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Aim:To study the enzymological characterization of a fibrinolytic enzyme(FⅡ_a)from Agkistrodon acutus venom.Methods:The fibrinogenolytic effect and theinfluences of several protease inhibitors,chelating agents,and metal ions onfibrinogenolytic activity were visualized by sodium dodecyl sulfate-polyacryla-mide gel electrophoresis.The metal content of FⅡ_a was determined by atomicabsorption spectroscopy.Results:After incubation with FⅡ_a(0.25 g/L),Aα-,Bβ-and γ-chains of fibrinogen disappeared within 5 min,30 min,and 8 h,respectively.The molecular weights of major degradation products were 45 000 and 41 000,which were different from those bands produced by plasmin.The fibrinogenolyticactivity of FⅡ_a was strongly inhibited by ethylenediamine tetraacetic acid(EDTA),ethyleneglycol tetraacetic acid(EGTA),dithiothreitol and cysteine,but not byphenyhnethyl-sulfonyl fluoride and soybean trypsin inhibitor.Zinc(3171±25rag&g),potassium(489±17 mg/kg)and calcium(319±13 mg/kg)were found in FⅡ_a.Zn~(2+),Ca~(2+)and Mg~(2+)could recover the fibrinogenolytic activity of FⅡ_a,which wasinhibited by EDTA.Only Ca2+ could recover the fibrinogenolytic activity inhibitedby EGTA.Conclusion:FⅡ_a can degrade the Aα-,Bβ-and γ-chains of fibrinogen.FⅡ_a is a metalloproteinase,and Zn~(2+),Ca~(2+),and disulfide bonds are necessary forits fibrinogenolytic activity.
Aim: To study the enzymological characterization of a fibrinolytic enzyme (FII_a) from Agkistrodon acutus venom. Methods: The fibrinogenolytic effect and the inluences of several protease inhibitors, chelating agents, and metal ions on fibrinogenolytic activity were visualized by sodium dodecyl sulfate-polyacryla-mide gel electrophoresis. The metal content of FⅡ_a was determined by atomic absorption spectroscopy. Results: After incubation with FⅡ_a (0.25 g / L), Aα-, Bβ- and γ-chains of fibrinogen within 5 min, 30 min, and 8 h, respectively The molecular weights of major degradation products were 45 000 and 41 000, which were different from those bands produced by plasmin. The fibrinogenolytic activity of FⅡ_a was strongly inhibited by ethylenediamine tetraacetic acid (EDTA), ethyleneglycol tetraacetic acid (EGTA), dithiothreitol and cysteine , but not byphenyhnethylsulfonyl fluoride and soybean trypsin inhibitor. Zinc (3171 ± 25ragg), potassium (489 ± 17 mg / kg) and calcium (319 ± 13 mg / kg) were found In FⅡ_a.Zn ~ (2 +), Ca ~ (2+) and Mg ~ (2+) could recover the fibrinogenolytic activity of FⅡ_a, which was inhibited by EDTA.Only Ca 2+ could recover the fibrinogenolytic activity inhibitedby EGTA.Conclusion: FⅡ_a can degrade the Aα-, Bβ- and γ-chains of fibrinogen. FⅡ_a is a metalloproteinase, and Zn ~ (2 +), Ca ~ (2 +), and disulfide bonds are necessary for fibrinogenolytic activity.