一种新型海洋纤溶酶的分离纯化与性质鉴定(英文)

Purification and Characterization of a Novel Fibrinolytic Enzyme from a marine animal,Urechis unicinctus

单环刺螠生活在近海泥沙地区的潮间带和低潮带,自1940年分类后曾归属于螠虫动物门,但是现在越来越认为它是多毛纲环节动物的一个分支。系统研究表明,单环刺螠的体腔液和内脏中含有高活性的溶栓物质,并从这两种组织中通过离心、过滤、硫酸铵盐析、超滤、Q.Sepharose Fast Flow、Sephadex G-75、Sephacry S-100等步骤分离到了一种纤溶酶UFE,SDS-PAGE聚丙烯酰胺凝胶电泳测得分子量为10.38kDa.利用尿激酶和PBS作对照的功能研究表明,UFE主要是直接降解纤维蛋白,但也表现部分激酶活性,这跟来自环节动物蚯蚓的蚓激酶一样,靠将纤维蛋白溶酶原转化为纤维蛋白溶酶降解纤维蛋白。研究表明,UFE是一种免疫原性小、纤溶能力强的具有潜在应用价值的溶栓剂。

Urechis unicirtctus living in the inter-tidal and hypotidal zone of the lower coastal mud-sand areas is a member of the group of worms classified as the Phylum Echiura since 1940, but increasingly thought to represent a clade within the polychaete annelids. A systematically analysis revealed that the highest thrombolytic activity was found in the haemocoelomic fluid and gut. Therefore the isolation of fibrinolytic enzyme （UFE）was undergone from the two tissues by centrifugation, filtration, ammonium sulfate precipitation, ultrafihration, and chromatographic steps, including Q. Sepharose Fast Flow, Sephadex G-75, and Sephacry S-100. UFE was finally purified approximately 11 fold with a yield of 29.82%. UFE appeared to be homogenous by sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE） with a low molecular weight of about 10.38 kDa. Functional studies showed that using urokinase （UK） as a positive control and phosphate buffer saline （PBS） as a negative control, UFE mainly hydrolyzed fibrin directly, but it also exihibited some UK activity. UFE appeared to degrade fibrin by transformation of plasminogen into plasmin, which was similar to lumbrokinase from different earthworms, whereas UK did not directly hydrolyze fibrin without the presence of active plasminogen. These suggest that UFE is a potent thrombolytic agent with less immunogenicity and strong fibrinolysis for treatment of thrombosis.