多酚与蛋白质相互作用的荧光内滤效应校正方法的选择

用荧光猝灭法研究猝灭剂与荧光供体的相互作用时，内滤效应会使分析结果不准确，需加以校正，其中物理与化学方法存在操作复杂、耗资大等缺点，而数学方法简便快捷，较易推广使用。采用荧光光谱法分析酚类物质与蛋白质的相互作用时内滤效应明显，然而目前针对酚类物质与蛋白质作用的荧光校正，国内外鲜见报道。以柿单宁级分(PT40)、A型连接ECG二聚体和EGCG为多酚化合物的代表，以中华眼镜蛇磷脂酶A₂(PLA₂)为蛋白模型，比较了四种数学公式的校正结果，以经典Stern-Volmer回归方程的K值、线性相关性及截距为评价标准，优选一种适用于多酚物质与蛋白质作用的荧光校正方法，并以PT40-PLA₂为例，分析校正前后的差异。结果表明，用Gauthier等建立的数学公式进行校正后，K值有所降低，线性相关性变好且截距更接近于1，此外用该式对PT40-PLA₂体系进行荧光校正后PT40与PLA₂相互作用的结合常数仅为校正前的60%，且对两者相互作用力类型的判断更加准确，推断该式更适合于多酚与蛋白质作用的荧光内滤效应校正。

Assessment of Inner Filter Effect Corrections in Fluorimetry of the Interaction Between Polyphenols and Proteins

Using persimmon tannin fraction (PT40), epicatechin-3-gallate-(4β→8, 2β→O→7)-epicatechin-3-gallate (A-type ECG dimer) and epigallocatechin-3-gallate (EGCG) as representatives of polyphenols and Chinese cobra snake venom phospholipase A₂ (PLA₂) as a model protein, different mathematical equations were compared to correct the inner filter effects produced by the fluorescence quenching of those polyphenols to PLA₂ based on the gradient, linearity and intercept of Stern-Volmer regression equation. The results revealed that correction by the equation developed by Gauthier et al made a significant reduction in gradients. Besides, the linearity was clearly improved and the intercepts were closer to 1 after correction in all cases. The binding constant of PT40 and PLA₂ declined by 60% and the inferred interaction forces were more convinced after correction by the above equation. Therefore, the equation developed by Gauthier et al was the most appropriate equation for correcting the inner filter effects when studying the interaction of polyphenols and protein using fluorescence quenching method.