S/R构型烟碱与胰蛋白酶作用机制的研究
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摘要
在模拟人体生理条件下,应用稳态荧光光谱和紫外可见吸收光谱手段辅助分子对接技术,重点考察了烟碱(NIC)两种对映体与胰蛋白酶(TYP)相互作用的荧光猝灭机理、构象变化及结合模式。结果表明,S-NIC与TYP碰撞结合形成激态复合物而猝灭TYP的内源性荧光,而R-NIC与TYP结合的猝灭机理为动静态结合的混合猝灭类型。R-NIC对TYP荧光猝灭的能力强于S-NIC,且结合常数(Ka)大于S-NIC,但二者在TYP活性空腔中均只有1个高亲和位点。热力学研究结果表明,S-NIC和R-NIC与TYP结合时活化能差异较大,R-NIC与TYP碰撞结合所需能量更小。同步和三维荧光光谱研究发现,NIC两种对映体均诱导了TYP的微环境和构象发生变化。分子对接结果表明,两种对映体均稳定结合于TYP分子的SER-195附近,且R-NIC与氨基酸残基SER-214形成氢键。
The aim of the present study was to determine the fluorescence quenching mechanism,binding mode and the changes of conformation of the trypsin(TYP)between two enantiomers of nicotine(NIC)and TYP under physiological conditions.The investigation was carried out using steady-state fluorescence and UV-vis absorption spectroscopic measurements,molecular docking method.The results demonstrated that the interaction of S-NIC with TYP is a process of dynamic quenching.However,the quenching mechanism between R-NIC and TYP is mainly dynamic quenching and static queching also exists.The quenching ability and effective association constant(K_a)values showed stronger biding of R-NIC to TYP than S-NIC.The obtained binding sites were found to be equal to one for the two NIC enantiomers.Activation energy for the interactions of S-NIC or R-NIC with TYP were evaluated,suggesting that the binding of R-NIC to TYP required less energy that S-NIC.Synchronous and three-dimensional fluorescence spectroscopy revealed that the binding of two NIC enantiomers to TYP resulted in an obvious change in micro-environmental and conformational of TYP.Furthermore,molecular docking study provided the same binding sites,the two enantiomers were situated near the SER-195 on TYP and the hydrogen bond between R-NIC and SER-214 was observed.
The aim of the present study was to determine the fluorescence quenching mechanism,binding mode and the changes of conformation of the trypsin(TYP)between two enantiomers of nicotine(NIC)and TYP under physiological conditions.The investigation was carried out using steady-state fluorescence and UV-vis absorption spectroscopic measurements,molecular docking method.The results demonstrated that the interaction of S-NIC with TYP is a process of dynamic quenching.However,the quenching mechanism between R-NIC and TYP is mainly dynamic quenching and static queching also exists.The quenching ability and effective association constant(K_a)values showed stronger biding of R-NIC to TYP than S-NIC.The obtained binding sites were found to be equal to one for the two NIC enantiomers.Activation energy for the interactions of S-NIC or R-NIC with TYP were evaluated,suggesting that the binding of R-NIC to TYP required less energy that S-NIC.Synchronous and three-dimensional fluorescence spectroscopy revealed that the binding of two NIC enantiomers to TYP resulted in an obvious change in micro-environmental and conformational of TYP.Furthermore,molecular docking study provided the same binding sites,the two enantiomers were situated near the SER-195 on TYP and the hydrogen bond between R-NIC and SER-214 was observed.
引文
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[2]Jacob P,Byrd G D.Use of gas chromatographic and mass spectrometric techniques for the determination of nicotine and its metabolites-Chapter 6[M].Amsterdam:Elesvier,1999:191-224.
[3]Perfetti T A,Coleman W M.Chiral-gas chromatography-selected ion monitoring-mass selective detection analysis of tobacco materials and tobacco smoke[J].Beitr?ge Zur Tabakforschung,1998,18(1):15-33.
