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_医学论文.docVIP

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_医学论文 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_医学论文 林熙 辛淑波 祈洁珍 梁秀霞 陈家树 邱鹏新 颜光美 【摘要】 【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? 【关键词】 蛇毒蝰蛇凝血因子Ⅹ激活物 　　Abstract: 【 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. 　　Key words: factor Ⅹ activator snake venom Daboia russelli siamensis 　　婴幼儿先天性心脏病体外循环(cardiopulmonary bypass, CPB)术后肺损伤是临床上常见的并发症[1]?其发生的原因与CPB诱导的大量炎症介质释放,CPB期间机械通气停止后肺塌陷后