【作者】 孟乐；

【导师】 杜林方；

【作者基本信息】 四川大学 ， 生物化学与分子生物学， 2005， 硕士

【摘要】 应用光谱学和生物信息学方法深入探讨了重金属离子Ag⁺和Hg²⁺与菠菜光系统Ⅱ外周33kD锰蛋白的相互作用。本文首先利用荧光光谱、紫外差吸收光谱以及圆二色谱分析了溶液中33kD蛋白与Ag⁺和Hg²⁺相互作用的动力学和热力学性质以及由金属离子结合所引起的蛋白构象变化。在不同pH条件下（2.0—9.0）,金属离子与33kD蛋白的相互作用有显著差异,表现为光谱学性质的不同。在所有pH条件下,Hg²⁺都能对蛋白内源荧光产生明显的淬灭效应,而Ag⁺在pH2.0条件下反而使蛋白荧光强度上升,说明Ag⁺和Hg²⁺对33kD蛋白内源荧光淬灭的方式或机制并不相同。金属离子对荧光光谱最大发射峰的位置影响不大,仅有±1nm的变化,表明金属离子的结合对33kD蛋白芳香族氨基酸残基的微环境仅有轻微影响。紫外差吸收光谱检测到明显的配体向金属的电荷转移（LMCT,ligand-to-metal charge transition）谱带。不同pH下Hg²⁺的LMCT带形状非常相似,但强度随着pH下降而显著上升;而Ag⁺体系则呈现出完全相反的现象,LMCT带的强度随着pH的下降而下降,当pH2.0时甚至出现了负信号,这种现象还鲜有报道。LMCT谱带的结果表明33kD蛋白中存在着多个Ag⁺或Hg²⁺的结合位点。对pH7.0条件下金属离子引起的33kD色氨酸荧光淬灭分析表明,由Ag⁺和Hg²⁺引起的色氨酸荧光淬灭的机制主要是静态淬灭或非辐射能量转移。进一步分析表明引起色氨酸荧光淬灭的Hg²⁺的结合位点只有一个,且Hg²⁺极有可能是和两个半胱氨酸的硫原子配位,Hg²⁺中心位于两个硫原子中间,配位趋向于直线;而Ag⁺的结合位点也只有一个,除了与两个半胱氨更多还原

【Abstract】 The interaction of heavy metal cations Ag+ and Hg2+ with 33 kD manganese stabilizing protein in spinach photosystem II was intensively studied by means of spectroscopy and bioinformatics. Firstly, we analyzed the kinetics, thermodynamics and the conformational changes of the protein induced by binding of metal ions through fluorescence spectra, Ultraviolet differential spectra and circular dichroism spectra. Under different pH values (2.0-9.0), the interaction of metal ions with 33 kD protein was very different as reflected by distinct spectral characteristics. The intrinsic fluorescence of the protein was quenched by Hg2+ in all pH conditions while Ag+ increased the fluorescence intensity at pH 2.0, suggesting different mechanisms for quenching induced by the two metal ions. And the maximum fluorescence emission was slightly influenced by binding of metal ions, which is indicative of little if any effect on the microenvironment of aromatic amino acid residues of metal ions. Distinguished ligand-to-metal charge transition (LMCT) bands were observed through UV differential spectra. The LMCT bands in Hg2+ systems exhibited the same shape and changing pattern under different pH conditions, and the absorption values increased as pH decreased. However, the LMCT bands in Ag+ system displayed distinct changing pattern as the function of pH; and when pH is 2.0, negative signals emerged, which is rarely reported before. The results of LMCTbands indicated that there exist many binding sites in 33 kD protein for Ag+ and Hg2+. Stern-Volmer analysis of the quenching data at pH 7.0 revealed that the quenching induced by Ag+ or Hg2+ was mainly due to static quenching or non-radioactive energy transfer. Further study showed that the number of Ag+ or Hg2+ binding sites for the tryptophan fluorescence quenching was only one, respectively. And the Hg center was very likely to coordinate with the thiol groups of the two cysteine residues; while in addition to cysteine residues, Ag+ also coordinated with tryptophan residue. Thermodynamic analysis showed that the Gibss’ free energy (AG) was negative, suggesting the spontaneity of the reaction. Fluorescence emission spectra of 8-Anilino-1-naphthalene-sulfonic acid added to 33 kD protein showed that the protein had an acid expansion under low pH values. CD spectra indicated the protein was dominated by β-sheet (about 41%) and only 4% α-helix. And incubation with Ag+ or Hg2+ resulted in slight conformational changes in secondary structure, exhibiting a little increase in unordered coil. Bioinformatic method was also introcuced to reveal the metal binding sites. According to the coordinate property of both metal ions, it can be speculated that the most possible binding sites for Ag+ and Hg2+ were the thiol groups in cysteine residues, the deprotonated carboxylic oxygens in glutamic and aspartic residues, the amide nitrogens and carboxylic oxygens in glutamine and asparagine residues, and a few deprotonated peptide oxygens. As for Ag+, in addition, side chains of aromatic amino acids, especially the tryptophan residue, were also potential binding sites.更多还原