缅甸蝰蛇毒纤溶酶FⅡaa分离及生物活性.docVIP

缅甸蝰蛇毒纤溶酶FⅡaa分离及生物活性 　 作者：陈慧珍， 梁秀霞， 邱鹏新， 陈家树 【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0.1 mL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、0.125 mg/mL)，生理盐水和粗毒(0.1 mg/mL)做对照。6 h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69 000 u。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 (0.2 g/L)保温后，Aα 和Bβ链分别在5 min和6 h后完全水解。γ-链在24 h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、0.125 mg/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6.0 μg。【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。 【关键词】 纤溶酶; 蛇毒; 蝰蛇 　Abstract： 【Objective】 To purify FⅡaa， a fibrinolytic enzyme from Burmese Russells Viper (Daboia Russelli Siamensis) venom， and investigate its fibrinolytic characterization. 【Methods】 FⅡaa was purified from Burmese Russells Viper venom by ion-exchange chromatography and gel-filtration. Molecular weight was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS). Fibrinolytic activity was determined using fibrin plates technique. Specific cleavage of fibrinogen and fibrin was shown on SDS. 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 w