• 关键词：LB  TB  荧光滴定  类蛋白  猝灭

• 基金项目：贵州省自然科学基金(No.黔(2004)3049);2007年度贵州师范大学研究生创新基金(No.研(2007)20);国家高技术研究发展(863)计划(No.2006AA06Z339)

• 康福星
• 1. 贵州省山地环境信息系统与生态环境保护重点实验室, 贵阳 550001; 2. 贵州师范大学, 地理与环境科学学院, 贵阳 550001

• 龙健
• 1. 贵州省山地环境信息系统与生态环境保护重点实验室, 贵阳 550001; 2. 贵州师范大学, 地理与环境科学学院, 贵阳 550001

• 潘响亮
• 中国科学院地球化学研究所环境国家重点实验室, 贵阳 550001

• 胡继伟
• 贵州师范大学, 地理与环境科学学院, 贵阳 550001

• 王倩
• 贵州省山地环境信息系统与生态环境保护重点实验室, 贵阳 550001

• 摘要：采用分子荧光技术和荧光滴定的方法解析了重金属Cd与2种不同类型胞外聚合物类蛋白的结合机理;并通过2种胞外聚合物类蛋白与Cd结合后荧光光谱的特征,进一步分析松散附着(Loosely Bound,LB)和紧密黏附(Tightly Bound,TB)2种胞外聚合物之间的结构差异.结果表明,在滴定的Cd浓度低于1×10^-4mo·lL^-1、pH值为4的条件下,Cd对LB具有显著的猝灭效应;结合Stern-Volmer分析,这种猝灭现象不仅与生色团的质子化效应有关,而且也和类蛋白与Cd结合位点的变化有关.同时,滴定过程中LB均出现不同程度的红移现象,而TB却出现不同程度的蓝移.这说明,Cd与2种类蛋白物质的结合机理方面具有显著差异.进一步三维光谱分析结果显示,在滴定前后LB中类蛋白荧光峰的结构与形态并未产生明显分化,由此说明,LB内荧光生色团性质和结构相对单一;而滴定后TB中肩峰的凸现说明,其类蛋白性质、结构与LB相比更具有多样性的特点.pH值为10的条件下,Stern-Volmer分析结果显示,LB和TB类蛋白荧光团的猝灭与2方面因素有关,一是与生成不产生荧光的EPS-Cd络合物有关,二是与分子之间的碰撞所导致的荧光猝灭有关.

• Abstract：Molecular fluorescence technology and fluorescence titration methods were applied to elucidate the mechanism of association between the protein-like regions of two extracellular polymeric substances (EPS) and the heavy metal Cd. After combining the protein-like EPSwith Cd, fluorescence spectra were used to further analyze the difference of configuration between the loosely bound extracellular (LB) and tightly bound extracellular polymeric substance (TB). An obvious quenching effect of LBtriggered by Cd concentrations less than 1×10^-4mol·L^-1 was observed at pH4. According to the Stern-Volmer equation, the phenomena that were quenched at low pHwere not only related to the protonation effect of fluorophores, but also linked with the change of binding sites between Cd and the protein-like EPS. At the same time, a red shift for LBand a blue shift for TBfluorescence peaks were observed during titration with Cd, which was attributed to the different mechanisms of association between the two EPSsamples and Cd. From analysis of the three-dimensional fluorescence spectrum of LB, the configuration of the fluorescence peak before and after titration with Cd showed no obvious change, which was attributed to single fluorophores in the protein-like EPS. Appearance of a shoulder peak after titration of TBshowed that the character and configuration of the protein-like region in TBis more diverse than in LB. Analysis of Stern-Volmer equation at pH10 showed that quenching of fluorescence peaks of the protein-like regions in LBand TBresulted from two aspects. On the one hand, they were related to the production of a non-fluorescent EPS-Cd complex (without producing fluorescence), and on the other hand it could be partially attributed to fluorescence quenching resulting from collisions between fluorescent molecules.

• Key words：LB  TB  fluorescence titration  protein-like EPS  quenching

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