title: Alteration of methotrexate binding to human serum albumin induced by oxidative stress. Spectroscopic comparative study.
abstract: Changes of oxidative modified albumin conformation by comparison of non-modified (HSA) and modified (oHSA) human serum albumin absorption spectra, Red Edge Excitation Shift (REES) effect and fluorescence synchronous spectra were investigated. Studies of absorption spectra indicated that changes in the value of absorbance associated with spectral changes in the region from 200 to 250nm involve structural alterations related to variations in peptide backbone conformation. Analysis of the REES effect allowed for the observation of changes caused by oxidation in the region of the hydrophobic pocket containing the tryptophanyl residue. Synchronous fluorescence spectroscopy confirmed changes of the position of the tryptophanyl and tyrosil residues fluorescent band. Effect of oxidative stress on binding of methotrexate (MTX) was investigated by spectrofluorescence, UV-VIS and (1)HNMR spectroscopy. MTX caused the fluorescence quenching of non-modified (HSA) and modified (oHSA) human serum albumin molecule. The values of binding constants, Hill's coefficients and a number of binding sites in the protein molecule in the high affinity binding site were calculated for the binary MTX-HSA and MTX-oHSA systems. For these systems, qualitative analysis in the low affinity binding sites was performed with the use of the (1)HNMR technique.
authors.affiliation: School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia, Chair and Department of Physical Pharmacy, 41-200 Sosnowiec, Jagiellońska 4, Poland. Electronic address: mmaciazek@sum.edu.pl.
authors.forename: M
authors.initials: M
authors.lastname: Maciążek-Jurczyk
authors.affiliation: School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia, Chair and Department of Physical Pharmacy, 41-200 Sosnowiec, Jagiellońska 4, Poland.
authors.forename: A
authors.initials: A
authors.lastname: Sułkowska
authors.affiliation: School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia, Chair and Department of Physical Pharmacy, 41-200 Sosnowiec, Jagiellońska 4, Poland.
authors.forename: J
authors.initials: J
authors.lastname: Równicka-Zubik
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000378:metabolism*; D006801:Humans; D008727:Methotrexate / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects*; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D066244:Proton Magnetic Resonance Spectroscopy; D012709:Serum Albumin / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D000964:Antimetabolites, Antineoplastic; D012709:Serum Albumin; D008727:Methotrexate
keywords: Fluorescence quenching; Human serum albumin oxidation; Methotrexate–human serum albumin interaction; Nuclear magnetic resonance study