色谱

色谱

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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

曾雄智1,皮建辉1,2,梁宋平1

  

  1. 1. The Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, Hunan Normal University, Changsha 410081, China; 2.Department of Bioengineering, Huaihua College, Huaihua 418008, China
  • 收稿日期:2007-04-17 修回日期:2007-07-02 出版日期:2007-11-30 发布日期:1984-12-25
  • 通讯作者: 梁宋平

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

ZENG Xiongzhi1, PI Jianhui1,2, LIANG Songping1   

  1. 1. The Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, Hunan Normal University, Changsha 410081, China; 2.Department of Bioengineering, Huaihua College, Huaihua 418008, China
  • Received:2007-04-17 Revised:2007-07-02 Online:2007-11-30 Published:1984-12-25
  • Contact: LIANG Songping

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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4.7 nm的蓝移以及7.4和8.0 nm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。

关键词: 单层小脂质体, 高效液相色谱, 敬钊毒素-I , 荧光谱

Abstract:

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.

Key words: fluorescence spectroscopy, Jingzhaotoxin-I (JZTX-I) , small unilamellar vesicles, high performance liquid chromatography (HPLC)