Investigation On The Binding Interaction Of Sulfonamides With Human Serum Albumin By Spectroscopic Technique

Sulfonamides that possess a p-aminobenzenesulfonamide framework are a common class of antibiotics used as antimicrobial agents to treat human and animal infections, which have the advantages of a broad spectrum antibiotic, more effective,convenience and safety. Sulfamethoxazole(SMZ) is a broad-spectrum antibiotics used to treat a variety of infectious diseases of livestock and poultry. Until now, it is still commonly used on clinical and veterinary medicine, which often combined with levofloxacin used to improve efficacy. In recent decades, it has been reported that some sulfa drugs, such as sulfamethizole(STZ), sulfisoxazole(SIZ) and sulfachlorpyridazine(SCP), have significant side effects. Because of its low price,character stability and high-efficiency, they are still widely used in animal husbandry and aquaculture. Abuse of sulfonamides or insufficient biodegradation, it will lead to the accumulation of these drugs in animal tissues, which may also cause allergy, carcinogenesis and formation of resistant bacteria in the human body and finally against to human health.Human serum albumin(HSA), being one of the most abundant proteins in human plasma, could combined with various exogenous and endogenous substances,which could affect ligand absorption, transport, distribution and metabolic processes in vivo.In this study, HSA was chosen as protein model, properties of three sulfonamides binding to human serum albumin and theirs effect on the structure of the protein were investigated with the application of multiple spectroscopy methods and molecular simulation. Besides, to research the synergy between SMZ and levofloxacinthe(LVFX), the binding interaction between SMZ or/and LVFX and HSA was investigated. The present work is expected to provide some insights into the distribution, transporting, metabolism and toxicity effect or efficacy of sulfonamides in body from the molecular level.The main contents are as follows:1. A simple introduction about the structure, physiological function and biological properties of protein was presented in the first chapter. Besides, theinvestigating methods about the interaction between small molecules and protein were showed in this chapter.2. The binding interaction between STZ and HSA was investigated by fluorescence spectroscopy, UV–vis absorption,circular dichroism(CD) spectroscopy and Fourier transform infrared(FT–IR) under simulative physiological conditions(pH 7.4). The results of fluorescence titration revealed that the mechanism of the fluorescence quenching of HSA was a static quenching procedure along with the formation of a SCP–HSA complex, and the interaction between STZ and HSA was mainly driven by hydrogen bonds and van der Waals forces. The binding constants Ka reached 105 L·mol-1, the number of binding sites was approximately equal to 1.The site marker displacement experiments suggested that the location of STZ binding to HSA was Sudlow’s site I in subdomain IIA. Analysis of UV–vis absorption, synchronous fluorescence, 8-anilino-1-naphthalenesulfonic acid(ANS)fluorescence, three-dimensional fluorescence, CD and FT–IR spectra demonstrated that the addition of STZ resulted in the conformational alteration of HSA with increase in the content of α-helix and β-turn, and decrease in β-sheet and random coil content. The determination of protein surface hydrophobicity(PSH) indicated that STZ binding to HSA caused an increase in the PSH.3. To probe the binding mechanism of SIZ or SCP with HSA and the protein conformational changes, the interaction between SCP and HSA was investigated under simulative physiological conditions(pH 7.4) by multispectroscopic methods including fluorescence, UV–vis absorption, Fourier transform infrared(FT–IR) and circular dichroism(CD) spectroscopy, coupled with molecular modeling technique.Fluorescence titration demonstrated that the mechanism of the fluorescence quenching of HSA was a static quenching procedure along with the formation of a SIZ–HSA or SCP–HSA. And the binding process was driven mainly by hydrogen bonds and van der Waals force, and the binding affinity of SIZ to HSA was slightly stronger than SCP. The primary binding site of SIZ or SCP in HSA(site I in subdomain IIA.) had been identified by site marker displacement experiments.Furthermore, the molecular docking results demonstrated the specific binding mode and binding site. Analysis of UV–vis absorption, synchronous fluorescence, ANSfluorescence, Fourier transform infrared(FT–IR) and circular dichroism(CD)spectra demonstrated that the addition of SIZ or SCP induced small conformational and micro-environmental changes in the structure of HSA. In addition, fluorescent phase diagram technical analysis, the changes in the SCP–HSA reaction conformational pattern showed a " two-state" model. The effect of SIZ on the protein structure was more remarkable than SCP.4. The synergy between SMZ and LVFX with HSA was researched in simulative physiological buffer(pH 7.4) by fluorescence, UV–vis absorption and molecular modeling technique. The results revealed that the fluorescence quenching of HSA by SMZ or LVFX were considered as a static quenching process. And the interaction between SMZ or LVFX and HSA were mainly driven by hydrogen bonds and hydrophobic force. The binding of SMZ or LVFX to HSA mainly took place in site I. The combinative ability for HSA-LVFX was greater than HSA-SMZ, and that for two-coexistin SMZ and LVFX with HSA was less than single with that.Analysis of synchronous fluorescence, CD spectra and determination of protein surface hydrophobicity(PSH) demonstrated that the addition of SMZ or LVFX resulted in the conformational alteration of HSA with a decrease in the content ofα-helix, which suggested a partial unfolding of the polypeptide chain of the protein and a increase in the PSH in the presence of SMZ. Therefore, data indicated that two-coexisting SMZ and LVFX may lead to the synergy.