Crude hemorrhagins in Agkistrodon halys ussuriensis venom were purified through DEAE-cellulose ion exchange chromatography. Acidic proteins in Agkistrodon halys ussuriensis venom were absorbed by DEAE-cellulose and basic protein flowed out. Basic protein solution was collected, concentrated and dialyzed. Acidic proteins absorbed in DEAE-cellulose were eluted by Tris-HCl buffer (pH7. 4) contained 0. 05M, 0. 1M, 0. 15M, 0. 2M and 0. 5M NaCl step by step. Each component was collected, concentrated, dialyzed and preserved respectively. Hemorrhagic activity for each component was tested and the part with hemorrhagic activity was crude hemorrhagin component. The hemorrhagin in crude hemorrhagin component was purified further through 12％ PAGE Preparation electrophoresis. The polyacrylamide gel was dyed using a kit named highly sensitive and rapid Coomassie brilliant blue staining kit (product No. C510041). Dyeing procedures were modified. The gel containing venom proteins was taken out and ground into gel suspension. The gel suspension was injected into mouse abdomen by subcutaneous injection. 1 hour later, the mouse was killed and the numbers of bleeding points was counted. Each bleeding point represents a kind of hemorrhagin. The results indicated that the part eluted by 0. 1M NaCl possessed hemorrhagic activity. In this part, there are 2 kinds of hemorrhagins.%用DEAE-纤维素离子交换层析、分段洗脱的方法将白眉蝮蛇毒成分了若干个组分,收集、浓缩、透析并确定出血毒素所在的组分.分段洗脱液含NaCl的Tris-HCl缓冲液(0.05 M、0.10 M、0.15 M、0.2 M、0.5 M, pH7.4).然后用12％ PAGE制备电泳方法进一步分离出血毒素,用生工生物工程(上海)股份有限公司生产的高灵敏快速考马斯亮蓝染色试剂盒(产品编号: C510041)染色.对染色和脱色方法进行改进,将含蛋白质的凝胶切下,磨细后注射到小鼠腹部皮下,观察小鼠腹部皮上的出血点,每一个出血点代表1种出血毒素.结果表明,浓度为0.10 M NaCL洗脱的组份含出血活性,对该部分电泳检测发现总共有2种出血毒素.
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