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

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。

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

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-[phospho-rac-(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.