Protein oxidation and aging. II. Difficulties in measuring alkaline protease activity in tissues using the fluorescamine procedure.

A current hypothesis explaining the aging process implicates the accumulation of oxidized protein in animal tissues. This is primarily based on a series of reports showing an age-dependent increase in protein carbonyl content and an age-dependent decrease in the activities of enzymes, especially of alkaline proteases, which preferentially degrade oxidatively modified protein. Recently, this hypothesis was strongly supported by the report of a novel effect of the spin-trapping compound N-tert-butyl-alpha-phenylnitrone (PBN) in reversing these age-dependent changes. However, we found that the reactive protein carbonyls could not be reliably measured in tissues by using the 2,4-dinitrophenylhydrazine procedure described in the PBN study. We now focus on the alkaline protease activity assay and show that alkaline protease activity cannot be reliably measured in crude tissue extracts by using the fluorescamine procedure also described in the PBN study. We were, however, able to reliably measure a protease activity in crude tissue extracts at alkaline pH by using a synthetic fluorogenic peptide substrate, but no effect of aging or PBN treatment was found on the protease activity in rat brain cortexes. Thus, the reported age-dependent changes in protein carbonyl formation and alkaline protease activity remain to be confirmed.