光谱法结合分子模拟技术研究维生素D_3与人血清蛋白的相互作用

Study on the interaction between vitamin D_3 and human serum albumin by a combination of spectroscopy and molecular modeling

在人体生理(pH 7.4)环境下,通过紫外-可见吸收光谱法、荧光光谱分析法、圆二色光谱分析法(CD)和分子模拟技术探究了维生素D_3(VD_3)与人血清蛋白(HSA)的相互作用。结果表明,VD_3与HSA通过形成基态复合物导致HSA内源荧光发生静态猝灭。结合常数为10~5 L·mol^(-1)数量级,结合位点数约等于1,表明VD_3与HSA有较强的结合作用且有一个结合位点。计算的热力学焓变(ΔH°)和熵变(ΔS°)分别为46.54kJ·mol^(-1)和59.26J·mol^(-1)·K^(-1),显示疏水作用是维持HSA-VD_3复合物稳定的主要驱动力。位点竞争实验显示,VD_3主要结合在HSA的Site Ⅰ位。分子模拟进一步证明了VD_3在HSA上的结合位点并展示了二者的结合模式。紫外、同步荧光和圆二色光谱结果显示,VD_3结合HSA导致HSA酪氨酸残基所在的微环境极性增强,疏水性降低,并且使HSA的α-螺旋含量降低,β-折叠含量升高,HSA的多肽链产生了局部伸展,二级结构发生了变化。

The interaction between vitamin D3（VD3）and human serum albumin（HSA）was investigated by ultraviolet-visible absorption（UV-vis）,fluorescence and circular dichroism（CD）spectroscopy coupled with molecular docking technique under simulated physiological conditions（pH 7.4）.The results indicated that the intrinsic fluorescence of HSA was quenched by the static quenching resulting from the formation of HSA-VD3 complex.The binding constant was in the order of magnitude of 105 L·mol^-1 and the number of binding sites was approximately equal to 1,indicating that a stronger affinity and a single class of binding sites existed between VD3 and HSA.The thermodynamic parameters of entropy change and enthalpy change were calculated to be 46.54 kJ·mol^-1 and 59.26 J·mol^-1·K^-1 respectively,suggesting that the main force of HSA-VD3 complex formation was hydrophobic interaction force.The site competitive experiments revealed that the VD3 was most likely bound to the hydrophobic pocket located in subdomain ⅡA（Sudlow site Ⅰ）.Furthermore,the molecular docking results corroborated the binding site and clarified the specific binding mode.The UV-vis absorption,synchronous fluorescence and CD spectra showed that the binding of VD3 to HSA resulted in an increase of the polarity and a decrease of hydrophobicity around the tyrosine residue,and caused a decrease inα-helix content and an increase inβ-sheet,leading to partial unfolding of the polypeptides of HSA and its second structural change.