短尾蝮蛇毒中磷脂结合抗凝蛋白的分离纯化及鉴定  

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

作　　者：黎渊弘[1] 黎肇炎[2] 林发全[3] 黄炳轮[2] 

机构地区：[1]广西医科大学第一附属医院药剂科,广西南宁530021 [2]广西医科大学蛇毒研究所,广西南宁530021 [3]广西医科大学第一附属医院检验科,广西南宁530021

出　　处：《中国药理学通报》2006年第7期831-835,共5页Chinese Pharmacological Bulletin

基　　金：广西科技厅科学基金资助项目(No9731027)54000

摘　　要：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)。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 thin-layer chromatography. Results A kind of protein was purified from Agkistrodon halys Brevicaudus Venom which was able to prolong APTT. The SDS- PAGE showed that it was dimer and its molecular weight was 24. 0 ×10^3 under non-redueing eondition and 14. 6 ×10^3 under reducing condition. The isoeleetrie point was pH 5.2 by the isoeleetrie focusing electrophoresis. Having arginine ester-hydrolyzing enzyme and binding Phospholipid aetivify, its effect on APTT was activity stronger with eoneentration inereasing. Conclusion It is a successful method of the purification of Phospholipid-binding antieoagulation protein from the Agkistrodon halys Brevieaudus Venom.

关 键 词：纯化 抗凝血 磷脂 蛇毒 

分 类 号：R282.74[医药卫生—中药学] R341.3[医药卫生—中医学]