多光谱法和分子对接模拟法研究茶碱和胃蛋白酶的相互作用  

Study on the Interaction Between Theophylline and Pepsin by Multispectral and Molecular Docking Simulation

作　　者：王晓霞[1,2] 马力通 孙吉盛[1] 聂智华 赛华征 成建国 段建国 WANG Xiao-xia;MA Li-tong;SUN Ji-sheng;NIE Zhi-hua;SAI Hua-zheng;CHENG Jian-guo;DUAN Jian-guo(School of Chemistry and Chemical Engineering,Inner Mongolia University of Science and Technology,Baotou 014010,China;Inner Mongolia Engineering Research Center of Comprehensive Utilization of Bio-coal Chemical Industry,Baotou 014010,China;School of Life Sciences,Tsinghua University,Beijing 100084,China)

机构地区：[1]内蒙古科技大学化学与化工学院,内蒙古包头014010 [2]生物煤化工综合利用内蒙古内蒙古自治区工程研究中心,内蒙古包头014010 [3]清华大学生命科学学院,北京100084

出　　处：《光谱学与光谱分析》2024年第3期714-721,共8页Spectroscopy and Spectral Analysis

基　　金：国家自然科学基金项目(52164013);内蒙古自治区人才开发基金项目(2021年度);内蒙古自治区科技计划项目(2020GG0158);内蒙古科技大学基本科研业务费专项基金项目(2024QNJS049)资助。

摘　　要：在模拟生理条件下,通过紫外可见吸收光谱法、傅里叶红外光谱、荧光光谱、三维荧光光谱、同步荧光光谱、圆二色谱等方法以及分子对接模拟法对茶碱(TPL)与胃蛋白酶(PEP)的结合机理进行了研究,在分子层面探讨TPL与PEP相互作用的机制,有助于对TPL的药理毒性、药效进行深入研究。根据Stern-Volmer方程,计算在298、 303和308 K三种温度下TPL对PEP的动态荧光猝灭速率常数Kq远大于最大动态荧光猝灭常数2.0×10^(10)L·(mol·s)^(-1),证明TPL对PEP的猝灭方式为静态猝灭。随着TPL浓度的不断升高,PEP的动态猝灭常数Ksv呈规律性下降趋势,TPL可以有效对PEP的内源荧光进行猝灭,进一步判定作用机制为静态猝灭。三维荧光图谱分析表明,体系中随着TPL浓度的不断升高,PEP中代表色氨酸残基与酪氨酸残基以及肽链骨架结构的荧光强度峰值明显降低,且峰位发生红移,表明TPL对PEP的二级结构产生影响。同步荧光图谱分析表明TPL与PEP结合时,主要集中在色氨酸的残基上。红外光谱表明TPL引起PEP内官能团伸缩振动,使PEP的二级结构发生改变。紫外吸收光谱表明随着混合体系中TPL浓度增大,吸收峰峰值逐渐增大,表明TPL可以改变PEP的二级结构。分子对接模拟法表明,TPL与PEP中氨基酸残基GLU13、 VAL30、 TRP39、 GLY76、 GLY78、 PHE117的结合作用力为范德华力,与氨基酸残基THR77、 GLY217形成氢键,与氨基酸残基TYR75、 LEU112、 ILE120、PHE111之间存在疏水作用力,证明两者主要通过范德华力与氢键结合,进一步证明TPL改变了PEP的二级结构。圆二色谱分析显示PEP中β-折叠的占比从50.2%下降至48.8%, α-螺旋结构的占比从8.1%上升到8.4%;β-转角的占比从18.3%上升到18.7%;无规则结构的占比从29.1%上升到29.2%,表明TPL改变了PEP的二级结构。实验研究结果有助于了解TPL与PEP的结合作用机制,为TPL的使用及研究提供了数据依据。In this study,the binding mechanism of theophyline(TPL)and pepsin(PEP)was studied for the first time by ultraviolet-visible absorption spectroscopy,Fourier infrared spectroscopy,fluorescence spectroscopy,three-dimensional fluorescence spectroscopy,synchronous fluorescence spectroscopy,circular dichography and molecular docking simulation method,and the mechanism of interaction between TPL and PEP was explored at the molecular level,which was helpful to conduct in-depth research on the pharmacotoxicity and efficacy of TPL.According to the Stern-Volmer equation,the dynamic fluorescence quenching rate constant Kq of TPL for PEP at three temperatures of 298,303,and 308 K is much greater than the maximum dynamic fluorescence quenching constant of 2.0×10^(10) L·(mol·s)^(-1),proving that TPL quenching PEP is static quenching.With the continuous increase of TPL concentration,the dynamic quenching constant Ksv of PEP shows a regular downward trend,and TPL can effectively quench the endogenous fluorescence of PEP and further infer that the quenching mechanism is static quenching.The three-dimensional fluorescence spectrum analysis showed that with the continuous increase of TPL concentration in the system,the peak fluorescence intensity representing tryptophan residues,tyrosine residues and peptide chain skeleton structure in PEP decreased significantly,and the peak position was redshifted,indicating that TPL a affected the secondary structure of PEP.Simultaneous fluorescence mapping analysis showed that when TPL binds to PEP,it is mainly concentrated on the tryptophan residues.Infrared spectroscopy showed is that TPL caused the functional groups in PEP to expand and vibrate,which changed the secondary structure of PEP.The ultraviolet absorption spectrum showed that the absorption peak and peak increased gradually with the increase of TPL concentration in the mixed system,indicating that TPL could change the secondary structure of PEP.The molecular docking simulation method shows that the binding force between TPL and t

关 键 词：茶碱 胃蛋白酶 多光谱法 分子对接模拟 

分 类 号：O657.3[理学—分析化学]