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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 new type of fibrinolytic active metal was isolated and purified from H. ammodendron by ammonium sulfate fractionation, DEAE-Sepharose FF anion exchange chromatography, CM-Sepharose FF cation exchange chromatography and Sephacryl S-100 HR gel filtration chromatography Protease, named NVMP, was detected by SDS-PAGE and MALDI-TOF MS, a single chain protein with a molecular mass of 28-32 kD. Isoelectric focusing showed an isoelectric point of 8.0. It was completely inhibited by EGTA, which showed that it was a typical metalloproteinase. The optimum temperature was 40 ℃ and the optimum pH was 6. Cu2 +, Co2 + and Zn2 + could block the enzyme activity, while Ca2 + and Mg2 + could enhance the protease activity. Peptide fingerprinting analysis revealed that NVMP is an unknown new protein, which can directly hydrolyze fibrin and indirectly hydrolyze fibrin by activating plasminogen.Therefore, NVMP can prevent and / The treatment of thrombotic diseases has some medicinal value.