光谱法和分子对接研究红斑红曲胺与牛血清白蛋白相互作用  

The Interaction between Rubropunctamine and Bovine Serum Albumin Using Spectrometry and Molecular Docking

作　　者：黄朝波 徐晗 杨明冠 李贞景 杨华[1,3] 王昌禄 周庆礼[1,2,3] HUANG Chao-bo;XU Han;YANG Ming-guan;LI Zhen-jing;YANG Hua;WANG Chang-lu;ZHOU Qing-li(State Key Laboratory of Food Nutrition and Safety,Tianjin 300457,China;Ministry of Education Key Laboratory of Food Nutrition and Safety,College of Food Engineering and Biotechnology,Tianjin University of Science and Technology,Tianjin 300457,China;College of Food Engineering and Biotechnology,Tianjin University of Science and Technology,Tianjin 300457,China)

机构地区：[1]省部共建食品营养与安全国家重点实验室,天津300457 [2]天津科技大学食品工程与生物技术学院,食品营养与安全教育部重点实验室,天津300457 [3]天津科技大学食品工程与生物技术学院,天津300457

出　　处：《光谱学与光谱分析》2019年第10期3102-3108,共7页Spectroscopy and Spectral Analysis

基　　金：国家自然科学基金项目(31271950);农业部农产品贮藏保鲜实验室开放基金项目(KF2018008)资助

摘　　要：近年来,随着对红曲色素的深入研究,其越来越多的功能活性被发现,但其某些致毒作用也使红曲色素的安全性受到了质疑。因此,阐明红曲色素在人体中与大分子的相互作用对深入研究其转运代谢及毒副作用具有重要作用。光谱法是研究溶液中小分子与蛋白质相互作用的一种有效方法,其具有灵敏度高、选择性强、用样量少、方法简单等优点,在研究中得到越来越广泛的应用。为探究红曲色素在体内的转运机制和血液中与转运蛋白的相互作用,本研究首次用红斑红曲胺(Rubropunctamine,Rub)作为红曲色素的典型代表与牛血清白蛋白(bovine serum albumin,BSA)相互作用。利用内源荧光光谱、同步荧光光谱探究不同浓度的Rub对BSA的荧光猝灭作用,采用Stern-Volmer方程、Lineweaver-Burk函数和Van’t-Hoff方程对不同温度下BSA与Rub作用后在λEX/λEM(280.0 nm/340.0 nm)(λEX/λEM表示荧光的激发波长和发射波长)的内源荧光强度值确定二者作用类型、结合位点数及相互作用机理,进一步利用圆二色谱定量测定了Rub的结合对BSA二级结构影响,最后运用软件Discovery Studio2.5对Rub与BSA的相互结合进行分子对接模拟。结果显示:(1)Rub对BSA具有较强的内源荧光猝灭效果,在λEX/λEM(280.0 nm/340.0 nm)的荧光强度下降306.1,发射波长由338.6 nm蓝移到331.8 nm,同步荧光显示荧光猝灭主要发生在色氨酸残基上。(2)Stern-Volmer方程计算得到动态猝灭速率常数K q为2.335×10^12 L·(mol·s)^-1,远大于此类型允许的最大扩散碰撞常数2.0×10^10 L·(mol·s)^-1,判定该猝灭是单纯的静态猝灭过程。利用Lineweaver-Burk函数计算得到静态猝灭速率常数K q随温度升高而减小,即该复合物在温度升高时变得不稳定。(3)利用等式lg[(F 0-F)/F]=lg K 0+n lg c Q得到两者结合常数可达10 3 L·mol^-1以上,结合位点数近似为1,且随着温度增加表观结合常数变小。(4)不同温度下In recent years,more and more functional activities have been discovered with the in-depth study of the monascus pigment,but some toxic effects of the monascus pigment have raised questions about its safety.Therefore,it is important to elucidate the interaction between monascus pigments and macromolecules in human body for further study of their transport,metabolism and toxic side effects.Spectroscopy is an effective method to study the interaction of small molecules with proteins in solution.It has been widely used in research for its high sensitivity,strong selectivity,low sample size,and simple method.In this study,rubropunctamine(Rub)was taken as the typical representative of monascus pigment to research the interaction of Monascus pigments with macromolecules bovine serum albumin(BSA).The fluorescence quenching effect of different concentrations of Rub on BSA was investigated by endogenous fluorescence spectroscopy and synchronous fluorescence spectroscopy.Then Stern-Volmer equation,the Lineweaver-Burk function and the Van’t-Hoff equation were used to determine the type of action,the number of binding sites and the interaction mechanism of BSA and Rub.The effect of Rub on the BSA secondary structure was quantitatively determined by circular dichroism spectrum.Finally,using the computer to perform the molecular docking simul-ation on the interaction of Rub and BSA.The results show:(1)Rub has a strong fluorescence quenching effect on BSA,and endogenous fluorescence spectrum shows that endogenous fluorescence decreases by 306.1 and emission wavelength shifts by 6.8 nm.Synchronous fluorescence shows that fluorescence quenching mainly occurs on tryptophan residues.(2)The dynamic quenching rate constant K q calculated by the Stern-Volmer equation is 2.335×10^12 L·(mol·s)^-1,which is much larger than the maximum diffusion collision constant allowed:2.0×10^10 L·(mol·s)^-1,and the annihilation is a pure static quenching process.(3)The binding constants reach above 10 3 L·mol^-1 which is calculated by the equ

关 键 词：荧光光谱 同步荧光 圆二色谱 分子对接 红斑红曲胺 牛血清白蛋白 

分 类 号：TS201.6[轻工技术与工程—食品科学]