广东眼镜蛇毒低分子量多肽的分离纯化与性质鉴定

Purification and Characterization of Low Molecular Weight Polypeptides from the Venom of Guangdong Cobra (Naja Naja Atra)

关键词:  眼镜蛇毒  低分子量多肽  纯化  神经毒素  心脏毒素  鉴定

Key words:  cobra venom  low molecular weight polypeptide  purification  neurotoxin  cardiotoxin  characterization

摘要:

目的 从广东眼镜蛇粗毒中分离纯化出色谱纯的低分子量多肽并对其部分性质进行鉴定。方法 采用化学沉淀法对蛇粗毒进行预处理，经Sephadex G-50凝胶过滤和UND Sphere S阳离子交换层析得到高纯度低分子量多肽，用SDS-PAGE及HPLC对产物纯度进行鉴定，并用SDS-PAGE和质谱法测定分子量，采用蛙心灌流法考察产物是否具有心脏毒性。结果 结果表明，经分离纯化后得到两种低分子量多肽GI2和GI3，均为单一组分，可达到色谱纯，SDS-PAGE法测得GI2分子量为14 300 Da，质谱法测得GI3分子量为6 725.8 Da，仅GI2具有心脏毒性，收率分别为9.5%和10.1%。结论 通过化学沉淀预处理，并经2次柱色谱可从广东眼镜蛇毒中分离得到两种色谱纯的低分子量多肽，且收率较高。

Abstract:

OBJECTIVE To isolate and characterize some properties of the chromatographically pure low molecular weight polypeptides from the venom of Guangdong cobra (Naja naja atra). METHODS Isolation and purification were carried out by chromatography on Sephadex G-50 and UND Sphere S column after the pretreatment by chemical precipitation. The purity of this product was determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and HPLC, and its molecular weight was observed by SDS-PAGE and mass spectrography. The cardiotoxicity of the polypeptides was investigated using the frog heart perfusion experiment. RESULTS After the chemical precipitation and Two-step column chromatography, the products, named ‘GI2’ and ‘GI3’, were single components. The molecular weight of GI2 is 14 300 Da observed by SDS-PAGE and GI3 is 6 725.8 Da determined by mass spectrography. The yields of the products were 9.5% and 10.1%, and only GI2 showed cardiotoxicity. CONCLUSION Two low molecular weight components could be obtained from the venom of Guangdong cobra after chemical precipitation and two-step column chromatographies, with high yield.