Detection of carbonyl groups on oxidized proteins using an immunochemical technique.
Halmes-NC; Keller-RJ; Hinson-JA; Pumford-NR
Toxicologist 1993 Mar; 13(1):117
Chemical toxicants may produce active oxygen species that react with amino acids on proteins forming carbonyl groups and other oxidative products. 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 using hydrogen peroxide and vanadyl as the metal catalyst. The resulting albumin-derived carbonyls were reacted with 2,4-dinitrophenylhydrazine, giving the corresponding hydrazone. The carbonyl-dinitrophenyl groups were detected by Western blot using antidinitrophenyl antisera. Gels stained with Coomassie blue show a decrease in albumin intensity with increasing vanadyl concentration. The immunoblot demonstrated a concentration-dependent increase in carbonyl formation, as well as fragmentation of the albumin into two distinct bands with molecular weights of 51 kDa and 45 kDa. This indicates albumin has two sites that are highly susceptible to oxidative cleavage. In a parallel experiment, dinitrophenylhydrazone adducts were detected using an established spectrophotometric method. The spectrophotometric results were consistent with those of the Western blot, showing more carbonyl groups formed with increasing vanadyl concentration. However, this immunochemical assay was greater than three orders of magnitude more sensitive than the spectrophotometric method in the detection of oxidative damage.
Analytical-methods; Immunochemistry; Carbonyls; Protein-chemistry; Blood-serum; Oxidative-processes; Analytical-processes
Pulmonary System Disorders
The Toxicologist. Society of Toxicology 32nd Annual Meeting, March 14-18,1993, New Orleans, Louisiana
University of Arkansas Med Scis Ltl Rock, Little Rock, Arkansas