Protein Oxidation in Aging, Disease, and Oxidative Stress

Abstract

The demonstration that oxidatively modified forms of proteins accumulate during aging, oxidative stress, and in some pathological conditions has focused attention on physiological and non-physiological mechanisms for the generation of reactive oxygen species (ROS) 1The abbreviations used are: ROS, reactive oxygen species; MeSOX, methionine sulfoxide reductase; PN, peroxynitrite; GS, glutamine synthetase. 1The abbreviations used are: ROS, reactive oxygen species; MeSOX, methionine sulfoxide reductase; PN, peroxynitrite; GS, glutamine synthetase. and on the modification of biological molecules by various kinds of ROS. Basic principles that govern the oxidation of proteins by ROS were established in the pioneering studies of Swallow (1Swallow A.J. Swallow A.J. Radiation Chemistry of Organic Compounds. Pergamon Press, New York1960: 211-224Google Scholar), Garrison (2Garrison W.M. Chem. Rev. 1987; 87: 381-398Crossref Scopus (632) Google Scholar, 3Garrison W.M. Jayko M.E. Bennett W. Radiat. Res. 1962; 16: 487-502Crossref Scopus (83) Google Scholar), and Scheussler and Schilling (4Schuessler H. Schilling K. Int. J. Radiat. Biol. 1984; 45: 267-281Crossref Scopus (199) Google Scholar) who characterized reaction products formed when proteins were exposed to ionizing radiation under conditions where only ⋅OH, O·̄2, or a mixture of both was made available. Results of these studies demonstrated that the modification of proteins is initiated mainly by reactions with⋅OH; however, the course of the oxidation process is determined by the availability of O2 and O·̄2 or its protonated form (HO⋅2). Collectively, these ROS can lead to oxidation of amino acid residue side chains, formation of protein-protein cross-linkages, and oxidation of the protein backbone resulting in protein fragmentation. In the meantime, it has been shown that other forms of ROS may yield similar products and that transition metal ions can substitute for ⋅OH and O·̄2 in some of the reactions. 2For review, see Donald C. Borg and Karen M. Schaich (1988) in Oxygen Radicals and Tissue Injury(Halliwell, B., ed) pp. 20–26, Proceedings of an Upjohn Symposium, Federation of American Societies for Experimental Biology, Bethesda, MD. 2For review, see Donald C. Borg and Karen M. Schaich (1988) in Oxygen Radicals and Tissue Injury(Halliwell, B., ed) pp. 20–26, Proceedings of an Upjohn Symposium, Federation of American Societies for Experimental Biology, Bethesda, MD.Oxidation of the Protein BackboneAs is illustrated in Fig. 1, oxidative attack of the polypeptide backbone is initiated by the⋅OH-dependent abstraction of the α-hydrogen atom of an amino acid residue to form a carbon-centered radical (Fig. 1, Reaction c). The ⋅OH needed for this reaction may be obtained by radiolysis of water or by metal-catalyzed cleavage of H2O2 (Reactions a and b). The carbon-centered radical thus formed reacts rapidly with O2 to form an alkylperoxyl radical intermediate (Reactiond), which can give rise to the alkylperoxide (Reactionf), followed by formation of an alkoxyl radical (Reactionh), which may be converted to a hydroxyl protein derivative (Reaction j). Significantly, many of the steps in this pathway that are mediated by interactions with HO⋅2 can be catalyzed also by Fe2+ (Reactions e, g, andi)2 or by Cu+ (not shown). The alkyl, alkylperoxyl, and alkoxyl radical intermediates in this pathway may undergo side reactions with other amino acid residues in the same or a different protein molecule to generate a new carbon-centered radical (Reaction 1) capable of undergoing reactions similar to those illustrated in Fig. 1.R1C⋅‖‖or R1OO⋅or R1O⋅+R2C‖‖H→R2C⋅‖‖+R1H or R1OOH or R1OHREACTION1Moreover, in the absence of oxygen, when Reactiond in Fig. 1 is prevented, the carbon-centered radical may react with another carbon-centered radical to form a protein-protein cross-linked derivative (Reaction 2).R1C⋅‖‖+R2C⋅‖‖→R1CCR2REACTION2Protein