左氧氟沙星与牛血清白蛋白相互作用的液滴荧光法研究

Investigation of Interaction between Levofloxacin and Bovine Serum Albumin by Fluorescence Analysis Based on Liquid Drop

采用液滴荧光技术与紫外-可见光度法研究了生理pH值条件下左氧氟沙星和牛血清白蛋白的相互作用机制。左氧氟沙星对牛血清白蛋白产生荧光猝灭,且猝灭过程是由于复合物形成而引起的静态猝灭。根据Frster偶极-偶极非辐射能量转移理论算出供体-受体的结合距离为2.68nm。由Linewear-Burk方程求出不同温度下反应时复合物的形成常数KLB和结合位点数n及对应温度下结合反应的热力学参数,证明二者主要靠疏水作用力结合。同时采用同步荧光分析技术,对蛋白质与药物结合时构象的变化进行了探讨。

The interaction between pharmaceutical and protein is an important pharmacokinetic characteristic. Most kinds of drugs must reach the receptor to perform the pharmacological function by plasma. Albumins can serve as a depot protein and a transport protein for numerous endogenous and exogenous compounds. It is of great significance to investigate the binding interaction between albumin and drugs, since it can not only help understand the transportation and distribution of drugs but also elucidate the mechanism. Under the physiological condition of body, the interaction between levofloxacin （LVFX） and bovine serum albumin （BAS） was investigated by fluorescence spectra and ultraviolet absorbance （UV） spectra based on liquid drop. The experimental results showed that LVFX quenches the fluorescence of BAS by forming a LVFX-BSA complex. According to Lineweaver-Burk equation, the apparent binding constants （KLB） between LVFX and BSA were 1. 694 9× 10^5 L· mol^-1 （291 K） and 2. 881 0× 10^5 L· mol^-1 （310 K）, and the binding sites （n） were 0. 884 9 （291 K） and 0. 808 9 （310 K）. Thermodynamic parameters could be evaluated from the thermodynamic second law, with △H （enthalpy） being 20. 94 kJ · mol^-1 and AS （entropy） being 172. 1 J · mol^-1. According to the relation between thermodynamic parameters and the interaction force, LVFX depended principally on the hydrophobic interaction to bind with BSA. The results showed that the quenching belonged to static fluorescence quenching with non-radiation energy transfer happening within single molecule. The binding locality was an area 2. 68 nm away from tryptonphan residue-212 in BAS according to Forster＇s non-radiation energy transfer mechanism. The conformational changes of BSA were evaluated by measuring the synchronous fluorescence intensity of protein amino acid residues, both before and after the addition of LVFX. A slightly stronger blue-shift of tryptophan fluorescence upon the addition of drug was observed, and the emission maximum of tyrosine kept its position. It was suggested that the environments of tryptophan residues in pure albumin solution are relatively polar. Binding of LVFX changes the environments to apolar ones. The shift in polarity is brought about by confirmation changes due to the interaction between albumin and ligand molecule.