缅甸蝰蛇毒纤溶酶FⅡaa的分离及生物活性  

Purification and Characterization of FⅡaa,a Fibrinolytic Enzyme from Burmese Russell's Viper (Daboia Russelli Siamensis)

作　　者：陈慧珍[1] 梁秀霞[2] 邱鹏新[2] 陈家树[2] 

机构地区：[1]中山大学东校区教学实验中心,广东广州510006 [2]中山大学中山医学院药理学教研室,广东广州510080

出　　处：《中山大学学报（医学科学版）》2010年第4期513-519,共7页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：广东省科技计划项目(2004B33001003)

摘　　要：【目的】从缅甸蝰蛇毒中分离纯化得到纤溶酶FⅡaa并研究其纤维蛋白溶解活性。【方法】缅甸蝰蛇蛇毒纤溶酶FⅡaa经过离子交换层析和凝胶层析分离程序得到,SDS-PAGE电泳测得其分子质量,用纤维蛋白平板法测得其纤维蛋白溶解活性,SDS-PAGE电泳测得其纤维蛋白原和纤维蛋白水解特异性。通过小鼠背部皮下注射FⅡaa测定FⅡaa的局部出血活性。取昆明种小白鼠42只,体质量(20±2)g,雌雄各半,随机分成7组,每组6只,皮下注射0.1mL的FⅡaa(5个剂量组:用生理盐水配制成浓度分别为0.025、0.05、0.075、0.1、0.125mg/mL),生理盐水和粗毒(0.1mg/mL)做对照。6h后处死小白鼠,从皮内侧面测量出血点的大小,计算最小出血剂量(MHD)。【结果】通过三步分离方法得到蛇毒纤溶酶FⅡaa,其分子质量是69000u。FⅡaa在纤维蛋白平板和加热纤维蛋白平板上均显示了纤维蛋白溶解活性。纤维蛋白原和FⅡaa(0.2g/L)保温后,Aα和Bβ链分别在5min和6h后完全水解。γ-链在24h后部分水解。FⅡaa水解纤维蛋白的过程与纤维蛋白原的水解过程相似。蛇毒纤溶酶FⅡaa(0.025、0.05、0.075、0.1、0.125mg/mL)的皮内出血点直径(mm)分别为7.7±1.0、9.9±1.4、10.5±1.8、12.7±1.4、13.5±1.3。最小出血剂量(MHD)为6.0μg。【结论】FⅡaa是一种可以直接降解纤维蛋白且具有出血活性的α-纤维蛋白酶。【Objective】 To purify FⅡaa,a fibrinolytic enzyme from Burmese Russell＇s Viper （Daboia Russelli Siamensis） venom,and investigate its fibrinolytic characterization. 【Methods】 FⅡaa was purified from Burmese Russell＇s Viper venom by ion-exchange chromatography and gel-filtration. Molecular weight was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）. Fibrinolytic activity was determined using fibrin plates technique. Specific cleavage of fibrinogen and fibrin was shown on SDS-PAGE. The local hemorrhagic activity was assayed by subcutaneous injection on the back of mouse. 42 Kunming strain mice （20 ± 2） g,bisexual each half,were randomly divided into 7 groups with 6 individual per groups. FⅡaa （0.025,0.05,0.075,0.1,0.125 g / L） were each injected subcutaneously in the back with the test samples in 0.1 mL of 0.9% saline,by control of 0.9% saline and crude venom and sacrificed after 6 hours and the diameters of hemorrhagic spots measured on the underside of the skin. 【Results】 FⅡaa was achieved by a three-step fraction with a molecular weight of 69 000. FⅡaa showed fibrinolytic activity whether on fibrin plate or heated fibrin plate. After incubated with FⅡaa （0.2 g / L）,Aα and Bβ of fibrinogen disappeared within 5 min and 6 h respectively. The γ-chain was partially degraded after 24 h. The process of fibrin hydrolysis by F Ⅱaa was similar to that observed with fibrinogen. FⅡaa （0.025,0.05,0.075,0.1,0.125 g/L） was injected subcutaneously,the diameters（mm） of hemorrhagic spots measured on the underside of the skin of which were 7.7 ± 1.0,9.9 ± 1.4,10.5 ± 1.8,12.7± 1.4,and 13.5 ± 1.3 mm,respectively. The minimum hemorrhagic dose （MHD） of FⅡaa was 6.0 μg. 【Conclusion】 FⅡaa was a α-fibrinogenase that hydrolyzed fibrin directly with hemorrhagic activity.

关 键 词：纤溶酶 蛇毒 蝰蛇 

分 类 号：R966[医药卫生—药理学] Q959.6[医药卫生—药学]