FragmentationThe generation of alkoxyl radicals (Fig. 1, Reactions hand g) sets the stage for cleavage of the peptide bond by either the diamide or α-amidation pathways. Upon cleavage by the diamide pathway (Fig. 2, Pathwaya), the peptide fragment derived from the N-terminal portion of the protein possesses a diamide structure at the C-terminal end, whereas the peptide derived from the C-terminal portion of the protein possesses an isocyanate structure at the N-terminal end. In contrast, upon cleavage by the α-amidation pathway (Fig. 2, Pathwayb), the peptide fragment obtained from the N-terminal portion of the protein possesses an amide group at the C-terminal end, whereas the N-terminal amino acid residue of the fragment derived from the C-terminal portion of the protein exists as anN-α-ketoacyl derivative. Upon acid hydrolysis, the peptide fragments obtained by the diamide pathway will yield CO2, NH3, and a free carboxylic acid, whereas hydrolysis of the fragment obtained by the α-amidation pathway yields NH3and a free α-ketocarboxylic acid.Figure 2Peptide bond cleavage by the (a) diamide and (b) α-amidation pathways.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Peptide bond cleavage can occur also as a result of ROS attack of glutamyl, aspartyl, and prolyl side chains. As described by Garrison (2Garrison W.M. Chem. Rev. 1987; 87: 381-398Crossref Scopus (632) Google Scholar), ⋅OH-dependent abstraction of a hydrogen atom from the γ-carbon atom of a glutamyl residue, followed by reactions analogous to Reactions d, f, and h in Fig. 1, will lead eventually to peptide bond cleavage by a mechanism in which oxalic acid is formed and the N-terminal amino acid of the peptide derived from the C-terminal portion of the protein will exist as anN-pyruvyl derivative (ReactionFR3).Figure FR3View Large Image Figure ViewerDownload Hi-res image Download (PPT)Based on the observation that the number of peptides formed during radiolysis of proteins is approximately equal to the number of prolyl residues, Schuessler and Schilling (4Schuessler H. Schilling K. Int. J. Radiat. Biol. 1984; 45: 267-281Crossref Scopus (199) Google Scholar) proposed that oxidation of prolyl residues would lead to peptide bond cleavage. This was verified by studies of Uchida et al. (5Uchida K. Kato Y. Kawakishi S. Biochem. Biophys. Res. Commun. 1990; 169: 265-271Crossref PubMed Scopus (111) Google Scholar) showing that oxidation of proline residues leads to the formation of 2-pyrrolidone and concomitant peptide bond cleavage (ReactionFR4). Because acid hydrolysis of 2-pyrrolidone yields 4-aminobutyric acid, the presence of 4-aminobutyric acid in protein hydrolysates is presumptive evidence for peptide bond cleavage by the proline oxidation pathway.Figure FR4View Large Image Figure ViewerDownload Hi-res image Download (PPT)Oxidation of Amino Acid Side ChainsAll amino acid residues of proteins are susceptible to oxidation by ⋅OH. However, the products formed in the oxidation of some residues have not been fully characterized. TableI lists some of the products formed during the oxidation of the residues that are most susceptible to oxidation.Table IAmino acids most susceptible to oxidationAmino acidsOxidation productsCysteineDisulfides, cysteic acidMethionineMethionine sulfoxide, methionine sulfoneTryptophan2-, 4-, 5-, 6-, and 7-Hydroxytryptophan, nitrotryptophan, kynurenine, 3-hydroxykynurinine, formylkynurininePhenylalanine2,3-Dihydroxyphenylalanine, 2-, 3-, and 4-hydroxyphenylalanineTyrosine3,4-Dihydroxyphenylalanine, tyrosine-tyrosine cross-linkages, Tyr-O-Tyr, cross-linked nitrotyrosineHistidine2-Oxohistidine, asparagine, aspartic acidArginineGlutamic semialdehydeLysineα-Aminoadipic semialdehydeProline2-Pyrrolidone, 4- and 5-hydroxyproline pyroglutamic acid, glutamic semialdehydeThreonine2-Amino-3-ketobutyric acidGlutamylOxalic acid, pyruvic acid Open table in a new tab Oxidation of Sulfur-containing Amino Acid ResiduesCysteine and methionine residues are particularly sensitive