IMMUNOCHEMICAL DETECTION OF OXIDIZED PROTEINS

An immunochemical assay was developed to detect carbonyl moieties that result from oxidative damage to proteins. Bovine serum albumin was re acted with hydroxyl radicals generated via a Fenton-like mechanism or by a radiolysis mechanism. The resulting albumin-derived carbonyls wer e reacted with 2,4-dinitrophenylhydrazine, giving the corresponding hy drazones, which were detected by Western blot using anti-dinitrophenyl antisera. The immunoblot demonstrated a concentration-dependent incre ase in carbonyl formation, as well as fragmentation of the albumin int o two distinct bands with molecular masses of 51 and 45 kDa when oxidi zed with the Fenton-like mechanism, and 62 and 46 kDa when oxidized by radiolysis. Analysis of the immunoblot using laser densitometry indic ated a linear relationship between carbonyl groups and increasing trea tment from radiolysis. This immunochemical assay was approximately 3 o rders of magnitude more sensitive than the spectrophotometric method a nd was able to determine the molecular mass of carbonyl-modified polyp eptides in the detection of oxidative damage.