蝰蛇(缅甸亚种)毒凝血因子Ⅹ激活物FⅤe-1的分离纯化与理化活性的研究(英文)

Purification,Biochemical Properties,and Activities of a Novel Factor Ⅹ Activator(FⅤe-1) from Daboia Russelli Siamensis(Myanmar) Venom

【目的】从蝰蛇(缅甸亚种)毒分离纯化一种凝血活性因子Ⅹ激活物FⅤe-1,并研究其理化性质、序列测定以及凝血活性。【方法】应用阳离子交换层析、分子凝胶过滤层析分离纯化蛇毒,采用MALDI质谱测定法测定分子质量,等电聚焦电泳测定等电点,Edman降解法测定蛋白N末端氨基酸序列,发色底物法和SDS-PAGE等方法测定FⅤe-1的酶学特征和凝血活性。【结果】从蝰蛇(缅甸亚种)毒分离得到的FⅤe-1为单体蛋白,相对分子质量为13 808,等电点4.6,0.5 mg FⅤe-1的凝血活性与1.5625 u的凝血酶或与54.93 ng RVVⅩ的凝血活性相当;可激活FⅩ,但对凝血酶和纤维蛋白原无作用;酶活性的适宜pH为6.5～7.5,适宜温度为25～60℃;为钙依赖性,凝血活性可被EDTA和DTT抑制;FⅤe-1的N端序列为NH2-N-L-Y-Q-F-G-E-M-I-N;具有呈剂量依赖性的促血浆凝固作用。【结论】从蝰蛇(缅甸亚种)毒纯化出一种FⅤe-1是一个凝血因子Ⅹ的激活物,可激活凝血因子Ⅹ,但对凝血酶和纤维蛋白原的作用微弱。

【 Objective】 To purify and characterize a novel factor Ⅹ activator,FⅤe-1 from Daboia russelli siamensis（Myanmar） venom.【Methods】 FⅤe-1 was purified by ion-exchange chromatography and gel filtration.The hemostatic activity of FⅤe-1 was determined based on chromogenic substrates.The fibrinogen-clotting activity of FⅤe-1 was also determined.Thermal stability,pH stability,enzyme activity,and inhibition of FⅤe-1 were determined by its remaining procoagulant activity.N-treminal sequence was determined by the method of automated Edman degradation.【Results】 FⅤe-1 was achieved by chromatography with a molecular weight of 13,808 and an isoelectric point of 4.6.The hemostatic activity of 0.5 mg FⅤe-1 was equal to that of 1.5625 u thrombin or that of 54.93 ng RVVⅩ.FⅤe-1 primarily activated FⅩ,but did not affect on prothrombin and fibrinogen.The suitable pH and temperature range of FⅤe-1 was 6.5-7.5 and 25-60 ℃,respectively.The activity of FⅤe-1 was enhanced by Ca2＋ and inhibited by EDTA and DTT.The N-terminal sequence of FⅤe-1 was NH2-N-L-Y-Q-F-G-E-M-I-N.【Conclusion】 FⅤe-1 is a factor X-activating enzyme,which could activate FⅩ to FⅩa,but have minimal effect on prothrombin and fibrinogen.