to oxidation by almost all forms of ROS. Under even mild conditions cysteine residues are converted to disulfides and methionine residues are converted to methionine sulfoxide (MeSOX) residues. Most biological systems contain disulfide reductases and MeSOX reductases that can convert the oxidized forms of cysteine and methionine residues back to their unmodified forms. These are the only oxidative modifications of proteins that can be repaired. Based on the observation that preferential oxidation of several exposed methionine residues in some proteins has little effect on their biological function, it was proposed that the cyclic oxidation-reduction of methionine residues serves as a “built-in” ROS scavenger system to protect such proteins from more extensive irreversible oxidative modifications (6Levine R.L. Mosoni L. Berlett B.S. Stadtman E.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15036-15040Crossref PubMed Scopus (870) Google Scholar). This proposition is supported by results of recent studies showing that a “knock-out” strain of yeast lacking MeSOX reductase is more sensitive to H2O2 toxicity than the wild-type strain and that, when grown in the presence of H2O2, the protein and free amino acid pool of the mutant strain contain higher levels of MeSOX than are present in the wild type strain. 3J. Moskovitz, B. S. Berlett, M. J. Poston, R. L. Levine, and E. R. Stadtman, unpublished results. Aromatic Amino Acid ResiduesAromatic amino acid residues are among the preferred targets for ROS attack. As shown in Table I, tryptophan residues are readily oxidized to formylkynurenine and kynurenine and to various hydroxy derivatives; phenylalanine and tyrosine residues yield a number of hydroxy derivatives; histidine residues are converted to 2-oxohistidine, asparagine, and aspartic acid residues.Reactions with PeroxynitriteWith the discovery that nitric oxide is a normal product of arginine metabolism and that it reacts rapidly with O·̄2 to form peroxynitrite (Reaction 5), the biological effects of peroxynitrite (PN) have been extensively studied.O·̄2+NO⋅→ONOO−REACTION5Methionine and cysteine residues of proteins are particularly vulnerable to oxidation by PN, and tyrosine and tryptophan residues are selective targets for PN-dependent nitration. The nitration of tyrosine residues may be of singular importance since nitration precludes the ability of tyrosine residues to undergo cyclic interconversion between phosphorylated and unphosphorylated forms (7Hunter T. Cell. 1995; 80: 225-236Abstract Full Text PDF PubMed Scopus (2577) Google Scholar) or between nucleotidylated and unmodified forms (8Chock P.B. Stadtman E.R. Bittar E.E. Bittar N. Principles of Medical Biology. 4. Jai Press, Greenwich, CT1996: 201-220Google Scholar). Accordingly, nitration would compromise one of the most important mechanisms of cellular regulation of key enzyme activities and of signal transduction networks (7Hunter T. Cell. 1995; 80: 225-236Abstract Full Text PDF PubMed Scopus (2577) Google Scholar). This possibility is underscored by the demonstration that nitration of tyrosine residues in model substrates prevents the phosphorylation of these residues by protein tyrosine kinases (9Kong S.-K. Yim M.B. Stadtman E.R. Chock P.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3377-3382Crossref PubMed Scopus (308) Google Scholar, 10Gow A.J. Duran D. Malcolm S. Ischiropoulos H. FEBS Lett. 1996; 385: 63-66Crossref PubMed Scopus (402) Google Scholar) and by the demonstration that nitration of tyrosine residues inEscherichia coli glutamine synthetase (GS) converts the enzyme to a form with regulatory properties similar to those obtained by in vivo enzyme-catalyzed adenylylation of a single tyrosine residue in each subunit of the enzyme (11Berlett B.S. Friguet B. Yim M.B. Chock P.B. Stadtman E.