短尾蝮蛇毒中磷脂结合抗凝蛋白的分离纯化及鉴定

目的从短尾蝮蛇毒中分离纯化一种抗凝蛋白,并对其生化性质进行研究。方法利用阳、阴离子交换、凝胶过滤的方法分离纯化这种抗凝蛋白,用活化部分凝血活酶时间(APTT)测定其抗凝活性,用SDS-PAGE测定其蛋白相对分子量,用等电聚焦法测定蛋白的等电点.用薄层析方法确定抗凝蛋白与磷脂酰胆碱结合。结果从短尾蝮蛇中分离纯化出的抗凝蛋白是二聚体,相对分子量为24.0×103(非还原)和14.6×103(还原)。等电点为pH 5.2。该蛋白具有精氨酸酯酶活性,能明显地延长活化的部分凝血活酶时间(APTT),其抗凝活性与磷脂结合有关。结论此方法成功地从短尾蝮蛇毒中纯化出一种抗凝蛋白。因其能够与磷脂结合,又具有明显的抗凝活性,因此把该蛋白称为磷脂结合抗凝蛋白(phospholip id-b ind ing anticoagu lation prote in,PBAP)。

The purification and identification of phospholipid-binding anticoagulation protein from agkistrodon halys brevicaudus venom

Aim To purify Phospholipid-binding anticoagulation protein(PBAP) from Agkistrodon halys Brevicaudus Venom and study the biochemical characterization.Methods The Phospholipid-binding anticoagulation protein was purified from Agkistrodon halys Brevicaudus Venom by Cation ion exchange chromatography on CM Sephadex C-25 and negative ion exchange chromatography on DEAE Sepharose CL-6B,gel filtration on Sephacryl S-200 and Sephadex G-75 chromatography.Its anticoagulant activities in vitro were assayed by activated partial thromboplastin time(APTT);its molecular weight was calculated by SDS-polyacrylamide gel electrophoresis(SDS-PAGE) and its isoelectric point was estimated by the isoelectric focusing electrophoresis.Binding experiments of anticoagulation protein to phospholipids vesicles were performed with thinlayer chromatography.Results A kind of protein was purified from Agkistrodon halys Brevicaudus Venom which was able to prolong APTT.The SDSPAGE showed that it was dimer and its molecular weight was 24.0×10~3 under non-reducing condition and 14.6×10~3 under reducing condition.The isoelectric point was pH 5.2 by the isoelectric focusing electrophoresis.Having arginine ester-hydrolyzing enzyme and binding Phospholipid activify,its effect on APTT was activity stronger with concentration increasing.Conclusion It is a successful method of the purification of Phospholipid-binding anticoagulation protein from the Agkistrodon halys Brevicaudus Venom.