荧光光谱法和分子模拟技术研究考马斯亮蓝G-250与牛血清白蛋白的相互作用  

Study on Interaction between Coomassie Brilliant Blue G-250 and Bovine Serum Albumin by Fluorescence Spectroscopy and Molecular Modeling

作　　者：王永刚[1] 杨光瑞[1] 马雪青[2] 冷非凡[1] 马建忠[1] 王晓力[3] 

机构地区：[1]兰州理工大学生命科学与工程学院,甘肃兰州730050 [2]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室国家口蹄疫参考实验室农业部畜禽病毒学重点开放实验室,甘肃兰州730046 [3]中国农业科学院兰州畜牧与兽药研究所,甘肃兰州730050

出　　处：《光谱学与光谱分析》2017年第8期2474-2479,共6页Spectroscopy and Spectral Analysis

基　　金：国家自然科学基金项目(31060041;31460032);甘肃省自然科学基金项目(1212RJYA008)资助

摘　　要：采用多种光谱技术结合圆二色谱技术研究了考马斯亮蓝G-250(CBBG-250)相互作用于牛血清白蛋白(BSA),探究了两者之间的作用机理。荧光光谱结果表明BSA与CBBG-250结合形式是静态猝灭,随CBBG-250浓度增加,其形成常数随温度逐渐减小,结合比为1∶1,根据Stern-Volmer曲线计算焓变值(ΔH)和熵变值(ΔS)分别为-4.38kJ·mol^(-1)和-6.16J·mol^(-1)·K^(-1),均小于零,证明该过程是一个自发过程。傅里叶红外光谱测定结果表明CBBG-250的加入引起BSA结构的变化,随着CBBG-250溶液浓度的增加,BSA中色氨酸微环境极性被改变,在1 600~1 700cm^(-1)(酰胺带Ⅰ)和1 600~1 500cm^(-1)(酰胺带Ⅱ)处的特征吸收带蓝移。其中1 650cm^(-1)移动至1 710cm^(-1),1 544cm^(-1)移动到1 573cm^(-1),显示BSAα-螺旋(1 650~1 658cm^(-1))和β-折叠(1 620~1 640cm^(-1),1 675cm^(-1))结构发生变化,且α-螺旋含量比结合前有所降低。圆二色谱分析结果表明,两者间通过氢键和范德华力为主的作用力使得BSA二级结构发生变化,α-螺旋含量由42.15%下降至1.27%,该结果进一步被分子建模对接技术证明。The interaction of coomassie brilliant blue G-250（CBBG-250）with bovine serum albumin（BSA）was investigated by the methods of fluorescence spectroscopy,Fourier transform infrared spectroscopy（FT-IR）and circular dichroism（CD）.Fluorescence data showed that the quenching of BSA by CBBG-250 was result of forming the complex of BSA-CBBG-250,and CBBG-250 effectively quenched the intrinsic fluorescence of BSA via static quenching.According to Stern-Volmer equation,the binding parameters between CBBG-250 and BSA were determined.The enthalpy change（ΔH）and entropy change（ΔS）were calculated to be-4.38 kJ＆#183;mol-1 and-6.16 J＆#183;mol-1＆#183;K-1,indicating that the hydrogen bonds and hydrophobic interactions played a dominant role in the binding.The conformational investigation revealed the α-helical structure was decreased and the polypeptides of BSA were slightly folded with the addition of CBBG-250 by synchronous fluorescence.Fourier infrared spectrum showed that the peak position of amide band Ⅰ（1 600～1 700 cm-1）and amide band Ⅱ（1 600～1 500 cm-1）of the BSA characteristic absorbing peaks was blueshifted.The amide band I moved from 1 650 to 1 710 cm-1,and amide band Ⅱ moved from 1 573 to 1 544 cm-1,which indicated the structure of alpha helix features（1 650～1 658 cm-1）and beta-fold（1 620～1 640 cm-1 and 1 645 cm-1）had changed.The content of α-helical structure decreased from 42.15%to 1.27%by circular dichroism.The main reasons were the microenvironment polarity of tryptophan changed with the addition of CBBG-250 increased,two interaction between BSA and CBBG-250 including the hydrogen bonding and van der Waals force occurred,and then the secondary structure of BSA was changed,which was confirmed by Molecular modeling.

关 键 词：光谱法 分子模拟 考马斯亮蓝G-250 牛血清白蛋白 相互作用 

分 类 号：O51[理学—低温物理]