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1776-1780Crossref PubMed Scopus (165) Google Scholar). The enzyme-catalyzed cyclic adenylylation and deadenylylation of GS is the basis of an exquisite mechanism for the feedback regulation of GS activity by diverse end products of glutamine metabolism (8Chock P.B. Stadtman E.R. Bittar E.E. Bittar N. Principles of Medical Biology. 4. Jai Press, Greenwich, CT1996: 201-220Google Scholar). In contrast, the nitration of tyrosine residues is an irreversible process and therefore locks the enzyme into a relatively inactive configuration.The ability of PN to nitrate tyrosine residues and oxidize methionine residues of proteins is dependent upon the availability of CO2. In the absence of CO2, PN is in equilibrium with an activated form (PN*) of unknown structure that reacts rapidly with methionine residues to form MeSOX (12Pryor W.A. Jin X. Squadrito G.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11173-11177Crossref PubMed Scopus (360) Google Scholar), but in the presence of CO2, PN is almost instantly converted to a derivative (possibly O=NOOCO2− or O2NOCO2−) that can nitrate aromatic compounds (13Lymar S.V. Hurst J.K. J. Am. Chem. Soc. 1995; 117: 8867-8868Crossref Scopus (493) Google Scholar, 14Lymar S.V. Jiang Q. Hurst J.K. Biochemistry. 1996; 35: 7855-7861Crossref PubMed Scopus (315) Google Scholar, 15Uppu R.M. Squadrito G.L. Pryor W.A. Arch. Biochem. Biophys. 1996; 327: 335-343Crossref PubMed Scopus (283) Google Scholar, 16Denicola A. Freeman B.A. Trujillo M. Radi R. Arch. Biochem. Biophys. 1996; 333: 49-58Crossref PubMed Scopus (500) Google Scholar). Accordingly, the nitration of tyrosine and the oxidation of methionine residues of proteins are mutually exclusive processes that are differentially regulated by the availability of CO2, as illustrated in SchemeFS1.Figure FS1View Large Image Figure ViewerDownload Hi-res image Download (PPT)Curiously, in the case of GS, there is little or no nitration of tyrosine residues in the complete absence of CO2, and no oxidation of methionine residues occurs in the presence of physiological concentrations of CO2 (i.e. 5% CO2, pH 7.4). Even so, the PN-dependent oxidation of methionine residues in the absence of CO2 and the nitration of tyrosine residues in the presence of CO2both convert GS to a form with regulatory properties similar to those obtained by enzyme-catalyzed adenylylation of a single tyrosine in each subunit of the enzyme (17Berlett B.S. Stadtman E.R. FASEB J. 1996; 10 (Abstr. 585): A1100Google Scholar).Generation of Protein Carbonyl DerivativesAs already noted, oxidative cleavage of proteins by either the α-amidation pathway (Fig. 2) or by oxidation of glutamyl side chains (Reaction FR3) leads to formation of a peptide in which the N-terminal amino acid is blocked by an α-ketoacyl derivative. However, as shown in Table I, direct oxidation of lysine, arginine, proline, and threonine residues may also yield carbonyl derivatives. In addition, carbonyl groups may be introduced into proteins by reactions with aldehydes (4-hydroxy-2-nonenal, malondialdehyde) produced during lipid peroxidation (Fig. 3 A) (18Schuenstein E. Esterbauer H. CIBA Found. Symp. 1979; 67: 225-244Google Scholar, 19Esterbauer H. Schaur R.J. Zolner H. Free Radical Biol. Med. 1991; 11: 81-128Crossref PubMed Scopus (5851) Google Scholar, 20Uchida K. Stadtman E.R. J. Biol. Chem. 1993; 268: 6388-6393Abstract Full Text PDF PubMed Google Scholar) or with reactive carbonyl derivatives (ketoamines, ketoaldehydes, deoxyosones) generated as a consequence of the reaction of reducing sugars or their oxidation products with lysine residues of proteins (21Kristal B.S. Yu B.P. J. Gerontol. 1992; 47: B104-B107Crossref Scopus (185) Google Scholar, 22Baynes J.W. Diabetes. 1996; 40: 405-411Crossref Scopus (3475) Google Scholar, 23Monnier V. Gerhardinger C. Marion M.S. Taneda S. Cutler R.G. Packer L. Bertram J. Mori A. Oxidative Stress and Aging. Birkhauser Verlag, Basel, Switzerland1995: 141-149Crossref Google Scholar) (glycation and glycoxidation reactions) (Fig.3 B). The presence of carbonyl groups in proteins has therefore been used as a marker of ROS-mediated protein oxidation, and several sensitive methods for the detection and quantitation of protein carbonyl groups have been developed (24Levine R.L. Williams J.A. Stadtman E.R. Schacter E. Methods Enzymol. 