sepharose and 3-nitrotyrosine

IgG is an important defence protein. To exhibit optimum function the molecule must maintain its native structure. Peroxynitrite is a potent oxidizing and nitrating agent produced in vivo under pathophysiological conditions. It can oxidize and/or nitrate various amino acids causing changes in the structure and function of proteins. Such proteins may be involved in the pathogenesis of many inflammatory diseases, including rheumatoid arthritis. In the present work, peroxynitrite-induced structural changes in IgG have been studied by UV-visible, fluorescence, CD, FT-IR, DLS spectroscopy and DSC as well as by SDS-PAGE. Peroxynitrite-modified IgG exhibited hyperchromicity at 280 nm, quenching of tryptophan fluorescence, increase in ANS fluorescence, loss of β-sheet, shift in the positions of amide I and amide II bands, appearance of new peak in FT-IR, attachment of nitro residues and increase in melting temperature, compared to native IgG. Furthermore, peroxynitrite-modified IgG exhibited an additional peak at 420 nm, quenching in tyrosine fluorescence and enhancement in dityrosine fluorescence compared to native IgG. Generation of nitrotyrosine, dityrosine and nitrotryptophan was also observed in peroxynitrite-modified IgG. Gross structural changes in IgG caused by peroxynitrite and observed in vitro may favour autoantibodies induction in vivo under similar conditions.

Topics: Arthritis, Rheumatoid; Calorimetry, Differential Scanning; Circular Dichroism; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Humans; Immunoglobulin G; Inflammation; Light; Microscopy, Fluorescence; Oxygen; Peroxynitrous Acid; Protein Structure, Secondary; Scattering, Radiation; Sepharose; Spectrophotometry; Spectroscopy, Fourier Transform Infrared; Temperature; Tryptophan; Tyrosine

We recently developed a sensitive method using biotin-N-maleimide (biotin-NM) as a probe to positively identify oxidized mitochondrial proteins. In this study, biotin-NM was used to identify oxidized cytosolic proteins in alcohol-fed mouse livers. Alcohol treatment for 6 wk elevated the levels of CYP2E1 and nitrotyrosine, a marker of oxidative stress. Markedly increased levels of oxidized proteins were detected in alcohol-fed mouse livers compared to pair-fed controls. The biotin-NM-labeled oxidized proteins from alcohol-exposed mouse livers were subsequently purified with streptavidin-agarose and resolved on 2-DE. More than 90 silver-stained protein spots that displayed differential intensities on 2-D gels were identified by MS. Peptide sequence analysis revealed that many enzymes or proteins involved in stress response, chaperone activity, intermediary metabolism, and antioxidant defense systems such as peroxiredoxin were oxidized after alcohol treatment. Smaller fragments of many proteins were repeatedly detected only in alcohol-fed mice, indicating that many oxidized proteins after alcohol exposure were degraded. Immunoblot results showed that the level of oxidized peroxiredoxin (inactivated) was markedly increased in the alcohol-exposed mouse livers and ethanol-sensitive hepatoma cells compared to the corresponding controls. Our results may explain the underlying mechanism for cellular dysfunction and increased susceptibility to other toxic agents following alcohol-mediated oxidative stress.

Topics: Animals; Bacterial Proteins; Biomarkers, Tumor; Biotin; Carcinoma, Hepatocellular; Central Nervous System Depressants; Computational Biology; Cytochrome P-450 CYP2E1; Cytosol; Electrophoresis, Gel, Two-Dimensional; Ethanol; Liver; Liver Extracts; Liver Neoplasms; Male; Mice; Mice, Inbred C57BL; Oxidation-Reduction; Peroxidases; Peroxiredoxins; Sepharose; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tyrosine