Spectroscopic analysis of the impact of oxidative stress on the structure of human serum albumin (HSA) in terms of its binding properties

• Published in: Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy Molecular and biomolecular spectroscopy, 2015-02, Vol.136, p.265-282
• Main Authors: Maciążek-Jurczyk, M., Sułkowska, A.
• Format: Article
• Language: English
• Subjects: Chloramine-T; Drug–drug–protein binding; Ellman’s reagent; Oxidation; Spectroscopy
• ISSN: 1386-1425
• DOI: 10.1016/j.saa.2014.09.034

[Display omitted] •We investigated the influence of oxidative stress on HSA structure.•HSA was oxidized by a chloramine-T (CT).•We evaluated of possible alterations in the binding of the drug to oHSA.•Modification of free thiol group in HSA was determined by the use of Ellman’s reagent. Oxygen metabolism has an important role in the pathogenesis of rheumatoid arthritis (RA). Reactive oxygen species (ROS) are produced in the course of cellular oxidative phosphorylation and by activated phagocytic cells during oxidative bursts, exceed the physiological buffering capacity and result in oxidative stress. ROS result in oxidation of serum albumin, which causes a number of structural changes in the spatial structure, may influence the binding and cause significant drug interactions, particularly in polytherapy. During the oxidation modification of amino acid residues, particularly cysteine and methionine may occur. The aim of the study was to investigate the influence of oxidative stress on human serum albumin (HSA) structure and evaluate of possible alterations in the binding of the drug to oxidized human serum albumin (oHSA). HSA was oxidized by a chloramine-T (CT). CT reacts rapidly with sulfhydryl groups and at pH 7.4 the reaction was monitored by spectroscopic techniques. Modification of free thiol group in the Cys residue in HSA was quantitatively determined by the use of Ellman’s reagent. Changes of albumin conformation were examined by comparison of modified (oHSA) and nonmodified human serum albumin (HSA) absorption spectra, emission spectra, red-edge shift (REES) and synchronous spectroscopy. Studies of absorption spectra indicated that changes in the value of absorbance associated with spectral changes in the region of 200–250nm involve structural alterations in peptide backbone conformation. Synchronous fluorescence spectroscopy technique confirmed changes of position of tryptophanyl and tyrosyl residues fluorescent band caused by CT. Moreover analysis of REES effect allowed to observe structural changes caused by CT in the region of the hydrophobic pocket containing the tryptophanyl residue. Effect of oxidative stress on binding of anti-rheumatic drugs, sulfasalazine (SSZ) and sulindac (SLD) in the high and low affinity binding sites was investigated by spectrofluorescence, ITC and 1H NMR spectroscopy, respectively. SSZ and SLD change the affinity of each other to the binding site in non- and modified human serum albumin. The presence of SLD causes t