眼镜蛇毒中降压因子的提取及生物活性的研究

Purification and Biological Activity of Hypotensive Factor from Naja naja atra Venom

目的:从广西眼镜蛇蛇毒中分离纯化血管紧张素转换酶抑制剂(angiotensin converting enzyme inhibitor,ACEI),命名为降压因子(hypotensive factor,HF),并测定其生物活性。方法:采用Sephacryl S-100凝胶过滤,CM Sepharose F.F.离子交换层析分离纯化HF,高效液相鉴定纯度,SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)测定其分子量,紫外分光光度法测定HF对血管紧张素转换酶(ACE)的抑制活性。用离体兔子十二指肠平滑肌测定HF增强缓激肽(bradykinin,BK)的效应。结果:纯化的广西眼镜蛇蛇毒HF经SDS-PAGE检测显示单一条带,测得其相对分子量约为8.2kDa,由12种氨基酸组成,蛋白回收率为5.70%。HF对ACE有明显的抑制作用,其抑制作用与剂量呈正相关。IC50为1.02μg/ml。HF能增强BK对离体兔子十二指肠平滑肌的收缩效应。结论:成功地从广西眼镜蛇蛇毒中纯化出降血压成分。该成分与血管紧张素转换酶抑制剂作用相似,对血管紧张素转换酶有明显的抑制作用。

Objective： To separate and purify the Angiotensin Converting Enzyme Inhibitor （ACEI） named hypotensive factor（HF） from Naja naja atra venom and determine its biological activity. Methods： HF of venom extract was isolated and purified by Sephacryl S-100 gel filtration, and CM Sepharose F.F. ionexc snake hange chromatography respectively. The purified product was identified by HPLC. The molecular weight was determined by SDS-polyacrylamid gel electrophoresis. The ACE inhibitor activity was determined by UV spectrophotometer. The effect of HF on contraction by BK was tested by using isolated intestinal smooth muscle from rabbit. Results： SDS-PAGE showed a single band of HF and a molecular weight of 8.2kDa. HF was a single polypeptide chain and consisted of 12 amino acids. Recovery rate of protein was 5.70%. ACE was inhibited by HF obviously. The inhibitory rate has a positive correlation with the concentration of HF. The IC50 was 1. 02μg/ml. HF could strengthen the contraction of isolated intestinal smooth muscle from rabbit by BK. Conclusion： The method has been proved to be successful for the purification of HF from Naja naja atra venom. HF has a similar effect as Angiotensin Converting Enzyme Inhibitor, and shows a significant inhibitory effect on ACE.