1994; 233: 346-357Crossref PubMed Scopus (2238) Google Scholar). As judged by the presence of carbonyl groups, it has been established that protein oxidation is associated with aging, oxidative stress, and a number of diseases.Figure 3Formation of protein carbonyls by glycation, glycoxidation, and by reactions with peroxidation products of polyunsaturated fatty acids (PUFA). A, reactions of sugars with protein lysyl amino groups (P-NH2).B, Michael addition of 4-hydroxy-2-nonenal to protein lysine (P-NH 2), histidine (P-His), or cysteine (PSH) residues. C, reaction of protein amino groups (PNH 2) with the lipid peroxidation product, malondialdehyde.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Oxidative Stress-induced Protein OxidationElevated levels of oxidized protein are present in animals and cell cultures following their exposure to various conditions of oxidative stress. Thus, exposure of animals or cell cultures to either hyperoxia, forced exercise, ischemia-reperfusion, rapid correction of hyponatremia, paraquat toxicity, magnesium deficiency, ozone, neutrophil activation, cigarette smoking, x-radiation, chronic alcohol treatment, or mixed function oxidation systems leads to an increase in the level of oxidized protein. 4For review, see E. R. Stadtman and B. S. Berlett (1997) Chem. Res. Toxicol. 10, 485–494. Protein Oxidation and AgingAging is associated with the accumulation of inactive or less active, more heat-labile forms of numerous enzymes (25Dreyfus J.C. Kahn A. Schapira F. Curr. Top. Cell. Regul. 1978; 14: 243-297Crossref PubMed Scopus (68) Google Scholar, 26Rothstein M. Mech. Ageing Dev. 1977; 6: 241-257Crossref PubMed Scopus (105) Google Scholar). The possibility that these age-related changes are due, at least in part, to oxidative modification is indicated by the facts. (a) In vitro exposure of enzymes to ROS elicits changes in catalytic activity, heat stability, and proteolytic susceptibility similar to those that occur during aging (27Takahashi R. Goto S. Arch. Biochem. Biophys. 1990; 277: 228-233Crossref PubMed Scopus (35) Google Scholar, 28Oliver C.N. Ahn B.-W. Moerman E.J. Goldstein S. Stadtman E.R. J. Biol. Chem. 1987; 262: 5488-5491Abstract Full Text PDF PubMed Google Scholar, 29Zhou J.Q. Gafni A. J. Gerontol. 1991; 46: B217-B221Crossref PubMed Scopus (18) Google Scholar, 30Oliver C.N. Fucci L. Levine R.L. Wittenberger M.E. Stadtman E.R. Heitanen E. Laitinen M. Hanninen O. Cytochrome P-450, Biochemistry, Biophysics, and Environmental Implications. Elsevier Biomedical Press, Amsterdam1982: 531-539Google Scholar). (b) Brief exposure of animals to oxidative stress leads to alterations in enzymes similar to that associated with aging (31Starke P.E. Oliver C.N. Stadtman E.R. FASEB J. 1987; 1: 36-39Crossref PubMed Scopus (91) Google Scholar, 32Starke-Reed P.E. Oliver C.N. Arch. Biochem. Biophys. 1989; 275: 559-567Crossref PubMed Scopus (342) Google Scholar). (c) Old animals are more susceptible than young animals to protein damage during oxidative stress, e.g.x-radiation, H2O2 (33Agarwal S. Sohal R.S. Biochem. Biophys. Res. Commun. 1993; 194: 1203-1206Crossref PubMed Scopus (52) Google Scholar, 34Sohal R.S. Agarwal S. Sohal B.H. Mech. Ageing Dev. 1995; 81: 15-25Crossref PubMed Scopus (149) Google Scholar). (d) There is an age-related increase in the carbonyl content of protein in human brain (35Smith C.D. Carney J.M. Starke-Reed P.E. Oliver C.N. Stadtman E.R. Floyd R.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10540-10543Crossref PubMed Scopus (1155) Google Scholar), gerbil brain (36Carney J.M. Starke-Reed P.E. Oliver C.N. Landum R.W. Cheng M.S. Wu J.F. Floyd R.