title: Exposure of hydrophobic moieties promotes the selective degradation of hydrogen peroxide-modified hemoglobin by the multicatalytic proteinase complex, proteasome.
abstract: The physiologically relevant stress of a flux of H2O2 increased hemoglobin (Hb) degradation in red blood cells (RBC) and increased the proteolytic susceptibility of Hb in vitro. After exposure to low H2O2 flux rates (6-32 microM/min) Hb exhibited increased exposure of hydrophobic (Trp, Met) and basic (Lys) amino acid R groups, increased hydrophobicity, and increased proteolytic susceptibility during subsequent incubation with RBC extracts, a partially purified preparation called Fraction II (which retains all of the proteolytic activities of RBC extracts), or the purified 670-kDa RBC multicatalytic proteinase complex proteasome. Hydrophobicity was measured by butyl-Sepharose hydrophobic interaction chromatography, by the free energy of transfer from water to ethanol, and by heat denaturation assays. Proteolytic susceptibility was measured by release of free alanine, by fluorescamine-reactive free amino groups, and by release of acid-soluble radioactivity from radiolabeled Hb. Low H2O2 flux rates also caused significant charge changes in Hb (isoelectric focusing gels) and extensive noncovalent aggregation (presumably due to increased hydrophobic interactions) but only limited covalent cross-linking (comparison of sodium dodecyl sulfate-polyacylamide gel electrophoresis (SDS-PAGE) and nondenaturing PAGE). Exposure to higher H2O2 flux rates (56-120 microM/min) caused progressive oxidative destruction of exposed hydrophobic amino acids, decreased hydrophobicity as judged by butyl-Sepharose chromatography and heat denaturation assays, increased hydrophilicity as judged by measurements of the free energy of transfer (delta G') from water to ethanol, and decreased proteolytic susceptibility during incubation with RBC extracts, Fraction II, or purified proteasome. High H2O2 flux rates also caused further charge changes and the extensive formation of covalently cross-linked Hb molecules. Linear regression analyses revealed correlations of 0.8-0.99 for the relationship between Hb hydrophobicity and proteolytic susceptibility for both Fraction II and proteasome. Inhibitor studies and SDS activation experiments indicate that proteasome is responsible for most of the Hb degradation during exposure of RBC to H2O2. Previous work yielded essentially identical conclusions for Hb exposed to hydroxyl radicals (R. E. Pacifici, Y. Kono, and K. J. A. Davies, J. Biol. Chem. 268, 15405-15411, 1993). Thus, nonspecific oxidation by .OH and site-specific (metal-catalyzed) oxidation by H2O2 both yield a more hydrophobic Hb molecule with increased proteolytic susceptibility. We propose that increased exposure of hydrophobic, and perhaps basic, amino acids is the general common cause for degradation of oxidized proteins.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Institute for Toxicology, University of Southern California, Los Angeles 90033.
authors.forename: C
authors.initials: C
authors.lastname: Giulivi
authors.forename: R E
authors.initials: RE
authors.lastname: Pacifici
authors.forename: K J
authors.initials: KJ
authors.lastname: Davies
mesh_terms: D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D005949:Glucose Oxidase / Q000494:pharmacology; D006454:Hemoglobins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007525:Isoelectric Focusing; D007700:Kinetics; D009097:Multienzyme Complexes / Q000378:metabolism*; D010108:Oxyhemoglobins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011489:Protein Denaturation
chemical_list: D006454:Hemoglobins; D009097:Multienzyme Complexes; D010108:Oxyhemoglobins; D006861:Hydrogen Peroxide; D005949:Glucose Oxidase; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex