부산대학교 도서관

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.freeradbiomed.2005.06.023 Byline: Daryoush Hamidi Alamdari (a), Elena Kostidou (a), Konstantinos Paletas (b), Maria Sarigianni (b), Anastasios G.P. Konstas (c), Alexia Karapiperidou (a), George Koliakos (a) Keywords: Oxidative stress; Protein modification; Carbonylation; ELISA method; Diabetes; Aqueous humor; Free radical Abbreviations: ROS, reactive oxygen species; DNPH, dinitrophenylhydrazine; ELISA, enzyme-linked immunosorbent assay; TCA, trichloroacetic acid; PBS, phosphate-buffered saline; BSA, bovine serum albumin; TMB, 3,3,5,5-tetramethylbenzidine; OPD, o-phenylenediamine; HRP, horseradish peroxidase Abstract: The oxidative modification of proteins has been shown to play a major role in a number of pathological processes. One such modification is the addition of the carbonyl groups to the amino acid residue in proteins. For the measurement of the carbonyl groups in low concentration protein samples, we have modified the ELISA (enzyme-linked immunosorbent assay) method that was developed by Buss et al. [Buss, I. H; Chan, T. P.; Sluis, K. B.; Domigan, N. M.; Winterbourn, C. C. Protein carbonyl measurement by a sensitive ELISA method. Free Radic. Biol. Med. 23:361-366; 1997 ]. In the modified method, protein samples diluted in phosphate-buffered saline were adsorbed to wells of an ELISA plate and then reacted with dinitrophenylhydrazine (DNPH). The protein-conjugated DNPH was probed by a commercial anti-DNPH antibody, and then a second antibody conjugated with horseradish peroxidase was added for quantification. The method was calibrated using oxidized albumin, and required only 5 [mu]g protein. This obviated the need to concentrate protein in experimental and clinical samples with low amounts of protein. In addition the effect of TCA on carbonyl measurement is eliminated. The standard curve was linear in the range of 0-3.36 nmol carbonyls/mg protein, which is the range within which clinical samples fell. The results correlated well with the colorimetric carbonyl assay. The method was used to analyze the amount of protein carbonyl in aqueous humor and diluted plasma samples. Author Affiliation: (a) Department of Biological Chemistry, Medical School, Aristotle University of Thessaloniki, P.O. Box 17034 54210, 54124 Thessaloniki, Greece (b) Laboratory for the Study of Metabolic Diseases, B' Medical Clinic, Medical School, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece (c) A' University Dept. of Ophthalmology, AHEPA Hospital, Thessaloniki, Greece Article History: Received 5 May 2005; Revised 24 June 2005; Accepted 27 June 2005