尖吻蝮蛇毒中一种具有抗补体活性金属蛋白酶的分离纯化及部分性质鉴定

Purification and Partial Characterization of a Metalloproteinase with Anticomplementary Activity from Agkistrodon acutus Venom

通过离子交换和凝胶过滤层析从湖南产尖吻蝮蛇毒中分离纯化出1个抗补体活性蛋白AACI-I.还原与非还原SDS-PAGE测得其表观分子质量分别为26 kD和22 kD,等电聚焦电泳显示其等电点为pH 8.9.它能抑制补体经典途径和替代途径的溶血,且该作用具有量效和时效性.AACI-I可依次水解纤维蛋白原的Aα、Bβ、γ链,此活性能被EDTA、EGTA和1,10-phenanthroline完全抑制,不受PMSF、SBTI作用影响.同时,该蛋白无精氨酸酯酶活性,提示AACI-I是金属蛋白酶.另外,AACI-I具有azocasein水解活性和水肿活性,当0.01μg/g AACI-I注射小鼠足趾,其诱导肿胀率约为(14.06±6.78)%,而小鼠皮下注射2.6μg/g AACI-Ⅰ后没有发现其有出血毒活性.

An anticomplementary protein AACI-I was purified from Agkistrodon acutus venom by a combination of ion-exchange and gel filtration chromatography.Its apparent molecular weight was estimated as 26 kD and 22 kD by SDS-PAGE under reducing and non-reducing conditions,respectively.Its isoelectric point was pH 8.9 by IEF.It inhibits serum complement haemolytic activity both via classical and alternative pathway in a dose,time-dependent fashion.AACI-I could cleave Aα,Bβ,γ chain of fibrinogen successively.This activity was inhibited by EDTA,EGTA,1,10-phenanthroline,but not PMSF or soybean trypsin inhibitor,while it has no arginine esterase activity,indicating that AACI-I was a metalloproteinase.Furthermore,AACI-I exhibited proteolytic activity toward azocasein and it has edema activity,as AACI-I was injected in the foot pad of mice with dose of 0.01 μg /g body weight.The ratio of swelling was calculated as（14.06 ± 6.78）%,and there was no hemorrhagic point after injected subcutaneously into mice with dose of 2.6 μg /g body weight.