Huang Fang, Dong Cheng-Yu, Zhang Li-Yang, Liu Ying
Guang Pu Xue Yu Guang Pu Fen Xi. 2014 Apr;34(4):1064-9.
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 x 10(4) L x mol(-1) (294 K) and 2.74 x 10(4) L x 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 (deltaH = -59.00 kJ x mol(-1)) and negative entropy (delta S = - 105.38 J x mol(-1) x 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 Förster 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 alpha-helical structures were decrease, but the numbers of beta-sheet structures, beta-turn structures and random structures were increase, respectively. This result indicated that LVFX induced unfolding of the polypeptides of HSA.
左氧氟沙星（LVFX）因其对革兰氏阳性菌和革兰氏阴性菌具有广泛的体外活性而被广泛应用于临床治疗。人血清白蛋白（HSA）是血浆中含量最丰富的蛋白质，约占人体血液中蛋白质总量的一半。在人体内使用的药物中，超过90%与HSA结合。因此，它常用于研究药物与血清白蛋白的相互作用，因为这种结合会显著影响药物的吸收、分布、代谢排泄、稳定性和毒性。因此，详细研究LVFX与HSA的相互作用对于了解LVFX的药代动力学行为非常重要。本文采用荧光、紫外、傅里叶变换红外（FT-IR）和分子模拟方法研究了LVFX与HSA的相互作用。结果表明，LVFX通过静态猝灭过程诱导了HSA的内源荧光猝灭，利用Stern-Volmer方程计算出有效结合常数（K(a)）在294 K时为9.44×10(4) L×mol(-1)，在310 K时为2.74×10(4) L×mol(-1)。根据范特霍夫方程，该反应的特征是负焓（ΔH = -59.00 kJ×mol(-1)）和负熵（ΔS = -105.38 J×mol(-1)×K(-(1))），表明LVFX-HSA复合物中的主要作用力是氢键和范德华力。通过置换测量，明确了LVFX在HSA位点I附近的特异性结合。根据Förster共振能量转移理论，得到Trp214与HSA之间的结合距离为3.66 nm。此外，分子对接研究进一步证实了LVFX与HSA之间的结合细节，与实验结果一致。从FT-IR光谱计算出蛋白质二级结构的变化。形成LVFX-HSA复合物后，α-螺旋结构的数量减少，但β-折叠结构、β-转角结构和无规结构的数量分别增加。该结果表明LVFX诱导了HSA多肽的去折叠。
