摘要
以荧光猝灭法与分子对接为主要研究手段,研究了在模拟生理条件下蒙花苷对胰蛋白酶荧光发射的猝灭作用,并探究了二者之间的相互作用类型与复合物的动力学稳定性。实验结果表明,pH 8.4时在不同温度下蒙花苷均可有效地猝灭胰蛋白酶的荧光发射,并且猝灭类型均为静态猝灭,即蒙花苷与胰蛋白酶间形成了稳定的复合物。使用双对数方程计算可知复合物为1∶1型,根据Van’t Hoff方程对不同温度下蒙花苷猝灭胰蛋白酶的光谱数据分析可知复合物的生成是熵增放热的自发过程。同步荧光光谱结果表明蒙花苷对胰蛋白酶的发光猝灭主要是通过影响其Trp残基的荧光发射实现的。分子对接研究表明蒙花苷可结合在胰蛋白酶表面由His57、Thr98、Gln175、Trp215、Gln192、Ser195等残基所形成的疏水性口袋中,并通过氢键、芳环堆积、疏水、静电吸引等弱作用与胰蛋白酶形成非共价复合物,蒙花苷与Trp215残基间存在明显的疏水与氢键作用,这些作用可减弱这个残基微环境的极性并最终导致其荧光发射的猝灭。30 ns分子动力学模拟表明胰蛋白酶与蒙花苷所形成的复合物具有良好的稳定性,验证了分子对接结果的稳定性和合理性。
The combination offluorescence quenching and molecular docking is adopted as the main method to study the quenching effect of linarin to the fluorescence emission of trypsin under simulated physiological conditions.The interaction and kinetic stability between the host andtheguest are also explored.Experimental results show that linarin can effectively quench trypsin’s fluorescence emission at different temperatureswhen pH is 8.4.The quenchingcan be classified asstatic quenching,due to the formation of stable complex.By using double logarithmic equation and Van't Hoff equation,the host-guest complex is determined as 1∶1 type and the complex formation is a spontaneous processwhich involves an increase in entropy.The results of synchronous fluorescence spectroscopy showed that the luminescence quenching of linarin to trypsin is achieved by affecting the fluorescence emission of its tryptophan residues.Molecular docking studies have shown that linarin can be accommodated on the surface of trypsin andthendocked into a hydrophobic pocket formed by His57,Thr98,Gln175,Trp215,Gln192 and Ser195 residues.Hydrogen bonding,aromatic ring stacking,hydrophobic forces and static attraction arelooked asdriving forces for the formation of the covalent complex of trypsin and linarin.There are obvious hydrogen bonding and hydrophobic interaction between the Trp215 residue and linarin.Theintermolecularinteractions between the host and the guestreduce the polarity of the micro-environment of Trp215and eventually lead to the fluorescence quenching.A 30 ns molecular dynamics simulation showed that the complex formed by trypsin and linarin has good stability,whichis in coincidence withthe molecular docking results.
作者
韩忠保
李天乐
苗瑞丹
刘丽艳
陈庆阳
于湛
HAN Zhong-bao;LI Tian-le;MIAO Rui-dan;LIU Li-yan;CHEN Qing-yang;YU Zhan(School of Chemistry and Chemical Engineering,Shenyang Normal University,Shenyang 110034,China;Provincial Key Laboratory for Separation and Analysis of Complex Systems in Liaoning Universities,Shenyang Normal University,Shenyang 110034,China)
出处
《化学研究与应用》
CAS
CSCD
北大核心
2020年第2期246-251,共6页
Chemical Research and Application
基金
国家自然科学基金(21205080)资助
辽宁省高校优秀人才支持计划(LJQ2015105)资助
2019年辽宁省教育厅基础研究项目资助
关键词
蒙花苷
胰蛋白酶
荧光猝灭
分子对接
分子动力学
linarin
trypsin
fluorescence quenching
molecular docking
molecular dynamics
作者简介
联系人:于湛(1978-),男,教授,主要从事光谱分析与质谱分析研究。E-mail:yuzhan@synu.edu.cn。
