Journal of Clinical and Cellular Immunology
Open Access

Abstract

Relevance of Nitroxidation of Albumin in Rheumatoid Arthritis: A Biochemical and Clinical Study

Zarina Arif, Mir Yasir Arfat, Jamal Ahmad, Asif Zaman, Shireen Naaz Islam and M Asad Khan

Objective: To study the role of peroxynitrite-modified human serum albumin (nitroxidized-albumin) in rheumatoid arthritis. Methods: Human serum albumin was exposed to peroxynitrite and changes in albumin structure were monitored by UV-visible, fluorescence and circular dichroism spectroscopy, thioflavin T, Congo red binding and attenuated total reflection-Fourier transformed infrared spectroscopy (ATR-FTIR). Antioxidant properties of nitroxidized-albumin were evaluated by free radical induced RBC hemolysis test. Markers of protein oxidation like carbonyl, thiol, dityrosine and RBC hemolysis were evaluated in RA patients’ sera. Binding of autoantibodies in RA sera (n=50) with nitroxidized-albumin was studied by direct binding, inhibition ELISA and electrophoretic mobility shift gel assay. Results: The nitroxidized-albumin indicated the generation of nitrotyrosine, nitrotryptophan, carbonyl, dityrosine and reduction in tyrosine and tryptophan fluorescence and α-helicity. Fluorescence emission intensities of thioflavin T and Congo red got augmented upon binding with nitroxidized-albumin. Furthermore, secondary and tertiary structures of nitroxidized-albumin were altered as evident by ATR- FTIR, far and near-UV CD. Autoantibodies in RA sera (or IgG purified from sera) showed enhanced binding with nitroxidized-albumin as determined by direct binding and inhibition ELISA. Protein carbonyls, dityrosine and RBC hemolysis were significantly high, but thiol was significantly low in RA sera compared to age- and sex- matched control. Conclusion: Endogenously available peroxynitrite can nitrate and oxidize albumin, leading to protein nitration/ oxidation and subsequent formation of crosslinks, aggregates and immunogenic nitroxidized-albumin. Therefore, nitroxidized-albumin may be a potential trigger for autoantibodies in RA patients.