Hydrophobicity as the signal for selective degradation of hydroxyl radical-modified hemoglobin by the multicatalytic proteinase complex, proteasome.

Red blood cells (RBC) and many other cell types exhibit increased rates of proteolysis during exposure to oxygen radicals and other activated oxygen species (oxidative stress). One of the major RBC proteins modified and proteolytically degraded during oxidative stress is hemoglobin (Hb). We now show that Hb undergoes a partial unfolding (or denaturation) during exposure to hydroxyl radicals (.OH), with an increase in hydrophobicity (hydrophobic interaction chromatography). At low .OH/Hb molar ratios, oxidatively modified Hb exhibits increased proteolytic susceptibility during incubation with RBC lysates, cell-free extracts, Fraction II, a 40-80% (NH4)2SO4 fraction, and purified proteasome (the 670-kDa RBC multicatalytic proteinase complex that we have previously called macroxyproteinase. At higher .OH/Hb molar ratios covalent cross-linking between Hb tetramers, and decreased proteolytic susceptibility are observed. The selective degradation of .OH-modified Hb is an ATP- and ubiquitin-independent process (in fact ATP is slightly inhibitory), and antibody precipitation studies, as well as inhibitor studies, indicate that proteasome is responsible for at least 60-70% of the activity in RBC. We propose that the mechanism of oxidation-induced proteolysis involves exposure of hydrophobic amino acid R groups during the partial Hb unfolding (or partial denaturation) that occurs at relatively low .OH/Hb molar ratios. Peptide bonds flanked by hydrophobic residues are preferred substrates for the proteasome complex, which degrades .OH-modified Hb in a processive process involving apparent serine-protease, sulfhydryl-protease, and metallo-peptidase activities. Highly denatured and covalently cross-linked Hb molecules, produced at high .OH/Hb molar ratios, are poorly degraded in RBC lysates and at all stages of proteasome purification. These cross-linked Hb tetramers have molecular sizes of 120-180 kDa and are presumably too large to fit in the proteasome active site(s). Recognition of exposed hydrophobic amino acid R groups provides a simple, energy-independent, and universal explanation for the proteasome-dependent proteolysis that accompanies oxidative stress.