Isolation and Characterization of a 32-kDa Fibrinolytic Enzyme (FE-32kDa) from Gloydius blomhoffii siniticus Venom: Fibrinolytic Enzyme from Gloydius blomhoffii siniticus Venom.

Abstract

Objectives:This study was undertaken to isolate a fibrinolytic enzyme from the snake venom of Gloydius blomhoffii siniticus and to investigate its enzymatic characteristics and hemorrhagic activity as a potential pharmacopuncture agent.Methods:The fibrinolytic enzyme was isolated by using chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fibrin plate assay. The characteristics of the enzyme were investigated using fibrin plate assay, protein hydrolysis analysis, and hemorrhage assay. Its amino acid composition was determined.Results:The fibrinolytic enzyme with the molecular weight of 32kDa (FE-32kDa) from Gloydius blomhoffii siniticus showed a fibrin hydrolysis zone at the concentration of 0.2 mg/mL in the fibrin plate assay. The fibrin hydrolysis activity of the enzyme was inhibited completely by ethylenediaminetetraacetic acid (EDTA), ethyleneglycoltetraacetic acid (EGTA), and 1, 10-phenanthroline, thiothreitol and cysteine, and partially by phenylmethanesulfonylfluoride (PMSF). Metal ions such as Fe2+ and Hg2+ inhibited the fibrin hydrolysis completely, but Zn2+ enhanced it. FE-32kDa hydrolyzed α-chain but did not hydrolyze β-chain and γ-chain of fibrinogen. High-molecular-weight polypeptides of gelatin were hydrolyzed partially into low-molecular-weight polypeptides, but the extent of hydrolysis was limited. FE-32kDa induced hemorrhage beneath back skin of mice at the dose of 2 μg.Conclusions:FE-32kDa is a α-fibrin(ogen)olytic metalloprotease that requires Zn2+ for fibrinolytic activity and causes hemorrhage, suggesting that the enzyme is not appropriate for use as a clinical pharmacopuncture.