EXPOSURE OF HYDROPHOBIC MOIETIES PROMOTES THE SELECTIVE DEGRADATION OF HYDROGEN PEROXIDE-MODIFIED HEMOGLOBIN BY THE MULTICATALYTIC PROTEINASE COMPLEX, PROTEASOME

The physiologically relevant stress of a flux of H2O2 increased hemogl obin (Hb) degradation in red blood cells (REC) and increased the prote olytic susceptibility of Hb in vitro. After exposure to low H2O2 flux rates (6-32 mu M/ min) Hb exhibited increased exposure of hydrophobic (Trp, Met) and basic (Lys) amino acid R groups, increased hydrophobici ty, and increased proteolytic susceptibility during subsequent incubat ion with RBC extracts, a partially purified preparation called Fractio n II (which retains all of the proteolytic activities of RBC extracts) , or the purified 670-kDa RBC multicatalytic proteinase complex protea some. Hydrophobicity was measured by butyl-Sepharose hydrophobic inter action chromatography, by the free energy of transfer from water to et hanol, and by heat denaturation assays. Proteolytic susceptibility was measured by release of free alanine, by fluorescamine-reactive free a mino groups, and by release of acid-soluble radioactivity from radiola beled Hb. Low H2O2 flux rates also caused significant charge changes i n Rb (isoelectric focusing gels) and extensive noncovalent aggregation (presumably due to increased hydrophobic interactions) but only limit ed covalent cross-linking (comparison of sodium dodecyl suIfate-polyac ylamide gel electrophoresis (SDS-PAGE) and nondenaturing PAGE). Exposu re to higher H2O2 flux rates (56-120 mu M/min) caused progressive oxid ative destruction of exposed hydrophobic amino acids, decreased hydrop hobicity as judged by butyl-Sepharose chromatography and heat denatura tion assays, increased hydrophilicity as judged by measurements of the free energy of transfer (Delta G') from water to ethanol, and decreas ed proteolytic susceptibility during incubation with RBC extracts, Fra ction II, or purified proteasome. High H2O2 flux rates also caused fur ther charge changes and the extensive formation of covalently cross-li nked Hb molecules. Linear regression analyses revealed correlations of 0.8-0.99 for the relationship between Hb hydrophobicity and proteolyt ic susceptibility for both Fraction II and proteasome. Inhibitor studi es and SDS activation experiments indicate that proteasome is responsi ble for most of the Hb degradation during exposure of RBC to H2O2. Pre vious work yielded essentially identical conclusions for Hb exposed to hydroxyl radicals (R. E. Pacifici, Y. Kono, and K. J. A. Davies, J. B iol. Chem. 268, 15405-15411, 1993). Thus, nonspecific oxidation by OH and site-specific (metal-catalyzed) oxidation by H2O2 both yield a mor e hydrophobic Hb molecule with increased proteolytic susceptibility. W e propose that increased exposure of hydrophobic, and perhaps basic, a mino acids is the general common cause for degradation of oxidized pro teins. Since peptide bonds flanked by hydrophobic or basic amino acids are the preferred substrates for proteasome, our results indicate tha t partial protein unfolding is the key to increased proteolysis during oxidative stress. Covalently cross-linked protein aggregates, generat ed during extreme oxidative stress conditions, are suggested to be poo r substrates for proteolysis due to stearic hinderance of their intera ction with proteasome. (C) 1994 Academic Press, Inc.