纤维蛋白酶谱检测蚯蚓纤溶酶组分

Determination of fibrinolytic enzymes from earthworm(Eisenia foetida) by fibrin zymography

目的建立鉴定蚯蚓纤溶酶粗酶液中活性组分的方法。方法根据蚯蚓纤溶酶能水解纤维蛋白的性质,建立了纤维蛋白-十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(Fibrin-SDS-PAGE),优化了电泳条件,电泳后酶所在部位为透明区带。结果分离胶中纤维蛋白终质量浓度为0.4×10^(-3)mg·L^(-1),电泳后胶板用2.5%TritonX-100复性30min,PBS缓冲液(pH7.2)保温30min,考马斯亮蓝R-250染色,7%醋酸脱色,可以得到清晰的酶谱。结论该方法灵敏度高,用2μg样品,就能准确检测出粗酶液中所有纤溶酶组分。

OBJECTIVE To establish a method for identifying the active fibrinolytic enzyme components from Eisenia foetida. METHODS Fibrin-SDS-PAGE containing fibrin as substrate in the separated gel was established for the determination of the fibrinolytic enzyme components. Electrophoresis conditions were optimized. The enzymes were identified as they showed clear zone. RESULTS The final concentration of fibrin in separated gel was 0.4×10-3 mg·L^-1. After electrophoresis, zymogram was obtained when the gel was renatured with 2.5 % TritonX-100 for 30 rain, incubated with PBS buffer（pH 7.2） for 30 rain, stained with Comassie Brilliant Blue R-250, restained with 7 % acetic acid. CONCLUSION This method is proved to be very sensitive, 2μg crude extract is enough and all the fibrinolytic enzymes could be detected exactly under this experimental conditions.