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目的:建立一种分离纯化长白山白眉蝮蛇血凝酶的方法。方法:先后采用分子筛层析法、离子交换色谱法、肝素-Sepharose亲和层析法从长白山白眉蝮蛇蛇毒中分离纯化得到一种具有有凝血活性的酶成分,并采用SDS-Page法、RP-HPLC法测定其纯度,凝胶电泳法(SDS-Page)考察其对牛纤维蛋白原的作用方式、高效空间排阻色谱法(HPSEC)测定其相对分子质量,等电聚焦电泳分析法(IEF)测定其等电点,Lowry法测定其蛋白浓度。结果:从长白山白眉蝮蛇蛇毒中分离纯化了一种凝血酶成分,SDS-Page显示为一条带,HPLC得到单一的色谱峰,该酶只作用于纤维蛋白原的α链,其相对分子质量为32.2 kD,等电点为5.21,此酶具有体外凝血活性。结论:该方法可用于长白山白眉蝮蛇血凝酶的分离纯化。
OBJECTIVE: To establish a method for the isolation and purification of hemagglutinin from Changbai Mountain Bai Mei vole. Methods: Enzyme components with thrombin activity were isolated and purified by molecular sieve chromatography, ion exchange chromatography and heparin-Sepharose affinity chromatography from Changbai Mountain, and SDS-PAGE, RP -HPLC method was used to determine its purity. SDS-PAGE was used to determine the mode of action on bovine fibrinogen. The relative molecular mass was determined by high performance liquid chromatography (HPSEC). The isoelectric focusing (IEF ) Isoelectric point determination, Lowry method for the determination of its protein concentration. Results: A thrombin component was isolated and purified from Chang snakehead snakehead snake venom. The SDS-PAGE showed a single band with a single chromatographic peak. The enzyme only acted on the α-chain of fibrinogen and its relative molecular mass was 32.2 kD, isoelectric point of 5.21, this enzyme has in vitro coagulation activity. Conclusion: This method can be used for the isolation and purification of capoeulent serpentinase from Changbai Mountain.