PROTEIN OXIDATION AND AGING .1. DIFFICULTIES IN MEASURING REACTIVE PROTEIN CARBONYLS IN TISSUES USING 2,4-DINITROPHENYLHYDRAZINE

A current hypothesis explaining the aging process implicates the accum ulation of oxidized protein in animal tissues. This hypothesis is base d on a series of reports showing an age-dependent increase in protein carbonyl content and an age-dependent loss of enzyme function, This hy pothesis is also supported by the report of a novel effect of N-tert-b utyl-a-phenylnitrone (PEN) in reversing these age-dependent changes. H ere we specifically study the method that was used to measure reactive protein carbonyls in tissues. This method uses 2,4-dinitrophenylhydra zine (DNPH) and includes a washing procedure. Our results indicate tha t reactive protein carbonyls in normal crude tissue extracts cannot be reliably measured by this method, although it does reliably measure r eactive carbonyls in purified proteins which have been oxidatively mod ified in vitro. The nucleic acids in tissues could be a major problem encountered in the assay. Using the streptomycin sulfate treatment com bined with a dialysis step, we were successful in removing most nuclei c acids from a crude tissue extract, but then the reactive carbonyl le vel in the crude tissue extract was too low to be reliably measured. T his streptomycin sulfate treatment procedure, however, had no effect o n the reactive carbonyl measurement of an oxidized protein sample. The unwashed free DNPH was another major problem in the assay because of its very strong absorption around 370 nm, where reactive carbonyls wer e quantitated. Nevertheless, on using the procedure described in the L iterature to measure total ''reactive carbonyls'' in rat liver and ger bil brain cortex, no change with age or PEN treatment was found. Then, we investigated a HPLC procedure which uses sodium dodecyl sulfate in the mobile phase but this was also found to be unsuitable for the rea ctive protein carbonyl assay in tissues. (C) 1995 Academic Press, Inc.