光谱法和分子对接模拟技术研究左氧氟沙星与人血清白蛋白的相互作用

左氧氟沙星(LVFX)是临床上普遍使用的一种抗生素，对革兰氏阳性菌及革兰氏阴性菌引起的各种感染都有一定的作用。人血清白蛋白(HSA)是血液循环系统中最丰富的运输蛋白，能与多种内源及外源性物质结合，起着储存和转运的作用。因此详细研究LVFX与HSA间的相互作用对了解LVFX的药代动力学行为具有重要意义。运用光谱法和分子对接模拟技术研究左氧氟沙星和人血清白蛋白的相互作用。结果表明，LVFX对HSA的荧光淬灭作用为形成复合物导致的静态猝灭，结合常数为9.44×104 L·mol-1 (294 K)和2.72×104 L·mol-1 (310 K)，结合位点数均为1，两者间的主要作用力为氢键和范德华力。取代实验表明，LVFX在HSA的Site Ⅰ，ⅡA 子域上有一个结合位点。根据Frster理论得到的LVFX和色氨酸(Trp)残基间的结合距离为3.66 nm，这一结果与分子对接模拟技术得到的结果相一致。紫外差谱，三维荧光光谱和红外光谱都进一步表明LVFX能够改变HSA的结构。采用傅里叶变换红外光谱法对LVFX与HSA作用前后HSA二级结构的变化进行了定量分析，结果表明，当加入LVFX后HSA的α-螺旋结构有所降低，β-折叠结构、β-转角结构和无规则卷曲有所上升，说明LVFX能使HSA的二级结构变得松散。

Study of Interaction between Levofloxacin and Human Serum Albumin by Multi-Spectroscopic and Molecular Modeling Methods

Levofloxacin (LVFX) is widely used in clinical treatment due to it has a broad spectrum of in vitro activity against Gram-positive and Gram-negative bacteria. Human serum albumin (HSA) is the most abundant protein in plasma and constitutes approximately half of the protein founds in human blood. And more than 90% of the drugs used in people are bound to HSA. So it is commonly used for the investigation of drug-serum albumin interaction because the binding will significantly influence the absorption, distribution, metabolism excretion, stability and toxicity of the drugs. Therefore, detailed investigating the interaction of LVFX with HSA is very important to understand the pharmacokinetic behavior of the LVFX. In this paper, the interaction of LVFX and HSA has been studied fluorescence, UV, Fourier transform infrared (FT-IR) and molecular modeling method. The results indicated that LVFX induced the intrinsic fluorescence quenching of HSA though a static quenching procedure, and the effective binding constants (K_a) were calculated to be 9.44×104 L·mol-1 (294 K) and 2.74×104 L·mol-1 (310 K) by used of the Stern-Volmer equation. According to the Vant’s Hoff equation, the reaction was characterized by negative enthalpy (ΔH=-59.00 kJ·mol-1) and negative entropy (ΔS=-105.38 J·mol-1·K-1), indicated that the predominant forces in the LVFX-HSA complex were hydrogen bonding and van der Waals forces. By displacement measurements, the specific binding of LVFX in the vicinity of Site Ⅰ of HSA was clarified. The binding distance of 3.66 nm between Trp214 and HSA was obtained by the Frster theory on resonance energy transfer. Furthermore, the binding details between LVFX and HSA were further confirmed by molecular docking studies, which were consistent with the experimental results. The alternations of protein secondary structure were calculated from FT-IR spectra. Upon formation of LVFX-HSA complexes, the amount of α-helical structures were decrease, but the numbers of β-sheet structures, β-turn structures and random structures were increase, respectively. This result indicated that LVFX induced unfolding of the polypeptides of HSA.