高效液相色谱-荧光检测法测定敬钊毒素-I的磷脂膜结合活性  

Determination of Jingzhaotoxin-I Phospholipid Membrane-Binding Activities by High Performance Liquid Chromatography with Fluorescence Detection

作　　者：曾雄智[1] 皮建辉[1] 梁宋平[1] 

机构地区：[1]湖南师范大学蛋白质化学与发育生物学教育部重点实验室,湖南长沙410081

出　　处：《色谱》2007年第6期825-829,共5页Chinese Journal of Chromatography

基　　金：国家自然科学基金重大项目资助(No.39990600)

摘　　要：敬钊毒素-I(JZTX-I)是一种能够抑制心肌钠通道失活的新型蜘蛛神经毒素,该文结合高效液相色谱与色氨酸荧光测定技术研究了JZTX-I的磷脂膜结合活性。脂质体共沉淀实验表明,JZTX-I具有不依赖于带负电荷磷脂组成的生物膜结合活性。当加入由酸性或中性磷脂构成的脂质体后,JZTX-I能够分别产生6.4和4.7nm的蓝移以及7.4和8.0nm的红移激发漂移,显示JZTX-I能够插入磷脂膜,同时该分子疏水表面的色氨酸残基处于一个运动受限的界面区域。荧光淬灭实验进一步证实,与脂质体结合能够减少该毒素分子表面色氨酸残基的溶剂暴露。该研究结果为阐明JZTX-I的离子通道门控调节机制提供了新的信息。Jingzhaotoxin-Ⅰ （JZTX-Ⅰ）, a 33-residue polypeptide with three disulfide bonds, was a novel spider neurotoxin preferentially inhibiting cardiac sodium channel inactivation. Its activities of phospholipid membrane-binding were studied by a combination of reversed-phase high performance liquid chromatography （HPLC） and fluorescence spectroscopy. Small unilamellar vesicles binding assays showed that the partitioning of JZTX-Ⅰ into lipid bilayer did not require negatively charged phospholipids. Further, JZTX-Ⅰ also exhibited a blue shift of 6. 4 nm or 4.7 nm as well as red-edge excitation shift of 7.4 nm or 8.0 nm in the presence of 75% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine （ POPE ）/25% 1-palmitoyl-2-oleoyl-sn-glycero-3- [ phosphorac-（1-glycerol）] （ sodium salt） （POPG） or 100% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine （POPC） vesicles respectively, suggesting that some tryptophan residues on the hydrophobic surface of the toxin were located within a motion restricted membrane interfacial region. Fluorescence quenching experiments suggested that some tryptophan residues of JZTX-Ⅰ were positioned within the membrane and protected from aqueous quenching agents. These findings should provide further insight into the molecular mechanism of the channel gating of JZTX-Ⅰ.

关 键 词：高效液相色谱 荧光谱 单层小脂质体 敬钊毒素-Ⅰ 

分 类 号：O658[理学—分析化学]