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An immunochemical assay was developed to detect carbonyl moieties that result from oxidative damage to proteins. Bovine serum albumin was reacted with hydroxyl radicals generated via a Fenton-like mechanism or by a radiolysis mechanism. The resulting albumin-derived carbonyls were reacted with 2,4-dinitrophenylhydrazine, giving the corresponding hydrazones, which were detected by Western blot using anti-dinitrophenyl antisera. The immunoblot demonstrated a concentration-dependent increase in carbonyl formation, as well as fragmentation of the albumin into two distinct bands with molecular masses of 51 and 45 kDa when oxidized with the Fenton-like mechanism, and 62 and 46 kDa when oxidized by radiolysis. Analysis of the immunoblot using laser densitometry indicated a linear relationship between carbonyl groups and increasing treatment from radiolysis. This immunochemical assay was approximately 3 orders of magnitude more sensitive than the spectrophotometric method and was able to determine the molecular mass of carbonyl-modified polypeptides in the detection of oxidative damage.
Keller et al. (Thu,) studied this question.