槲皮素与β-乳球蛋白B的结合机制及其溶解性能

Binding between Quercetin and β-Lactoglobulin B: Mechanism and Improved Solubility of Quercetin

为解决槲皮素水溶性差的问题,采用β-乳球蛋白B作为载体,基于多光谱、等温滴定量热仪和分子模拟等实验手段,探究槲皮素和β-乳球蛋白B的结合机制,以及结合后β-乳球蛋白B的二级结构和槲皮素溶解度的变化。结果表明:槲皮素可与β-乳球蛋白B结合,该结合过程中氢键和范德华力起主要作用;三维荧光、同步荧光以及紫外-可见吸收光谱表明,槲皮素的结合使β-乳球蛋白B的二级结构发生了改变;圆二色谱和傅里叶变换红外光谱结果表明,槲皮素的结合诱导该蛋白中部分α-螺旋转变为β-折叠;分子模拟结果表明,槲皮素在β-乳球蛋白B上的结合位点位于一个由α-螺旋与β-转角所形成的空穴中,在该结合位点中有8个氨基酸残基参与了与槲皮素的结合,其中第125位的苏氨酸残基提供了较强的氢键,其余残基则提供了范德华力;基于高效液相色谱检测发现在与β-乳球蛋白B结合后,槲皮素的溶解度增大至原来的1 844倍左右。综上所述,以β-乳球蛋白B为载体结合的槲皮素水溶性显著提高。

In order to improve the poor water solubility of quercetin, the mechanism of quercetin binding to β-lactoglobulin B was studied by various spectroscopies, isothermal titration calorimetry and molecular simulation, and changes in the secondary structure of β-lactoglobulin B and quercetin solubility were determined after the binding reaction. The results showed that quercetin was able to bind to β-lactoglobulin B through hydrogen bonding and van der Waals forces. Threedimensional fluorescence spectra, synchronous fluorescence spectra and UV-visible absorption spectra demonstrated that quercetin quenched β-lactoglobulin B fluorescence strongly in the static mode. Furthermore, circular dichroism and Fourier transform infrared spectra indicated that quercetin induced partial transition of α-helix to β-structures in β-lactoglobulin B.Moreover, molecular simulation results showed that the binding site of quercetin on β-lactoglobulin B was located in a cavity formed by α-helix and β-turn, and 8 amino acid residues were involved in the binding site. In combination with quercetin,the threonine residue at position 125 provided strong hydrogen bonds, while the remaining residues provided van der Waals forces. Besides, the solubility of quercetin was increased by about 1 844 times after its conjugation to β-lactoglobulin B. In summary, the water solubility of quercetin can be significantly improved by conjugating it to β-lactoglobulin B.