长白山白眉蝮蛇毒类凝血酶的分离纯化

目的 建立一种分离纯化长白山白眉蝮蛇蛇毒类凝血酶的方法.方法 将长白山白眉蝮蛇蛇毒溶解后离心,取上清进行阴离子交换层析,所得蛋白进行分子筛层析.提纯的蛋白用SDS-聚丙烯酰胺凝胶电泳进行纯度鉴定及相对分子量测定.测定酶的N-末端氨基酸序列.结果 经SDS-聚丙烯酰胺凝胶电泳呈单一区带,相对分子量为35.5kDa.N-末端氨基酸序列分析表明其N-末端氨基酸序列与立止血中巴曲酶N-末端氨基酸序列基本一致.提取率约为5mg类凝血酶/g蛇毒.结论 用上述方法可制得高纯度的类凝血酶.

Purification of Thrombin-like Enzyme from the Snake Venom of Agkistrodon Blomhoffii Ussurensis of Changbai Mountain

Objective To purify and characterize the thrombin-like enzyme from the snake venom of Agkistrodon blomhoffii ussurensis of Changbai mountain. Methods Thrombin-like enzyme of Agkistrodon blomhoffii ussurensis of Changbai mountain venom has been purified to homogeneity via two steps consisting of DEAE - Sephadex A - 50 and Sephadex G- 75 column chromatography in series. The molecular weight was confirmed by SDS- polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the enzyme was confirmed. Results The purified thrombin-like enzyme was proved to be homogenous by SDS - polyacrylamide gel electrophoresis. The molecular weight was about 35.5kDa by SDS- polyacrylamide gel electrophoresis. N-terminal amino acid sequence analysis showed that thrombin-like enzyme from Agkistrodon blomhoffii ussurensis of Changbai mountain venom had the same N-terminal amino acid residues as the batroxobin. Approximately 5mg thrombin-like enzyme was obtained from each gram of the snake venom. Conclusions The results showed this kind of method can be used successfully to purify thrombin-like enzyme from Agkistrodon blomhoffii ussurensis of Changbai mountain venom.