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3633-3636Crossref PubMed Scopus (640) Google Scholar), eye lens (37Garland D. Russell P. Zigler J.S. Simic M.G. Taylor K.S. Ward J.F. von Sontag V. Oxygen Radicals in Biology and Medicine. Plenum Publishing Corp., New York1988: 347-353Crossref Scopus (44) Google Scholar), rat hepatocytes (32Starke-Reed P.E. Oliver C.N. Arch. Biochem. Biophys. 1989; 275: 559-567Crossref PubMed Scopus (342) Google Scholar), whole body protein of flies (38Sohal R.S. Agarwal S. Dubey A. Orr W.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7255-7259Crossref PubMed Scopus (319) Google Scholar), and human red blood cells (28Oliver C.N. Ahn B.-W. Moerman E.J. Goldstein S. Stadtman E.R. J. Biol. Chem. 1987; 262: 5488-5491Abstract Full Text PDF PubMed Google Scholar). (e) The carbonyl content of protein in cultured human fibroblasts increases exponentially as a function of the age of the fibroblast donor (28Oliver C.N. Ahn B.-W. Moerman E.J. Goldstein S. Stadtman E.R. J. Biol. Chem. 1987; 262: 5488-5491Abstract Full Text PDF PubMed Google Scholar). (f) There is an inverse relationship between regimens that lead to an increase in life span and regimens that lead to an increase in protein carbonyl content and vice versa (39Sohal R.S. Ku H.-H. Agarwal S. Forster M.J. Lal H. Mech. Ageing Dev. 1994; 79: 121-133Crossref Scopus (689) Google Scholar). For example, diet (caloric) restriction of rats (39Sohal R.S. Ku H.-H. Agarwal S. Forster M.J. Lal H. Mech. Ageing Dev. 1994; 79: 121-133Crossref Scopus (689) Google Scholar) and mice (40Youngman L.D. Park J.-Y.K. Ames B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9112-9116Crossref PubMed Scopus (214) Google Scholar) leads to an increase in life span and to a decrease in the level of protein carbonyls. When compared at the same chronological age, strains of short lived houseflies contain higher levels of oxidized proteins than their longer lived cohorts (41Sohal R.S. Ku H.-H. Agarwal S. Biochem. Biophys. Res. Commun. 1993; 196: 7-11Crossref PubMed Scopus (103) Google Scholar).Protein Oxidation and DiseaseAccumulation of oxidized protein (protein carbonyls) is associated with a number of diseases, including amyotrophic lateral sclerosis, Alzheimer's disease, respiratory distress syndrome, muscular dystrophy, cataractogenesis, rheumatoid arthritis, progeria, and Werner's syndrome.4Although the level of carbonyl has not been directly determined, there is reason to believe that oxidative modification of proteins is implicated also in atherosclerosis, diabetes, Parkinson's disease, essential hypertension, cystic fibrosis, and ulcerative colitis.4Accumulation of Oxidized ProteinThe intracellular level of oxidized protein reflects the balance between the rate of protein oxidation and the rate of oxidized protein degradation. This balance is a complex function of numerous factors that lead to the generation of ROS, on the one hand, and of multiple factors that determine the concentrations and/or activities of the proteases that degrade oxidatively damaged protein, on the other. As illustrated in Fig. 4, many different physiological and environmental processes lead to the formation of ROS. Collectively, these processes can promote the generation of a battery of ROS, including a number of free radicals (⋅OH, O·̄2, R⋅, ROO⋅, RO⋅, NO⋅, RS⋅, ROS⋅, RSOO⋅, and RSSR·̄), various non-radical oxygen derivatives (H2O2, ROOH,1O2, O3, HOCl, ONOO−, O=NOCO2−, O2NOCO2−, N2O2, NO2+, and highly reactive lipid- or carbohydrate-derived carbonyl compounds,viz. 4-hydroxy-2-nonenal, malondialdehyde ketoamines, ketoaldehydes, and deoxyosones. Any one of these ROS is capable of promoting the modification of proteins. However, as shown in Fig. 4, their abilities to do so are dependent upon the concentrations of a myriad of enzymic and non-enzymic factors (antioxidants) that can either inhibit the formation of ROS or facilitate their conversion to inactive derivatives. For example, the O·̄2 formed by several pro-oxidant systems shown in Fig. 4 is readily converted to H2O2 by the action of superoxide