[4]俞银姣.吸烟的危害与尼古丁替代疗法[J].药物不良反应杂志,2008,10(5):346-351.
[5]张国文,王琳.芒果苷与胰蛋白酶相互作用的光谱法研究[J].南昌大学学报:工科版,2012,34(2):103-106.
[6]Zhang H,Zhou Q,Cao J,et al.Mechanism of cinnamic acid-induced trypsin inhibition:a multi-technique approach[J].Spectrochim Acta A,2013,116(12):251-257.
[7]Zhang H M,Wang Y Q,Zhou Q H.Investigation of the interactions of quercetin and morin with trypsin[J].Luminescence,2009,24(5):355-362.
[8]Wu D,Yan J,Wang J,et al.Characterisation of interaction between food colourant allura red AC and human serum albumin:Multispectroscopic analyses and docking simulations[J].Food Chem,2015,170:423-429.
[9]Sharma A,Anandakumar S,Ilanchelian M.In vitro investigation of domain specific interactions of phenothiazine dye with serum proteins by spectroscopic and molecular docking approaches[J].Rsc Adv,2014,4(68):36267-36281.
[10]Li X,Wang G,Chen D,et al.β-Carotene and astaxanthin with human and bovine serum albumins[J].Food Chem,2015,179:213-221.
[11]Li X,Hao Y.Probing the binding of(+)-catechin to bovine serum albumin by isothermal titration calorimetry and spectroscopic techniques[J].J Mol Struct,2015,1091:109-117.
[12]Ishtikhar M,Rabbani G,Khan R H.Interaction of 5-fluoro-5′-deoxyuridine with human serum albumin under physiological and non-physiological condition:A biophysical investigation[J].Colloid Surface B,2014,123:469-477.
[13]Zhao L,Liu J,Guo R,et al.Investigating the interaction mechanism of fluorescent whitening agents to human serum albumin using saturation transfer difference-NMR,multi-spectroscopy,and docking studies[J].Rsc Adv,2017,7(44):27796-27806.
[14]Ricchelli F.Principles of Fluorescence Spectroscopy[M].New York:Plenum Press,1999:456-457.
[15]Rakotoarivelo N V,Perio P,Najahi E,et al.Interaction between Antimalarial 2-Aryl-3 H-indol-3-one Derivatives and Human Serum Albumin[J].J Phys Chem B,2014,118(47):13477-13485.
[16]王金菊,刘保生,边刚,等.光谱法结合分子对接模拟技术研究头孢他啶与胰蛋白酶的相互作用机理[J].发光学报,2017,38(9):1256-1263.
[17]周磊,迟燕华,蒋志强,等.对甲氧基苯乙酮与人血清白蛋白相互作用的研究[J].西南科技大学学报,2007,22(3):10-13.
[18]Ross P D,Subramanian S.Thermodynamics of protein association reactions:forces contributing to stability[J].Biochemistry,1981,20(11):3096-3102.
[19]Vaya I,Lhiaubet-Vallet V,Jimenez M C,et al.ChemInform abstract:Photoactive assemblies of organic compounds and biomolecules:Drug—protein supramolecular systems[J].Chem Soc Rev,2014,43(12):4102-4122.
[20]Lloyd J B F.Synchronized excitation of fluorescence emission spectra[J].Nature,1971,231(20):64-65.
[21]Miller J N.Recent developments in fluorescence and chemiluminescence analysis.Plenary lecture[J].Analyst,1984,109(3):191-198.
[22]Jing K,Guo X,Diao X,et al.Synthesis and characterization of dipicolinate sensitized LaF 3:Tb3+,nanoparticles and their interaction with bovine serum albumin[J].J Lumin,2015,157(3):184-192.
[23]Gorinstein S,Goshev I,Moncheva S,et al.Intrinsic tryptophan fluorescence of human serum proteins and related conformational changes[J].J Protein Chem,2000,19(8):637-42.