Study On The Interaction Between New Environmental Pollutants And Human Serum

In recent years, pharmaceuticals and perfluorinated compounds, as two representative classes of the new emerging contaminants, have attracted much concern for their global distribution and potential threats to the environment and humans. Most pollutants have great potential to interact with serum proteins after entering into body, which could result in the alteration of protein’s structure and function, and thus induce the damages to cells, or a few pollutants may be transported to other organ and injure the organ by way of serum proteins. Because the interaction between pollutants and serum albumin determines the distribution within the body of pollutants and their characterization of biological toxicity, it becomes a research focus to study the interaction mechanism of pollutants and serum albumin.In this paper, we established a new strategy to probe the binding modes of doxycycline (DC, tetracyclines), cefodizime (CEF, cephalosporin) and perfluorononanoic acid (PFNA), perfluoroundecanoic acid (PFUnA) and perfluorotridecanoic acid (PFTriA) with human serum albumin (HSA) in physiological solution by multi-spectroscopic methods and molecular docking at a molecular level. Furthermore, comparing with existing research on their chemical cousin, then we analyzed the interaction law of three classes of pollutants with HSA.The binding of DC to HSA was analyzed by means of multi-spectroscopy and molecular modeling to obtain the accurate binding constants, the number of binding sites and binding distance and so on. The results showed that DC was bound at subdomain IIA of HSA and there was only one binding site. According to thermodynamic results (ΔH=-83.55kJ·mol-1, ΔS=-176.31J·mol-1·K-1), the interaction of DC with HSA was driven mainly by hydrogen bond and van der Waals force. The three dimensional fluorescence and synchronous fluorescence showed that the hydrophobicity of Trp and Tyr residues microenvironment increased and the conformation of the polypeptide backbone came loose after its complexation with DC, and the alternations of α-helix content were quantitatively calculated from FT-IR from 57.3% to 40.0%. Furthermore, molecular docking offered the specific chemical groups at a molecular level and explanation with their interactions in complex stabilization. Summarizing and analyzing the existing researches on the binding tetracyclines to serum albumin (SA), we discovered that the interactions of semisynthetic tetracyclines such as DC, rolitetracycline (RTC), demeclocycline (DMCT) and methacycline (METC) with SA were obvious stronger than natural tetracycline (TC), oxytetracycline (OTC) and chlorotetracycline (CTC). And the thermodynamic and molecular docking results showed that DC, RTC, DMTC and METC were bound in hydrophobic pocket of subdomain IIA in SA mainly through hydrogen bond and van der Waals force, while TC, OTC and CTC were mainly driven by electrostatic effect to bind at subdomain IIIA of SA. Furthermore, we discovered that all seven tetracyclines could make the conformation of SA extend and hydrophobicity of microenvironment increase.The interaction between CEF and HSA was studied under the simulative physiological conditions. On the basis of the thermodynamic results, site marker competitive experiments and molecular docking, it was considered that CEF was bound to site I (subdomain IIA) of HSA mainly by hydrogen bonds and van der Waals force, as well as hydrophobic effect, polar forces, etc. The calculated binding distance (r=3.3nm) indicated that the non-radioactive energy transfer came into being in the interaction between CEF and HSA. In addition, three-dimensional fluorescence results revealed that the binding of CEF can lead to HSA backbone conformational changes and hydrophobicity of Trp and Tyr residues microenvironment decreases. And circular dichroism (CD) spectra exhibited an increase of α-helix content with addition of CEF. Comparing CEF (third generation) with previous studies of ceftriaxone (CRO)(third generation), cefotaxime (CFT)(third generation), cefuroxime axetil (CFA)(second generation) and cefazolin (CFZ)(first generation) and analyzing, we discovered that the order of binding constant with SA was CEF～CRO> CFT～CFA> CFZ. CFA bound to SA mainly through electrostatic force while other substances interacted with SA by hydrogen bonds and van der Waals force, but all interactions increased the polarity of chromophores microenvironment. Molecular docking indicated that the specific binding of CEF, CFZ and CRO was located in the vicinity of site I (subdomain IIA) of HSA mainly through hydrogen bond.This study was designed to examine the interaction of PFNA, PFUnA and PFTriA with HSA by multi-spectroscopy, molecular docking and isothermal titration calorimetry (ITC), and then compared with previous researches of homologue perfluorooctanoic acid (PFOA) and perfluorodecanoic acid (PFDA). The fluorescence quenching results revealed that the binding mechanisms of three perfluorocarboxylic acids with HSA were static quenching as well as dynamic quenching and Foster non-radioactive energy transfer. Through site marker competitive experiments and molecular docking, subdomain IIA of HSA had been assigned to possess the high-affinity binding site of PFNA, PFUnA and PFTriA through mutiple forces, such as polar force, hydrophobic interaction and halogen-bond. Compared with previous ITC research on the interaction of PFOA with HSA, PFNA of this paper first bound with polar amino acid residues on the hydrophilic surface of HSA by electrostatic force and halogen-bond, then inserted itself into the interior of the hydrophobic part of protein molecule by hydrophobic interaction; while PFOA was exactly opposite, first binding with amino acid residues of hydrophobic cavity by halogen-bond, then polar amino acid residues of hydrophilic surface. As further revealed by three-dimensional fluorescence, synchronous fluorescence and CD spectra, the binding of PFNA, PFUnA and PFTriA with HSA can cause polypeptide backbone loose and α-helical stability decrease.