荧光光谱法和分子对接模拟技术研究白藜芦醇与胃蛋白酶的相互作用

白藜芦醇（Resveratrol, RES）属于非黄酮类多酚化合物，存在于葡萄科、百合科等多种植物体内，是一种具有多种生物活性和药理作用的天然活性物质，被广泛应用于食品和药品领域。研究表明多酚在生物体消化吸收过程中，会与消化酶（如胃蛋白酶、胰蛋白酶等）相互作用，使多酚类物质和消化酶的生物活性发生改变，进而影响多酚物质和其他营养物质的消化吸收，而RES与胃蛋白酶(Pepsin, PEP)的分子间相互作用机制未见报道。采用荧光光谱、紫外-可见吸收光谱、红外光谱和分子对接模拟等技术研究不同温度下RES与PEP相互作用的结合特性，为阐明RES和PEP的作用机制提供重要信息，同时为RES在食品和药品领域的应用提供理论参考。荧光光谱实验结果表明，PEP的荧光强度随着RES浓度的增加呈现出有规律的降低，表明RES对PEP有荧光猝灭作用。加入RES前后，PEP的紫外吸收光谱发生明显变化，初步判断RES与PEP的相互作用属于静态荧光猝灭类型；根据Stern-Volmer方程计算得到不同温度下最小猝灭速率常数K_q值远大于猝灭剂对生物大分子的最大扩散碰撞常数2.0×1010 L·mol-1·s-1，且猝灭常数(K_SV)与温度呈负相关关系，进一步验证了RES与PEP静态荧光猝灭类型结论。化学计量结合的值数目大约等于1，表明一个RES分子只能结合一个PEP分子。根据Van’t Hoff 方程以及热力学方程计算得到结果显示，ΔG＜0，说明RES与PEP的结合过程可以自发进行；ΔH＜0和ΔS＜0，表明RES与PEP之间结合作用力类型主要是氢键和范德华力。RES与PEP相互作用的同步荧光光谱和三维荧光光谱图表明，在RES的作用下，PEP的构象和微环境发生变化，色氨酸或酪氨酸残基所处微环境极性增强，疏水性减弱，蛋白构象变得疏松。红外光谱显示RES能使PEP的二级结构中α螺旋含量降低，β折叠含量增加，β转角和无规则卷曲变化不明显，这可能会影响PEP的活性。分子对接模拟实验结果显示RES与PEP中的残基Asp-32，Gly-34，Ser-35，Asn-37，Tyr-75，Gly-76，Thr-77，Ile-128，Ala-130及Gly-217有范德华力作用，与残基Ile-128及Asp-215产生超共轭效应，与残基Ser-36，Asn-37，Ile-128及Thr-218形成氢键，各种作用力使RES与PEP形成较稳定的复合物。

Study on Interaction between Resveratrol and Pepsin by Fluorescence Spectroscopy and Molecular Modeling

Resveratrol (RES) is a non-flavonoid polyphenols found in many plants, such as Vitaceae and Liliaceae. It is a natural active substance with variety biological and pharmacological functions and widely used in food and pharmaceutical field. Studies have shown that polyphenols had an interaction with digestive enzymes (such as pepsin, trypsin, etc.) in the process of digestion and absorption of organisms, resulting in changes in the biological activity of polyphenols and digestive enzymes and affecting the digestion and absorption of polyphenols and other nutrients. However, the mechanism of interaction between RES and pepsin (PEP) has rarely been reported. The attempt of this paper was to investigate the binding characteristics between RES and PEP at different temperatures by fluorescence spectroscopy, UV-Vis absorption spectroscopy, infrared spectroscopy (FT-IR) and molecular modeling technique. The experimental results provided important information for elucidating the action mechanism of RES and PEP. Fluorescence data revealed that the fluorescence intensity of PEP decreased regularly with the increase of RES concentration, indicating that RES had a fluorescence quenching effect on PEP. After RES was added, the UV-vis spectra of PEP changed significantly. The K_q value (the minimum quenching rate constant) at different temperatures were all much larger than 2.0×1010 L·mol-1·s-1(the maximum diffusion collision constant of the quenching agent on biological macromolecules). Moreover, Stern-Volmer quenching constant (K_SV) gradually decreased with the increase in temperature. These results verified quenching mode between PEP and RES to be static. The value of the stoichiometric binding number approximately equals 1, suggesting that one molecule of RES combined with one molecule of PEP. The thermodynamic parameters indicated that RES could spontaneously bind with pepsin mainly through the hydrogen bonds and Van der Waals forces. Synchronous fluorescence and three-dimensional fluorescence results provided data concerning conformational and some micro-environmental changes of pepsin. According to the results from FTIR analyses of PEP, the content of β-sheet increased accompanying with significantly decrease of α-helix, and no obvious change of β-turn and random coil upon binding with RES. The presence of RES loosened the skeleton of pepsin. These secondary structure changes might lead to changes of the physiological function of pepsin, such as the enzyme activity. Finally, molecular docking further suggested that RES molecule binded within the active pocket of PEP mainly via the van der Waals forces and hydrogen bonds. There were the van der Waals forces between RES and residues Asp-32, Gly-34, Ser-35, Asn-37, Tyr-75, Gly-76, Thr-77, Ile-128, Ala-130 and Gly-217 of PEP, super conjugation between RES and residues Ile-128 and Asp-215 of PEP, and hydrogen bonds between RES and Ser-36, Asn-37, Ile-128 and Thr-218 of PEP. Various forces make RES and PEP form a more stable complex.