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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∶1FLRX-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=-107.99kJ·mol~(-1)和ΔS=-240.99J·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siteⅠ(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。
Fleroxacin (FLRX) is a fluoroquinolone-containing antibiotic that has been implicated in its effect on human serum albumin (HSA) and its mechanism of action, in particular on the secondary structure of HSA and the effect of internal light filtering (which affects the accuracy of fluorescence data Correction studies have reported less. The interaction between FLRX and HSA was explored using multispectral and molecular modeling techniques. Fluorescence spectra showed that quenching of HSA by FLRX was due to the static quenching induced by the formation of 1: 1 FLRX-HSA ground state complex with a binding constant at 105 L · mol -1. ΔH = -107.99 kJ · mol -1 and ΔS = -240.99 J · mol -1 K -1 during the binding of FLRX to HSA as determined by the Van’t Hoff equation, indicating that FLRX The main interactions with HSA are hydrogen bonding and van der Waals forces. Simultaneous fluorescence spectroscopy, infrared spectroscopy and three-dimensional fluorescence spectroscopy showed that the intermediate complex produced by static quenching changed the conformation of HSA. Through the Fourier deconvolution and peak fitting of the amide Ⅰ band of the infrared spectrum before and after the action of HSA and FLRX, different sub-peaks representing the secondary structure of HSA were obtained and the secondary structure of each sub-peak was assigned. According to the sub-peaks The integral area of each secondary structure calculated relative percentage content. The results showed that after the combination of FLRX and HSA, the α-helix decreased from 51.5% to 33.2%, the β-sheet reduced from 30.3% to 20.7% and the β-turn increased from 15.6% to 33.6%. Substitution experiments showed that the binding site of FLRX to HSA is at site I (subdomain IIA) of HSA. The experimental results of molecular docking show that FLRX can be well integrated in the hydrophobic cavity of sub-region ⅡA through a variety of forces such as hydrogen bonding, hydrophobic interaction and van der Waals forces. The credible data obtained from the experiments will help elucidate the mechanism of action of FLRX and HSA and also help understand the effect of FLRX on protein function during storage and transportation.