多光谱法结合分子对接研究柠檬黄与牛血清白蛋白的相互作用

采用荧光光谱法、同步荧光光谱法、三维荧光光谱法、紫外光谱法以及分子对接法研究了柠檬黄与牛血清白蛋白(BSA)的相互作用。柠檬黄与BSA相互作用的荧光光谱分析表明柠檬黄能有效猝灭BSA的内源荧光，根据Stern-Volmer方程计算得到柠檬黄对BSA的荧光猝灭常数K_SV随着温度的升高而逐渐降低，表明柠檬黄对BSA的荧光猝灭过程属于静态猝灭；通过静态猝灭双对数公式计算结合常数K_A为4.335×107 L·mol-1(293 K)，结合位点数n约为1，说明柠檬黄与BSA有很强的结合能力，且形成了一个结合位点；根据Van’t Hoff方程确定柠檬黄与BSA结合过程中的热力学参数ΔH=-154.5 kJ·mol-1，ΔS=-387.8 J·mol-1·K-1，ΔG＜0，两者之间的作用力主要为氢键和范德华力，且该结合过程是自发进行的；根据Förster非辐射能量转移理论计算得到结合距离r为3.310 nm，说明柠檬黄与BSA相互作用过程中发生了非辐射能量转移；同步荧光光谱分析表明，随着柠檬黄浓度的增加，Tyr残基和Trp残基的荧光强度都逐渐降低，表明Tyr残基和Trp残基均参与了柠檬黄与BSA的作用过程；三维荧光光谱分析表明柠檬黄的加入引起peak 1和peak 2的峰强度显著降低，同时peak 2的发射波长发生了变化，表明BSA的肽链结构发生了改变；随着柠檬黄浓度的增加，BSA的紫外吸收峰峰值逐渐增大；光谱分析结果表明柠檬黄与BSA的结合使BSA的构象发生改变，从而改变Trp残基和Tyr残基周围微环境，导致其发光效率降低；分子对接表明柠檬黄结合在BSA的Ⅲb亚域，柠檬黄周围的氨基酸残基主要包括：Phe506，Thr507，Ala527，Leu528，Met547，Gly571，Pro572，Leu574，Val575，Thr578。柠檬黄与BSA间主要通过范德华力与极性不带电荷的Thr507，Thr578残基作用。苯环磺酸基O与Thr507残基侧链-OH的H形成氢键。柠檬黄周围也存在非极性氨基酸残基，因此，疏水作用也可能是柠檬黄与BSA间非共价作用方式之一。该研究有助于了解柠檬黄与BSA的作用机制，为揭示柠檬黄在生物体内的分布、代谢及毒理作用机制等提供参考。

Interaction Between Tartrazine and Bovine Serum Albumin Using Multispectral Method and Molecular Docking

The interaction between tartrazine and bovine serum albumin (BSA) was investigated by fluorescence spectrometry, synchronous fluorescence spectrometry, three-dimensional fluorescence spectrometry, ultraviolet spectrometry and molecular docking. The analysis of fluorescence spectrum of tartrazine-BSA showed that tartrazine could effectively quench the endogenous fluorescence of BSA, and the fluorescence quenching constant K_SV decreased with the increase of temperature, so the quenching mechanism was static quenching on the basis of stern-volmer equation; According to the static quenching double logarithm formula, the binding constant (K_A) was calculated to be 4.335×107 L·mol-1 (293 K) and the number of binding point (n) was approximately equal to 1, which indicated that tartrazine had a strong binding ability with BSA and formed a binding site; The thermodynamic parameters obtained by van’t Hoff’s law (ΔH=-154.5 kJ·mol-1, ΔS=-387.8 J·mol-1·K-1, ΔG＜0) revealed that the main forces between tartrazine and BSA were hydrogen bond and van der Waals force, and the binding process was spontaneous; The binding distance (r) between tartrazine and BSA was calculated to be 3.310 nm based on the theory of Förster’s non-radiation energy transfer, which indicated that energy was likely to be transfered from BSA to tartrazine; With the increase of the concentration of tartrazine, the synchronous fluorescence intensity of Tyr and Trp residues decreased; The three-dimensional fluorescence spectra analysis showed that the intensities of peak 1 and peak 2 decreased significantly with the addition of tartrazine, and the emission wavelength of peak 2 changed, indicating that the peptide chain structure of BSA changed, and at the same time, the UV absorption peak of BSA increased gradually; The results of spectral analysis showed that the combination of tartrazine and BSA changed the conformation of BSA, thus changed the microenvironment around Trp and Tyr residues, resulting in the decrease of luminous efficiency; The results of molecular docking further illustrated that tartrazine was interacted with amino acid residues on subdomain Ⅲb of BSA and the amino acid residues around tartrazine mainly included: Phe506, Thr507, Ala527, Leu528, Met547, Gly571, Pro572, Leu574, Val575, Thr578; Tartrazine could interact with Thr507 and Thr578 residues by van der Waals force, with Thr507 by hydrogen bond and with other nonpolar amino acid residues by hydrophobic force. This research was helpful to understand the mechanism of interaction between tartrazine and BSA and reveal the distribution, metabolism and toxicological mechanism of tartrazine in vivo.