荧光光谱法探究pH值对叶黄素与牛血清白蛋白相互作用的影响

Effect of pH on the Interaction of Lutein with Bovine Serum Albumin Studied by Fluorescence Spectroscopy

采用荧光光谱法研究不同pH值(3.0、5.2、7.4)条件下叶黄素与牛血清白蛋白(bovine serum albumin,BSA)的相互作用,利用数学模型计算得到二者的猝灭常数和热力学参数等,分析叶黄素与BSA的荧光猝灭现象及蛋白构象的变化,最后通过位点竞争实验和分子模拟技术确定了二者的结合位点。荧光光谱表明,不同pH值条件下叶黄素对BSA均产生荧光猝灭效应,在298 K时,pH 7.4的猝灭常数最大,结合常数较大;热力学参数和分子对接结果表明,叶黄素与BSA间的作用力主要是疏水相互作用;同步荧光光谱表明BSA的构象在叶黄素与pH值的共同作用下发生改变;位点竞争性实验和分子对接实验表明,pH 7.4和pH 5.2时,二者的结合位点在亚结构域ⅡA和ⅢA之间,但更接近于Sudlow’s site Ⅱ;pH 3.0时,结合位点不在亚结构域ⅡA及ⅢA附近。结果表明,pH值对叶黄素和BSA的相互作用有影响,这为蛋白质与生物活性成分的相互作用提供一定的理论依据。

The interaction between lutein and bovine serum albumin(BSA)was investigated using fluorescence emission spectroscopy under different pH(3.0,5.0 and 7.4)conditions.The quenching constant and thermodynamic parameters were calculated using a mathematical model.The fluorescence quenching and conformation changes of BSA were analyzed,and the binding sites were determined by site marker competitive experiments and molecular docking.Fluorescence spectra showed that lutein had a fluorescence quenching effect on BSA under different pH conditions.The quenching constant was the highest and the binding constant was larger at pH 7.4 and 298 K.The thermodynamic parameters and molecular docking showed that the binding of lutein with BSA was mainly driven by hydrophobic interaction.Synchronous fluorescence spectra indicated that lutein and the change of pH jointly induced the conformational changes of BSA.The results of site marker competitive experiments and molecular docking showed that the binding site of lutein and BSA was located between subdomainsⅡA andⅢA,but closer to Sudlow’s siteⅡat pH 7.4 and 5.2.At pH 3.0,the binding site was neither near subdomainⅡA nor near subdomainⅢA.Altogether,the results revealed that pH influenced the binding of lutein and BSA,which may provide a theoretical basis for understanding the interaction between proteins and bioactive components.