长白山白眉蝮蛇蛇毒中类凝血酶的分离纯化方法研究

目的:研究长白山白眉蝮蛇蛇毒中类凝血酶的简单分离纯化方法。方法:采用DEAE-Sephadex A-25及Sephadex G-25层析的方法,比较二者对长白山白眉蝮蛇蛇毒中类凝血酶简单分离纯化的效果。结果:从长白山白眉蝮蛇蛇毒中分离出类凝血酶,SDS-PAGE电泳显示为一条带,分子量大约为35.5kDa,达到电泳纯。理化性质研究表明,此类凝血酶具有体外凝血活性,体外凝血酶比活力为12.57IU.mg-1,用N-苯甲酰-L-精氨酸乙酯盐酸盐测得该酶的精氨酸酯酶比活力为137.65IU.mg-1。用蛋白酶抑制剂和乙二胺四乙酸对该酶进行抑制实验,结果表明该酶属于丝氨酸蛋白酶,而不是金属蛋白酶。结论:本方法可用于长白山白眉蝮蛇蛇毒中类凝血酶的分离纯化。

Isolation and Purification of Thrombin-like Enzyme from the Venom of Agkistrodon halys Pallas

OBJECTIVE:To study a simple method for the isolation and purification of thrombin-like enzyme (TLE) from the venom of Agkistrodon halys Pallas. METHODS:DEAE-Sephadex A-25 vs. Sephadex G-25 chromatography in the isolation and purification of TLE from the venom of Agkistrodon halys Pallas was analyzed. RESULTS:TLE was isolated from the venom of Agkistrodon halys Pallas. SDS-PAGE electrophoresis appeared as a strap with its molecular weight at about 35.5 kDa, meeting electrophoresis purity standard. Physical-chemical character assay showed that the TLE has hemostasia activity in vitro with its specific activity at about 12.57IU·mg-1, while the arginine esterase activity was about 137.65IU·mg-1 measured by BAEE method. PMSF and EDTA were used in the inhibition experiment on this enzyme, and the enzyme was proved to be serine protease other than metalloproteinase. CONCLUSION:The methods can be used for the isolation and purification of thrombin-like enzyme (TLE) from the venom of Agkistrodon halys Pallas.