PROTEIN OXIDATION AND AGING .2. DIFFICULTIES IN MEASURING ALKALINE PROTEASE ACTIVITY IN TISSUES USING THE FLUORESCAMINE PROCEDURE

A current hypothesis explaining the aging process implicates the accum ulation of oxidized protein in animal tissues. This is primarily based on a series of reports showing an age-dependent increase in protein c arbonyl content and an age-dependent decrease in the activities of enz ymes, especially of alkaline proteases, which preferentially degrade o xidatively modified protein. Recently, this hypothesis was strongly su pported by the report of a novel effect of the spin-trapping compound N-tert-butyl-alpha-phenylnitrone (PEN) in reversing these age-dependen t changes. However, we found that the reactive protein carbonyls could not be reliably measured in tissues by using the 2,4-dinitrophenylhyd razine procedure described in the PBN study. We now focus on the alkal ine protease activity assay and show that alkaline protease activity c annot be reliably measured in crude tissue extracts by using the fluor escamine 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 activit y in rat brain cortexes. Thus, the reported age-dependent changes in p rotein carbonyl formation and alkaline protease activity remain to be confirmed. (C) 1995 Academic Press, Inc.