多光谱法和分子对接模拟法研究黄腐植酸和牛血清白蛋白的相互作用

在模拟生理环境中，使用荧光光谱法、紫外光谱法、圆二色谱法、同步荧光光谱法、三维荧光光谱法与分子对接模拟法研究黄腐植酸和牛血清白蛋白(BSA)之间相互作用。在荧光光谱法研究中，经Stern-Volmer方程计算得到298，303和308 K温度下的动态荧光猝灭速率常数K_q和猝灭常数，证明BSA与黄腐殖酸(FA)相互作用的猝灭过程为静态猝灭；同时根据计算得出的结合位点数n都在1附近，FA与BSA体系相互作用比为1∶1；利用静态猝灭双对数方程计算三个温度下的热力学参数，焓变ΔH<0，熵变ΔS＜0，得出结论，FA与BSA之间的主要作用力为氢键和范德华力；ΔG＜0，说明作用过程为自发过程。采用Förster’s偶极-偶极非辐射能量转移理论，计算出结合距离r=6.340 nm，表明BSA与FA之间存在非辐射能量转移。分子对接模拟结果表明FA与BSA残基的结合作用力具有氢键和范德华力，同时二者之间还存在疏水作用力，多种力共同作用使FA与BSA能够稳定结合。通过对FA与BSA相互作用的紫外-可见吸收光谱分析，发现BSA最大吸收峰发生了较为明显的红移，表明FA使BSA的二级结构发生改变。通过研究FA与BSA相互作用的同步荧光光谱，得到FA使BSA中的色氨酸（Trp）残基周围的微环境极性增强，疏水性减弱，亲水性增强，使BSA的蛋白质构象发生了一定程度的改变。通过研究FA与BSA相互作用的三维荧光光谱，峰1（peak 1）与峰2（peak 2）的最大发射波长峰都发生了红移，证明FA与BSA发生了相互作用，FA使BSA周围环境的极性增大，疏水性减小，亲水性增加，BSA蛋白质构象发生变化。最后采用圆二色谱法进行分析，利用软件计算得出该实验相互作用体系下α-螺旋（α-Helix）减少2.3％、β-折叠（β-sheet）增加7.7％、β-转角（β-Turn）增加0.6％和无规则结构（Random coil）含量减少1.2％，β-折叠（β-sheet）含量增加最为明显, 强有力地说明了FA使BSA结构发生了改变。

Study on the Interaction Between Fulvic Acid and Bovine Serum Albumin by Multispectral and Molecular Docking

In this paper, the interaction between fulvic acid (FA) and bovine serum albumin (BSA) was studied by fluorescence spectroscopy, ultraviolet spectroscopy, circular dichroism, synchronous fluorescence spectroscopy, three-dimensional fluorescence spectroscopy and molecular docking simulation in the simulated physiological environment. In the fluorescence spectroscopy study, the dynamic fluorescence quenching rate constant K_q and quenching constant at 298, 303 and 308 K are calculated by the Stern-Volmer equation, which proves that the quenching process of the interaction between BSA and FA is static quenching. At the same time, according to the calculated binding sites n, the interaction ratio between FA and BSA is 1∶1. The thermodynamic parameters at three temperatures are calculated by static quenching double logarithm equation, enthalpy change ΔH<0, entropy change ΔS<0, it is concluded that the main interaction force between FA and BSA is hydrogen bond and van der Waals force, ΔG<0, indicating that the interaction process is spontaneous. Based on Förster’s dipole-dipole non-radiative energy transfer theory, the binding distance rang 6.340 nm is calculated, indicating a non-radiative energy transfer between BSA and FA. The molecular docking simulation results show that the binding force between FA and BSA residues is hydrogen bond and van der Waals force, and there is a hydrophobic force between them. The interaction of multiple forces makes FA and BSA combine stably. Through the UV-Vis absorption spectrum analysis of the interaction between FA and BSA, it is found that the maximum absorption peak of BSA has an obvious red-shift, indicating that FA changes the secondary structure of BSA. By studying the synchronous fluorescence spectrum of the interaction between FA and BSA, it was found that FA enhanced the polarity of the microenvironment around the tryptophan (Trp) residue in BSA, weakened its hydrophobicity and enhanced its hydrophilicity, which changed the protein conformation of BSA to a certain extent. Through the study of the three-dimensional fluorescence spectrum of the interaction between FA and BSA, the maximum emission wavelengths of peak 1 (peak 1) and peak 2 (peak 2) were red-shifted, which proved that FA interacted with BSA. FA increased the polarity of the environment around BSA, decreased its hydrophobicity, increased its hydrophilicity, and changed the protein conformation of BSA. Finally, circular dichroism was used for analysis, and the software was used to calculate that under the experimental interaction system, α-helix (α-Helix) decreased by 2.3%, β-sheet increased by 7.7%, β-Turn increased by 0.6%, and irregular structure (Random coil) content decreased by 1.2%. The content of β-sheet increased most obviously, which strongly indicated that FA changed the structure of BSA.