单环刺螠纤溶酶的分离纯化及溶栓活性的初步研究

Purification and Biological Activity of Fibrinolytic Enzyme from Urechis unicinctus

从单环刺螠中分离提取出具有溶栓功效的纤溶酶(UFE),并对其理化性质进行初步研究。单环刺螠组织液经Sephadex G-75凝胶过滤层析、DE52离子交换层析和Sephacryl S-100HR凝胶过滤层析三步分离纯化,获得单一活性组分,分别观察pH值、温度和金属离子对该酶活性的影响,并对其体外溶栓活性进行研究。经SDS-PAGE凝胶电泳和高效液相色谱表征,该酶为单一蛋白,其分子量约为26 kD。该酶的最适pH为8.0,最适温度约为50℃,能被Cu2+、Fe2+和Fe3+抑制,Mg2+激活。纤维蛋白平板实验证实该酶有激酶活性,体外溶栓实验表明其溶栓效果良好。经过三步分离纯化流程可得单一组分的单环刺螠纤溶酶,为其进一步研究和开发提供了实验依据。

To purify and study the fibrinolytic enzyme from the Urechis unicinctus(UFE).Using column chromatography separation consisting of Sephadex G75,Sephacryl S-100HR gel filtration and DE52 ion-exchange gel.It is a single constituent determined by sodium duodecyl sulfate-polyacrylamide gel electrophoresis and high performance liquid chromatography.And the Urechis unicinctus fibrinolytic enzyme was found to have an approximately molecular weight of 26 kD.Its optimal pH is about 8.0 and optimal temperature is probably 50 ℃.Cu2+,Fe2+ and Fe3+ are inhibitors of it,but Mg2+ is activator.The result of fibrinolytic test in vitro shows that UFE owns kinase activity and apparent fibrinolytic effection.UFE can be purified through three steps of purification flow,and the results offered reliable information for the further research and development.