibrinolysis zone was formed a indicating its fibrinolytic activity. The fibrinolytic activity was inhibited completely by phenylmethanesulfonylfluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA) and partially by thiothreitol and cysteine. Metal ions such as Hg2+ and Fe2+ inhibited the fibrinolytic activity completely, but Mn2+ did not. FE-27kDa preferentially hydrolyzed α-chain of fibrinogen and slowly hydrolyzed β-chain, but did not hydrolyze γ-chain. High-molecular-weight polypeptides of gelatin were hydrolyzed partially into polypeptides with molecular weights of more than 45 kDa. A dosage of more than 10 μg of FE- 27kDa per mouse was required to induce hemorrhage beneath the skin. Conclusion: FE-27kDa was a serine proteinase consisting of two heterogeneous polypeptides, hydrolyzed fibrin, fibrinogen, and gelatin, and caused hemorrhage beneath the skin of mouse. This study suggests that the potential of FE-27kDa as pharmacopuncture agent should be limited due to low fibrinolytic activity and a possible side effect of hemorrhage.