五步蛇毒蛋白C激活剂的纯化与活性分析  

Purification and activity analysis of protein C activator from the Agkistrodon acutus venom

作　　者：张根葆[1] 张毅[2] 孔岩[1] 许敏[1] 李曙[1] 

机构地区：[1]皖南医学院蛇毒研究室,安徽芜湖241002 [2]河南工业大学国际学院

出　　处：《蛇志》2008年第4期249-251,共3页Journal of Snake

基　　金：安徽省教育厅自然科学基金(2005kj295);安徽省高校青年教师科研资助项目(2007jq1172)

摘　　要：目的研究五步蛇毒蛋白C激活剂(PCA)组分的分离纯化及其抗凝活性。方法借助DEAE-Cel-lulose、CM-Sephadex C-50和Sephadex G-75柱层析,以离子交换和凝胶过滤法从皖南产五步蛇粗毒中分离纯化PCA组分;以SDS-PAGE凝胶电泳检测其纯度和分子量,发色底物法(Chromogenic Substrate Assay)测定PCA组分的生色反应能力;测定PCA对正常血浆KPTT和PT的影响。结果从五步蛇粗毒中纯化的PCA组分经SDS-PAGE测定为单一区带,相对分子质量约为18.5 kD,等电点pH4.9;发色底物法显示其具有生色反应能力;该PCA1 mg/L在体外明显延长KPTT,但PT不受影响。结论采用离子交换和凝胶过滤法,可从皖南产五步蛇毒中分离纯化出高纯度的PCA组分,该组分可抑制内凝途径影响血液凝固过程。Objective To investigate the purification and anticoagulation activity of protein C activator（PCA） fromAgkistrodonacutus venom（AAV）. Methods Isolation and purification of the PCA fraction from AAV were carried out by a combination of DEAE-Cellulose, CM-Sephadex C-50 and SP-Sephadex G-75 column chromatography. Homogenicity and molecular weight of the fraction were determined in SDS-polyacrylamide gel electrophoresis （SDS- PAGE）. Anticoagulant activity of the PCA fraction were measured by KPTT, PT of normal rabbit plasma and color producing reaction ability were performed with Chromogenic substrate assay. Results The purified PCA fraction showed a single band in SDS-PAGE and the molecular mass was about 18.5 kD. The isoelectric point of PCA was pH 4. 9. The fraction showed a color producing reaction ability,and prolong obviously KPTT on normal rabbit plasma（1 mg/ L） in vitro. Conclusion The purified PCA fraction from AAV in Wannan area, using ion exchange and gel filtration method, possessed distinct anticoagulant activity by inhibiting endogenous blood clotting pathway.

关 键 词：五步蛇毒 蛋白C激活剂 柱层析 

分 类 号：R996.3[医药卫生—毒理学]