光谱学与光谱分析, 2017, 37 (1): 327, 网络出版: 2017-02-09   

多光谱法和分子模拟技术研究氟罗沙星与人血清白蛋白的相互作用

Spectroscopic and Molecular Modeling Studies on Binding of Fleroxacin with Human Serum Albumin
作者单位
1 中央民族大学生命与环境科学学院, 北京 100081
2 中央民族大学北京市食品环境与健康工程技术研究中心, 北京 100081
摘要
氟罗沙星(FLRX) 是一种含氟喹诺酮类抗菌素, 有关它对人血清白蛋白(HSA)的影响及作用机理, 特别是对HSA二级结构的影响及内滤光(影响荧光数据的准确性)校正的研究报道较少。 采用多光谱法和分子模拟技术探究了FLRX 与HSA的相互作用。 荧光光谱结果表明, FLRX对HSA的猝灭是由于形成结合常数在105 L·mol-1水平上的1∶1 FLRX-HSA基态复合物引起的静态猝灭作用。 由Van’t Hoff方程确定的FLRX与HSA结合过程中的ΔH=-107.99 kJ·mol-1和ΔS=-240.99 J·mol-1·K-1, 表明FLRX与HSA之间的主要作用力是氢键和范德华力。 同步荧光光谱、 红外光谱和三维荧光光谱结果表明, 静态猝灭过程所产生的中间复合物使HSA的构象发生改变。 通过对HSA与FLRX作用前后红外光谱酰胺Ⅰ带进行傅里叶去卷积和分峰拟合, 获得代表HSA二级结构的不同子峰, 对各子峰进行二级结构归属, 根据各子峰的积分面积计算出各二级结构的相对百分含量。 结果表明: FLRX与HSA结合后, α-螺旋从51.5%减小到33.2%, β-折叠从30.3%减小到20.7%, β-转角从15.6%增加到33.6%。 取代实验显示FLRX与HSA的结合位点在HSA的site Ⅰ(亚域ⅡA)。 分子对接实验结果表明, FLRX可以通过氢键、 疏水作用和范德华力等多种作用力很好的结合在亚域ⅡA的疏水腔中。 实验获得的可信数据将有助于阐明FLRX与HSA的作用机制, 也有助于理解FLRX在储运过程中对蛋白质功能的影响。
Abstract
Fleroxacin (FLRX) is a new member of the class of fluoroquinolones, its effects on human serum albumin (HSA) and the mechanism of action are poorly understood, Especially, the secondary structural alterations of HSA induced by FLRX and the inner filter effect, which resulted in a spurious decrease in the observed fluorescence intensity and affected the binding parameters calculated from it are not considered. In this paper, binding of FLRX to HSA has been studied using multi-spectroscopy and molecular modeling methods. Fluorescence spectra revealed that the observed fluorescence quenching of HSA by FLRX was due to a 1∶1 complex formation by a static quenching process with a constant of 105 L·mol-1. The thermodynamic parameters (ΔH and ΔS) were calculated to be -107.99 kJ·mol-1 and -240.99 J·mol-1·K-1 via the Van’t Hoff equation, which indicated that hydrogen bond and van der Waals force were the dominant intermolecular force. From the synchronous fluorescence, FT-IR and three dimensional fluorescence spectra, it was evident that the binding of FLRX to HSA induced a conformational change in the protein, and the alterations of secondary structure were quantitatively calculated by the evidence from FTIR spectra with reductions of α-helices of about 18.3%, decreases of β-sheet structures of about 9.6%, and increases of β-turn structures of about 18.0%. Site marker competitive experiments showed that phenylbutazone and FLRX shared a common binding site Ⅰ corresponding to the subdomain ⅡA of HSA. The binding details between FLRX and HSA were further confirmed by molecular docking studies, which revealed that FLRX was bound at subdomain ⅡA through multiple interactions, such as hydrogen bond, hydrophobic and van der Waals, etc. The accurate and full basic data in the work is beneficial to clarify the binding mechanism of FLRX with HSA and is helpful for understanding its effect on protein function during the blood transportation process.

董澄宇, 徐佳, 周珊珊, 刘颖. 多光谱法和分子模拟技术研究氟罗沙星与人血清白蛋白的相互作用[J]. 光谱学与光谱分析, 2017, 37(1): 327. DONG Cheng-yu, XU Jia, ZHOU Shan-shan, LIU Ying. Spectroscopic and Molecular Modeling Studies on Binding of Fleroxacin with Human Serum Albumin[J]. Spectroscopy and Spectral Analysis, 2017, 37(1): 327.

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