Ring Technology Used In Antibacterial Drug Testing And Drug And Protein Interaction Mechanism Of Self-assembly

Based on the significance of determination of antimicrobial, the characteristic and application of self-ordered ring (SOR) fluorescent microscopic imaging technique, in this paper we developed the methods for the determination of tosufloxacin tosylate (TSFX), levofloxacin (LVFX) and lomefloxacin (LMX), and employed the developed methods to detect the content of above drugs in tablet, capsule and injecta, the content of TSFX in rabbit serum, LVFX in chiken serum, muscle, liver, manure and LMX in rabbit serum, muscle, liver and kidney after dosing, respectively. Besides, The interactions of TSFX and BSA, ATLL and BSA, NF and HSA were studied by fluorescence spectra, UV-vis absorption spectra and FT-IR spectra and molecular modeling methods.In the pH10.50NH3-NH4C1buffer solution and poly(vinyl alcohol)-124(PVA-124), with Mn2+and cetyltrimethyl ammonium bromide (CTMAB) as sensitizer, a highly sensitive and simple method is developed for the determination of TSFX by the SOR fluorescent microscopic imaging technique. When the droplet volume is0.2μL, TSFX in the range of4.05x10-14-4.28x10-13mol/ring (2.02x10-7-2.14x10-6mol/L) can be detected, and the limit of detection (LOD) can reach4.05x10-15mol/ring (2.02x10" mol/L). This developed methods was applied to determine the content of TSFX in the tablet and in the rabbit serum of different time after dosing. The recovery is90.0-105.0%and relative standard deviations (RSDs) are less than3.3%.Based on the SOR fluorescent microscopic imaging technique on a hydrophobic glass slide with Mn2+and CTMAB as sensitizer, PVA-124and NH3-NH4Cl buffer (pH9.30) as the medium, a method has been developed for determining LVFX residues in chicken muscle, chicken liver, chicken manure, the concentrations in chicken serum and the content of LVFX in tablet and capsule. When the droplet volume is0.2μL, LVFX in the range of5.66xl0-14-1.00x10-13mol/ring can be detected, and the limit of detection (LOD) can reach5.66x10-15mol/ring (2.83x10-8mol/L). It is a viable method for the determination of LVFX in these samples with the recoveries of90.0-105.0%and RSDs0.8-4.0%. The results indicate that the method applied to chicken tissue, manure and serum is reliable and applicable.With Al3+and CTMAB as sensitizer, PVA-124and NH3-NH4Cl (pH9.60) as the medium, a simple and sensitive SOR technique was successfully developed to determination LMX. When the droplet volume is0.2μL, LMX in the range of9.87x10-14-1.47x10-12mol/ring can be detected, and the detection limit can reach9.87x10-15mol/ring (4.93x10-8mol/L). This developed methods was applied to determination LMX content in pharmaceutic preparation(tablet, capsule and injecta), concentrations in rabbit serum and residues in rabbit tissue, urine. It was proved to be a viable method for analysis of LMX in those samples with the recoveries90.6-106.3%and RSDs less than4.2%.The interaction of tosufloxacin tosylate (TSFX) and bovine serum albumin (BSA) was studied by fluorescence spectroscopy, UV-vis spectroscopy and FT-IR spectroscopy. The results indicated that the intrinsic fluorescence of BSA was quenched by TSFX through a static quenching mechanism, and the effective binding constants (Kα) were obtained to be2.58x104L/mol (298K) by means of the modified Stern-Volmer equation. Thermodynamic parameters showed that electrostatic interaction was mostly responsible for the binding of TSFX to BSA. The binding distance (r) between TSFX and Trp-212was determined to be3.90nm according to Foster non-radiative energy transfer theory. BSA had a single class of binding site at Sudlow'sites I in subdomain IIA for TSFX. The effects of TSFX on the conformation of BSA were analyzed by synchronous fluorescence spectra and three-dimensional fluorescence spectra, and the results exhibited that the hydrophobicity of tryptophan microenvironment was decreased. In FT-IR spectra, Fourier self-deconvolution, secondary derivative and the curve-fitting process were carried out to obtain the components of BSA secondary structure. At298K when the molar ratio of TSFX to BSA changed from0:1to10:1, the a-helix decreased from48.5%to38.6%, β-sheet changed from23.3%to18.3%, while β-turn had an increase from15.3%to24.1%, random structure increased from12.9%to 19.0%. This indicated that TSFX induced unfolding of the polypeptides of BSA.The interaction of acetylisovaleryltylosin tartrate (ATLL) and bovine serum albumin (BSA) without or with Zn2+and Cu2+has been studied by fluorescence spectroscopy, FT-IR spectroscopy and UV-vis spectroscopy. The fluorescence of BSA was quenched by ATLL through a static quenching mechanism. The effective quenching constant (Ka) of ATLL to BSA decreased with Zn2+and increased with Cu2+. Thermodynamic parameters revealed that hydrogen bonds and hydrophobic forces played significant roles in the binding of ATLL to BSA. The polarity of tryptophan and tyrosine residues changed on addition of ATLL regardless with or without Zn2+and Cu2+. FT-IR spectra showed that ATLL changed a-helix and β-sheet of BSA into β-turn and random structure. And the adding of Zn2+and Cu2+further loosen the polypeptides of BSA. The UV-vis spectra indicated that the effects of Zn2+on ATLL binding to BSA may through a competition binding, and Cu2+possibly formed Cu2+-ATLL complex via metal ion bridge.The interaction of nitrofurazone (NF) and human serum albumin (HSA) has been studied by fluorescence spectroscopy, UV-vis spectroscopy, FT-IR spectroscopy and molecular modeling methods. The results showed that the fluorescence of HSA was quenched by NF in a static quenching mechanism. Thermodynamic parameters revealed that hydrogen bonds and van der Waals force played the major role during the interaction. The calculated binding distance (r) indicated that the non-radioactive energy transfer coming into being in the interaction between NF and Trp-214of HSA. HSA had a single class of binding site at Sudlow'sites I in subdomain IIA for NF, which was verified by the displacement experiment. The molecular modeling study further confirmed the specific binding sites of NF on HSA, such as the interaction between N11and Lue283; N14and Lue283, Ser287;07and Ser287; predominately through hydrogen bonds. Three-dimensional fluorescence spectra indicated that the polarity around the tryptophan residues decreased and the conformation of HSA changed after adding NF. FT-IR spectra showed that NF could induce the polypeptides of HSA unfolding because it changed α-helix and β-sheet into β-turn and random structure of HAS with the content of α-helix reducing froom54.2％to45.8%, β-sheet reducing froml8.5%to15.7%,β-turn increasing from21.7%to23.8%and random structure from5.6%to14.7%.