一种新型具有纤溶活性的沙蚕金属蛋白酶的分离纯化及鉴定

Purification and Characterization of a Novel Fibrinolytic Metalloprotease from Nereis virens

通过硫酸铵分级盐析、DEAE-Sepharose FF阴离子交换色谱、CM-Sepharose FF阳离子交换色谱和Sephacryl S-100 HR凝胶过滤色谱,从沙蚕体内分离纯化出一种新型的具有纤溶活性的金属蛋白酶,命名为NVMP.采用SDS-PAGE和MALDI-TOF MS质谱检测,该酶是一种分子质量为28～32 kD的单链蛋白,等电聚焦电泳显示其等电点为8.0.NVMP酶活性被EGTA完全性抑制,表明其是一种典型的金属蛋白酶,最适温度为40℃,最适pH为6,Cu2+、Co2+和Zn2+可阻断其酶活性,而Ca2+和Mg2+可增强蛋白酶活性.经肽指纹图谱分析发现,NVMP是一种未知的新蛋白.NVMP可直接水解纤维蛋白,也可通过激活纤溶酶原转变成纤溶酶的方式,间接水解纤维蛋白.因此,NVMP对预防和治疗血栓性疾病具有一定的药用价值.

A novel fibrinolytic metalloprotease from Nereis virens,named NVMP,was purified by using ammonium sulfate precipitation,anion exchange chromatography on DEAE-Sepharose FF,cation exchange chromatography on CM-Sepharose FF and gel-filtration chromatography on Sephacryl S-100 HR.NVMP consisted of a single polypeptide chain with a molecular weight of 28~32 kD,which was determined by SDS-PAGE and Matrix-assisted laser desorption/ionization time-of-flight mass spectrum（MALDI-TOF MS）.The isoelectric point of NVMP determined by isoelectric focusing electrophoresis（IEF） was 8.0.NVMP activity was strongly inhibited by EGTA,indicating that it was a metalloprotease and the maximum activity of the enzyme was observed at 40 ℃ and pH 6.0.The enzymatic activity of this metalloprotease was inhibited by Cu2＋,Co2＋ and Zn2＋,but enhanced by Ca2＋ and Mg2＋.The peptide mass fingerprinting（PMF） revealed that NVMP was a newly discovered protein.More importantly,NVMP degraded fibrin either directly or indirectly through generating plasmin from activation of plasminogen.NVMP may thus have a medical value for preventing and treating thrombosis.