DITYROSINE AND TYROSINE OXIDATION-PRODUCTS ARE ENDOGENOUS MARKERS FORTHE SELECTIVE PROTEOLYSIS OF OXIDATIVELY MODIFIED RED-BLOOD-CELL HEMOGLOBIN BY (THE 19-S) PROTEASOME

Cells exposed to oxidative stress have been shown previously to exhibi t both protein oxidation and increased proteolysis. Experiments conduc ted with purified proteins in vitro have indicated that oxidatively mo dified proteins may be selectively degraded by intracellular proteases , but a definitive cause-and-effect relationship has not been demonstr ated previously in intact cells. Several investigators have proposed t hat oxidatively modified proteins are selectively degraded within cell s, but the possibility that oxidants may activate intracellular protea ses (directly or indirectly) to catalyze the indiscriminate degradatio n of undamaged proteins has not been discounted. Armed with the knowle dge that dityrosine is a specific product of protein oxidation, we und ertook a series of experiments to test the hypothesis that oxidized pr oteins undergo selective intracellular degradation. Our results demons trate that dityrosine is produced in the hemoglobin molecule when red blood cells are exposed to a continuous flux of hydrogen peroxide (H2O 2). The dityrosine so produced is only released from the hemoglobin by proteolysis and is stable to prolonged incubation with cell extracts. Inhibitors of proteolysis have no effect on dityrosine production but do effectively prevent dityrosine release. Proteasome (the 670-kDa mu lticatalytic proteinase complex, that we have previously called macrox yproteinase or MOP (Pacifici, R. E., Salo, D. C., and Davies, K. J. A. (1989) Free Radical Biol. & Med. 7, 521-526; Salo, D. C., Pacifici, R . E., Lin, S. W., Giulivi, C., and Davies, K. J. A. (1990) J. Biol. Ch em. 265, 11919-11927; Pacifici, R. E., and Davies, K. J. A. (1991) Ger ontology 37, 166-180) appears responsible for dityrosine release durin g the selective degradation of oxidatively modified proteins in red bl ood cells and red cell extracts. We conclude that the elevated rates o f proteolysis observed in response to oxidative stress do, indeed, ref lect selective degradation of oxidatively modified (damaged) proteins. Despite a relatively low production rate, dityrosine has a high fluor ometric quantum yield and is, of course, a specific product of protein oxidation. As an apparently stable metabolic end product, dityrosine may prove to be an extremely valuable (cellular or urinary) marker or index of organismal oxidative stress.