dismutase. This H2O2 together with H2O2 produced by various oxidases and metal-catalyzed oxidation systems is readily degraded by catalase, glutathione peroxidase, thiol-specific antioxidant enzymes, and other peroxidases. However, if in the course of metabolism the concentrations of these antioxidant activities become insufficient to decompose all of the H2O2 formed, the H2O2 may undergo metal ion-catalyzed cleavage by the Fenton reaction to generate the even more toxic ⋅OH. This reaction is dependent upon the availability of iron and copper, which is determined by the concentrations of metal-binding proteins (ferritin, transferrin, lactoferrin, and ceruloplasmin), and of multiple factors (iron-responsive elements, etc.) that control the intracellular concentrations of these proteins, as well as factors that influence the binding and/or the release of metal ions from these binding proteins. The level of ROS is also a function of the concentrations of vitamins (A, C, and E) and of metabolites (uric acid, bilirubin, etc.) that are capable of either scavenging free radicals directly or of facilitating the regeneration of metabolites that do so. Finally, metal ion chelators can either suppress or enhance the rates of ROS generation by forming complexes with iron or copper that inhibit their ability to catalyze ROS formation or alter their redox potentials and therefore their ability to undergo cyclic interconversion between oxidized and reduced states. Furthermore, other divalent cations (Mg2+, Mn2+, and Zn2+) may compete with Fe(II) or Cu(I) for binding to metal binding sites on proteins and thereby prevent site-specific generation of ⋅OH, which is likely the most important mechanism of protein damage (42Stadtman E.R. Free Radical Biol. Med. 1990; 9: 315-325Crossref PubMed Scopus (1015) Google Scholar). In addition, Mn(II) is able to inhibit the reduction of Fe(III) to Fe(II) (43Nakamura K. Oliver C. Stadtman E.R. Arch. Biochem. Biophys. 1985; 240: 319-329Crossref PubMed Scopus (38) Google Scholar) and thus prevent its ability to promote formation of ⋅OH by the Fenton reaction as well as the generation of other forms of ROS as illustrated in Fig. 1.Figure 4Accumulation of oxidized protein is dependent upon the balance between pro-oxidant, antioxidant, and proteolytic activities. MSR, methionine sulfoxide reductase;GPx, glutathione peroxidase; CAT, catalase;RSH-Px, thiol-specific peroxidase; NOS, nitric oxide synthetase; SOD, superoxide dismutase; GST, glutathione transferase.View Large Image Figure ViewerDownload Hi-res image Download (PPT)As noted above, the accumulation of oxidized protein reflects not only the rate of protein oxidation but also the rate of oxidized protein degradation, which (as shown in Fig. 4) is also dependent upon many variables, including the concentrations of proteases that preferentially degrade oxidized proteins and numerous factors (metal ions, inhibitors, activators, and regulatory proteins) that affect their proteolytic activities. For example, oxidized forms of some proteins (e.g. cross-linked proteins (44Friguet B. Szweda L. Stadtman E.R. Arch. Biochem. Biophys. 1994; 311: 168-173Crossref PubMed Scopus (157) Google Scholar, 45Grune T. Reinheckel T. Joshi M. Davies K.J.A. J. Biol. 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They are intended to call attention to the extraordinary complexity of ROS biochemistry. Numerous other factors not discussed are certainly important in determining the steady-state level of oxidative damage under varying physiological and environmental conditions. It is our belief that during aging there is a progressive accumulation of errors at the level of DNA that affect any one or more of the factors that govern the dynamics of protein oxidation and oxidized protein degradation. This leads to a shift in the balance between these processes in favor of oxidized protein accumulation and attendant loss of biological function. Two observations are consistent with this hypothe