蕲蛇蛇毒中一个新的类凝血酶的分离纯化与表征

Purification and Characterization of a Novel Thrombin-like Enzyme from Agkistrodon acutus Venom

使用DEAE SephadexA 5 0、POROS 5 0HS及POROS 2 0PI等色谱分离技术 ,从蕲蛇粗毒中分离提纯得到 1个具有凝血活力的组分 ,经SDS PAGE和PAGE检测均为一条带 ,非还原条件下相对分子质量 2 4 .3kDa,还原条件下相对分子质量 33.0kDa ;其凝血活力为 91 .0NIHu mg.该组分不具有激活因子ⅩⅢ的活性 ,肝素不影响该组分的凝血活性 ,EDTA部分抑制其活性 ,苯甲基磺酰氟 (PMSF)则会产生不可逆抑制作用 .该酶N 末端氨基酸序列为VIGGNGXDINEHRFLVAFF 。

A novel thrombin-like enzyme was identified and isolated from Agkistrodon acutus venom by anion-exchange chromatography (DEAE-Sephadex A-50), followed by cation-exchange (POROS 50HS) and anion-exchange chromatography (POROS 20PI). The enzyme with fibrinogen-clotting activity was purified to homogeneity determined by SDS-PAGE and PAGE, the molecular weight of which was estimated by SDS-PAGE to be 24.3?kDa under nonreduced conditions and 33.0?kDa under reduced conditions. It shows fibrinogen-clotting activity of 91.0 NIH u/mg on human citrate plasma. The clotting activity of the protease could be completely inhibited by PMSF and partially inhibited by EDTA but not influenced by heparin, a thrombin inhibitor. The enzyme has no activity to activate coagulation factor ⅩⅢ. The N-terminal amino acid sequence of the purified enzyme was identified as VIGGNGXDINEHRFLVAFF.