分子对接结合荧光光谱法探究单宁酸与血清蛋白结合的pH依赖性

pH Dependence of Binding of Tannic Acid to Serum Protein by Molecular Docking and Fluorescence Spectroscopy

探明单宁酸(TA)与牛血清蛋白(BSA)/人血清蛋白(HSA)之间的相互作用机制,对于开发蛋白基活性成分载体和递送机制研究具有重要意义。血清白蛋白(BSA/HSA)是常用可溶性载体蛋白,含有多个疏水腔作为活性成分结合位点。本文采用荧光发射光谱研究不同pH值(7.4,5.0,4.0)条件下TA对BSA/HSA荧光猝灭作用,并通过数学方程计算猝灭常数和热力学参数,分析荧光猝灭机理,最后通过位点Marker试验和分子对接技术确定结合位点。结果表明:在不同pH值条件下TA对BSA/HSA荧光均产生猝灭作用,当生理pH 7.4时,猝灭率最高。非线性拟合得到TA与血清蛋白的结合常数在105~107L/mol数量级,说明结合能力很强,pH值显著影响结合常数(P<0.05),当生理pH 7.4时,结合常数最大。热力学参数与分子对接表明BSA/HSA与TA主要通过氢键和疏水相互作用结合。位点Marker试验和分子对接表明,在pH 7.4时,BSA/HSA与TA结合位点均位于结构域ⅡA和ⅢA之间的疏水腔内,Sudlow’s site Ⅰ位点附近。试验证实TA与BSA/HSA的结合存在pH依赖性,对设计开发蛋白-TA载体提供了理论参考,对TA应用有指导意义。

It is important to elucidate the interaction mechanisms between tannic acid(TA) and bovine serum albumin(BSA)/human serum albumin(HSA) for developing protein-based carriers of active components and exploring delivery mechanisms. Serum albumin(BSA/HSA) is a commonly used soluble protein-based carrier containing multiple hydrophobic cavities, which was binding sites of active components. The fluorescence quenching effect of BSA/HSA by TA under different conditions was studied by fluorescence emission spectroscopy, the quenching constants and thermodynamic parameters were calculated by mathematical equation to analyze the fluorescence quenching mechanism. Finally, the binding sites were determined by site Marker experiments and molecular docking technique. The results showed that fluorescence quenching of BSA/HSA by TA occur under different pH conditions, the quenching rate is highest at pH 7.4. The binding constants of TA and serum proteins obtained by nonlinear fitting are magnitude of 105-107 L/mol, which indicated that the binding ability is very strong and pH significantly affect the binding constants(P <0.05), the binding constant is highest at pH 7.4. Thermodynamic parameters and molecular docking indicated that the binding of TA to BSA/HSA mainly through hydrogen bonding and hydrophobic interactions, site Marker experiments and molecular docking indicated that in pH 7.4, the binding sites are located in the hydrophobic cavity between domain ⅡA and domain ⅢA, near the Sudlow’s site Ⅰ. The paper confirmed that the binding of TA to BSA/HSA is pH dependent, which provides a theoretical reference for the design and development of protein-TA carriers and provides meaningful guidance for TA applications.