Studies Of The Interaction Mechanisms Between Sinafloxacin And Bio-macromolecules

Protein and DNA are important bio-macromolecules in the life. They have respective biological function, and have been playing many vital roles for all kinds of biological phenomena. Exploring the interaction mechanisms on these bio-macromolecules with small molecules or ions, especially for those targeting-drug molecules, at the molecular level is of current interest in many research areas such as biology, clinical medicine, medicinal chemistry, chemistry and so on. Quinolones gets more and more use in the clinic for its advantages of high activity, broad spectrum and the convenience in the taking, et al. But its pharmacology and toxicology still have to be studied further. At the time, it also has some disadvantages, so develop new quinolones which has the high activity and low side-effect is the point of research. There are many metal ions in the organism, and they participate in many important vital action. Metal ions can compound with quinolone drug, and also can combine with bio-macromolecules such as protein. Sinafloxacin is a novel fourth-generation quinolone antimicrobial drug, which has in vitro and in vivo activities against a wide range of Gram-positive and Gram-negative bacteria. In a previous study, it was observed that sinafloxacin had more potent antibacterial activity but lower cytotoxicity than ciprofloxacin, levofloxacin and gatifloxacin. This paper studied the interaction mechanisms of sinafloxacin with bio-macromolecules, and it was consisted of two parts.In the first part, bovine serum albumin (BSA) has been selected for investigative object, and the binding characteristics and mechanism of sinafloxacin to BSA have been investigated by using fluorescence spectroscopy and absorbance spectra. Sinafloxacin can quench the endogenous fluorescence of BSA regularly and the quenching was not initiated by dynamic collision but from compound formation, it was a static quenching process. The interaction association constants of BSA and sinafloxacin were determined from the double reciprocal Lineweaver-Burk plot. The transfer efficiency of energy and distance are obtained in view of the theory of F(o|¨)rster energy transfer. From thermodynamic coordination it can be judged that the binding force between sinafloxacin and BSA is mainly electro-static force. The effect of sinafloxacin on the conformation of BSA was analyzed by synchronous fluorescence spectrometry and three-dimensional fluorescence spectrometry. The spectroscopy analysis shows that can sinafloxacin react with BSA and form a drug-protein compound. Furthermore, it can affect the configuration of the protein. The interaction between sinafloxacin and BSA in the presence of metal ions (Cu²⁺, Fe³⁺, Zn²⁺, Mg²⁺) was also studied. In the second part, calf thymus DNA has been selected for investigative object, and the binding characteristics and mechanism of sinafloxacin to DNA have been studied by fluorescence spectroscopy and UV-vis absorption spectroscopy. The variations in the spectroscopy characteristics of sinafloxacin in an aqueous medium upon addition of DNA were observed. The maximum absorption wavelength of sinafloxacin did not exhibit red-shift and the absorption has little change in intensity and shape after adding DNA into the system. The fluorescence intensity of sinafloxacin at 469nm is decreased dramatically, but there is no shift in the emission wavelength. The results show that the Stern-Volmer quenching constant K_SV is inversely correlated with temperature. It was proved that the fluorescence quenching of sinafloxacin by DNA is a result of the formation of sinafloxacin-DNA complex. Quenching constants were determined using the Stern-Volmer equation to provide a measure of the binding affinity between sinafloxacin and DNA .Salt concentration effect, KI quenching, fluorescence polarization, DNA denaturation experiments, melting temperature (T_m) curves and viscosity measurements were also carried out to investigate the binding mechanism. In our experiments, the fluorescence of sinafloxacin -DNA system in a gradual increasing of NaCl concentration was studied and the results showed that the fluorescence intensity ratio had no significant change when the concentration of NaCl was within 0-0.2M range, which indicated that the interaction between sinafloxacin and DNA was not surface-binding mode. The experiments showed that single stranded DNA (ssDNA) could quench the fluorescence of sinafloxacin linearly, and the quenching of the fluorescence from sinafloxacin by double stranded DNA (dsDNA) was a little smaller than that by ssDNA under the same conditions. Fluorescence polarization measurements were performed on steady state sinafloxacin-DNA solutions. Only a slight increase was obtained with the addition of DNA. In aqueous solutions, iodide quenched the fluorescence of sinafloxacin efficiently. The K_SV values between sinafloxacin and iodide anions with the presence of DNA decreased slightly, which indicated that sinafloxacin could be partly protected. The addition of sinafloxacin caused no appreciable the melting temperature(T_m) and viscosity change in our experiments. From the experiment evidences, the major binding mode of sinafloxacin with DNA was evaluated to be the groove binding. The influence of Mg²⁺ on the binding between sinafloxacin and DNA was studied. The spectroscopy analysis shows that Mg²⁺ and DNA both can quench the endogenous fluorescence of sinafloxacin, and thefluorescence intensity of sinafloxacin quenched by DNA was aggravated in the presence of Mg²⁺. This means that there may be a ternary complex formed among Mg²⁺, sinafloxacin and DNA, Mg²⁺ plays a stimulative role as a bridge in the interaction between sinafloxacin and DNA.