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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 order to obtain the prothrombin activator of the snake venom (Bothrops atrox) venom and study its basic properties, SP Sepharose Fast Flow, DEAE-Sepharose Fast Flow and SP Sepharose High Performance chromatography were used to separate and purify the venom of Agkistrodon blugu A single-component prothrombin activator (FIIA). Reducing SDS-PAGE results showed that its molecular mass was approximately 72 kD and its isoelectric point was 6.67. HPSEC showed a purity of >95%. The enzyme was A N-linked glycoprotein, N-terminal amino acid sequence ALVLIAFAQYLQQCP, accession number: B3A0N1. Its activity can be inhibited by EDTA-Na2, PMSF has no effect on its activity, and does not require Ca2+, FVa, and prothrombin activation process. The participation of phospholipids is P-I metalloproteinase. The activation of prothrombin is similar to FXa. The new thrombin activator purified and identified in this study provides a reference for its pharmaceutical research and clinical application.