一种新的蛇毒凝血酶原激活物的分离纯化及特征

Purification and Characterization of FⅡA,a Novel Prothrombin-activator from Bothrops atrox Venom

为获得矛头蝮蛇(Bothrops atrox)蛇毒凝血酶原激活物并研究其基本性质,采用SP SepharoseFast Flow,DEAE-Sepharose Fast Flow和SP Sepharose High Performance等层析方法从巴西矛头蝮蛇蛇毒中分离纯化得到1种单一组分的凝血酶原激活物(prothrombin activator,FⅡA).还原性SDS-PAGE结果显示,其分子质量约为72 kD,等电点为6.67.HPSEC显示纯度大于95%.该酶是1种N连接的糖蛋白,N末端氨基酸序列为ALVLIAFAQYLQQCP,获得登录号为:B3A0N1.其活性可被EDTA-Na2抑制,PMSF对其活性无影响,对凝血酶原的激活过程无需Ca2+、FⅤa、磷脂的参与,为P-Ⅰ金属蛋白酶,对凝血酶原的激活方式与FⅩa相似.本研究纯化与鉴定的新凝血酶原激活物为其药学研究及临床应用提供参考.

In this study,a novel prothrombin-activator from the venom of Bothrops atrox designated FⅡA was isolated.FⅡA was purified using a SP Sepharose Fast Flow anion-exchange chromatography,DEAE-Sepharose Fast Flow cation-exchange chromatography followed by SP Sepharose High Performance anion-exchange chromatography.FⅡA was a single-chain polypeptide with a molecular mass 72 kD as measured by reduced SDS-PAGE.Its pI was 6.67 as measured by IEF.Its N-terminal amino acid sequence was ALVLIAFAQVLQQCP and the accession number was B3A0N1 in UniProtKB Database.The enzymatic activity of FⅡA was inhibited by the chelating EDTA,whereas the serine protease inhibitor PMSF had no effect on its activity.Interestingly,its enzymatic activity is not requiring cofactors such as Ca2＋,FⅤa or phospholipid.The results suggest that FⅡA is a P-Ⅰmetalloproteinase and its pathways for prothrombin activation is similar to FⅩa.