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

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.

Purification and Characterization of a Novel Fibrinolytic Metalloprotease from Nereis virens

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.