江浙蝮蛇毒纤溶酶的分离纯化及性质研究

PURIFICATION AND CHARACTERIZATION OF FIBRINOLYTIC ENZYME FROM AGKISTRODON HALYS PALLAS VENOM

目的:江浙蝮蛇毒纤溶酶的分离纯化及部分生物化学和药理性质的研究。方法:应用DEAE Sepharose CL-6B阴离子交换柱和HiPrep Sephacryl S 100柱分离纯化纤溶酶,以高效液相色谱仪和聚丙烯酰胺凝胶电泳检测纯度、相对分子质量,应用等电聚焦电泳测定等电点,然后测定氨基酸组分及部分药理性质。结果:分离得到相对分子质量为59 100,等电点为4.98,SDS-PAGE为一条带,高效液相色谱分析为单一峰的纤溶酶。该组分属于金属蛋白酶类。氨基酸组成分析表明它含较多的酸性氨基酸。高浓度的纤溶酶对肿瘤细胞CEN2有抑制作用,有微弱的出血毒性。结论:分离纯化得到了一种有微弱出血毒,具有较强抗凝活性的单一组分的酸性金属蛋白酶。

Objective：To investigate the purification method of a fibrinolytic enzyme（FLE） from the venom of Agkistrodon blomhoffi brevicaudus and research some biochemistral characterizations. Methods： The fibrinolytic enzyme（FLE） was purified from the venom of Agkistrodon blomhoffi brevicaudus by ion-exhange chromatography on a DEAE Sepharose CL-6B column, followed by gel filtration chromatography on HiPrep Sephacryl S 100. The molecular weight was estimated with SDS-PAGE. The pI was determined through isoelectric focusing. Amino acid composition assay was carried out on a Hitachi 835-50 mould automatic amino acid analyzer. A series of pharmacological experiments were carried out. Result： The purified FLE was shown to be homogeneous in SDS-PAGE, which acted as an anticoagulant in vitro. Its molecular weight was estimated to be 59,100 D and isoelectric point was 4.98 . According to the result of amino acid composition assay, it has a number of acidic amino acid, which is consistent with the pI we detected . Through the research of pharmacology, we found it possessed low haemorrhagic activity . FLE belongs to metallic protein and can inhabit the growth of cancer in high concentration . Conclusion： We separate a fibrinolytic enzyme（FLE） which is a acid protein and has low haemorrhagic activity from the venom of Agkistrodon blomhoffi brevicaudus.