五步蛇毒纤溶组分FⅨcaⅠ的分离纯化和生物活性的测定  

Purification and biological activity of FⅨcaⅠ,a fibrinolytic component from Agkistrodon acutus venom

作　　者：杨亚萍[1] 刘涛[2] 陈家树[2] 林熙[3] 

机构地区：[1]暨南大学分析测试中心,广东广州510632 [2]中山大学医学院药理学教研室,广东广州510080 [3]暨南大学医学院药理学系,广东广州510632

出　　处：《中国药理学通报》2011年第11期1550-1555,共6页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No81000209);教育部科学技术研究重点资助项目(No210255);暨南大学科研培育与创新基金资助项目(No21609304)

摘　　要：目的从五步蛇(安徽产)毒分离纯化一种具有纤溶活性的组分FⅨcaⅠ,并研究其理化性质和生物活性。方法应用DEAE-Sephadex A-50阴离子交换层析、Sephadex G-75凝胶层析、Chelating Sepharose Fast Flow金属离子螯合亲和层析和Sephadex G-50凝胶层析四步分离纯化目的组分FⅨcaⅠ;纤维蛋白平板法和SDS-PAGE测定FⅨcaⅠ的生物活性;通过小鼠皮下注射不同剂量的FⅨcaⅠ,测量皮下出血斑的面积并求出最小出血剂量。结果从五步蛇毒分离纯化的FⅨcaⅠ组分为单体蛋白,相对分子量23 ku,等电点为4.8。FⅨcaⅠ可降解纤维蛋白和纤维蛋白原,优先降解α链,呈时效、量效关系。蛇毒纤溶酶FⅨcaⅠ最小出血剂量为54.9μg,为非出血性蛇毒纤溶酶。结论从五步蛇(安徽产)毒分离纯化的FⅨcaⅠ是一种分子量较小,比较稳定,纤溶活性强,可直接降解纤维蛋白且非出血性的新蛇毒纤溶酶。Aim To purify FⅨcaⅠ,a fibrinolytic component from Agkistrodon acutus venom（Anhui province）,and illustrate its biochemical and biological characters.Methods FⅨcaⅠ was purified from Agkistrodon acutus venom by a combination of negative ion exchange chromatography on DEAE-Sephadex A-50,gel filtration on Sephadex G-75,Znion affinity chromatography on Chelating Sepharose Fast Flow and gel filtration on Sephadex G-50.Fibrinolytic activity was determined by using fibrin plate method.Specific cleavage of bovine fibrinogen and fibrin was shown on SDS-PAGE.The local hemorrhagic activity of FⅨcaⅠ was measured by injecting subcutaneously at the back of mouse with different concentrations of FⅨcaⅠ.The areas of hemorrhagic spots were measured and the min-imum hemolytic dose（MHD） was calculated.Results FⅨcaⅠ was achieved by a four-step fraction with a molecular weight of 23 ku and the isoelectric point was 4.8.FⅨcaⅠ could degrade fibrin and fibrinogen.The α chain was degraded preferentially,which showed time-effect and dose-effect relationship.The MHD of FⅨcaⅠ was 52.9 μg.Conclusion FⅨcaⅠ is successfully isolated and purified by this method.It is stable and has small molecular weight.FⅨcaⅠ can degrade fibrin and fibrinogen,and hydrolyzed fibrin directly with no hemorrhagic activity.

关 键 词：五步蛇 FⅨcaⅠ 蛇毒 纤溶活性 纯化 金属蛋白酶 

分 类 号：R-332[医药卫生] R282.74