Studies Of The Interaction Of Several Antimicrobial Drugs With Bovine Serum Albumin Using Spectroscopy

Exploring the inieractions between small molecules and bovine serum albumin at the molecular is of current interest in many research areas such as clinical medicine, biology, chemistry and so on. In recent decades, a large number of domestic and foreign scholars have made a lot of reports about this study. However, most of these reports are all about the interaction between a single drug and bovine serum albumin. The interactions of combination of several antimicrobial drugs with bovine serum albumin were studied, and the chemometrics method was applied in complicated biochemical system. This method can be used to solve some problems and obtain the equilibrium concentration and pure spectra of each component, from which the concentrtation trends of each component and the binding constant between drugs and BSA can be obtained. More importantly, the pure spectra of the new compound of the reaction can be obtained by chemometrics, which can not be obtained by conventional means. This thesis includes the following five parts:Chapter one:The structure and function of serum albumin was introduced briefly. Then the research methods and contents of interaction of several kinds of small molecules with serum albumin were reviewed in turns. The application of chemometrics in the field of protein research was also discussed. Finally, a brief overview about the interactions of antibacterial drugs with BSA was given.Chapter two:The interactions of the two floroquinolones, ciprofloxacin (CPFX) and Gatifloxacin (GTFX) with BSA were studied by spectrofluorimetry and UV-vis spectrometry with the aid of the chemometrics method - multivariate curve resolution-alternating least squares (MCR-ALS). CPFX and GTFX quenched the BSA fluorescence mainly through a static quenching mechanism, the quenching constant of BSA-GTFX is larger than that of BSA-CPFX. But when the drug mixture was involved, the quenching constant was larger than individual drug. Site marker displacement experiments with warfarin and diazepam demonstrate that both the two FQ bound to site I of BSA. The application of the MCR-ALS for the resolution of the expanded matrix data of fluorescence and UV-vis spectra produced the estimates of pure spectra and equilibrium concentration profiles. The resolved results combined with the experimental phenomena indicated that GTFX will interact with CPFX and BSA to form a ternary complex when they coexist.Chapter three:The interaction of gold nanoparticles (GNPs) and ciprofloxacin (CPFX) with bovine serum albumin was studied by spectroscopy. GNPs quenched the fluorescence of BSA and CPFX through a static quenching mechanism. The binding constant of the interaction of CPFX (GNP) with BSA in the presence of GNP (CPFX) decreased, competitive effect may exist between CPFX and GNP. Besides, the interaction of CPFX-capped GNP with BSA was also studied, and the chemometrics method - parallel factor analysis (PARAFAC) was applied to resolve the three-dimensional data. The results demonstrated that the existence of GNP weaken the binding ability of CPFX and BSA, which made the release of CPFX easily and increased the efficacy of CPFX.Chapter four:The interactions of the two antibiotics, ciprofloxacin (CPFX) and cefradine (CF) with BSA were studied by fluorescence spectrometry. Both CPFX and CF quenched the BSA fluorescence mainly through a static quenching mechanism, the quenching constant of BSA-CF is larger than that of BSA-CPFX. Site marker displacement experiments indicated that the binding site of CPFX and CF on BSA was siteⅠ. From the comparison of the quenching of BSA fluorescence in the binary and ternary systems, it was found that CF made the binding of CPFX with BSA stronger despite its lower affinity to the BSA molecule. Similarly, CPFX made the formation of the complex BSA-CF easier in subdomainⅡA. The fluorescence spectra of the interaction of BSA, CPFX and CF indicated that CF will interact with CPFX and BSA to form a ternary complex. Besides, florescence contour plots revealed that the conformation and micro-environment of BSA were changed by the binding of CPFX/CF or their mixture. And the conformational changes of BSA were related to the order of CPFX and CF added to BSA solution.Chapter five:The interaction between tetracyclines (chlorotetracycline (CTC) and oxytetracyline (OTC)) and bovine serum albumin (BSA) was investigated by fluorescence and UV-vis spectroscopy. The influence of some metal ions on the interaction was also studied. When tetracycline were added into the solution containing BSA, the fluorescence intensity of BSA decreased with the increasing of the drug concentrations, indicating their interactions were involving fluorescence quenching. The Stern-Volmer quenching constant Ksv, binding constant K and corresponding thermodynamic parameters AH, AG, and AS were calculated. The main sorts of acting force between the drug and BSA were found to be electrostatic force. The distances, r, between donor (BSA) and acceptor (CTC and OTC) were calculated according to fluorescence resonance energy transfer (FRET).