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. 1997 Apr;20(2):81–89. doi: 10.1007/s11357-997-0008-y

Age-associated, oxidatively modified proteins: A critical evaluation

Sataro Goto 1, Akihiro Nakamura 1
PMCID: PMC3456151  PMID: 23604294

Abstract

Reactive oxygen species have been implicated in oxidative modifications of proteins, in many cases represented as carbonyls, which can lead to a variety of diseases and the age-associated decline of physiological functions. Considerable progress, as well as controversy, about oxidatively modified proteins and aging has unfolded in the last few years. In this article we critically evaluate changes in protein carbonyl content as a marker of the oxidative stress associated with age and other relevant issues on the degradation of oxidatively modified proteins.

A definitive conclusion on the age-related increase of protein carbonyls is currently viewed as having to await further confirmation using detailed analysis with new methodologies. Controversial methodological measurements and characterizations of protein carbonyls are discussed, emphasizing the merits of immunoblot analysis using two-dimensional gel electrophoresis. The degradation of oxidatively modified proteins has not yet been studied in depth in relation to their possible accumulation in old tissues. Recent efforts to establish a causal relation between the effect of oxidative stress on proteins and physiological declines with age are discussed briefly.

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References

  • 1.Harman D. In: Free radical theory of aging: History in Free Radicals and Aging. Emerit I., Chance B., editors. Basel, Switzerland: Birkhauser Verlag; 1992. pp. 1–10. [Google Scholar]
  • 2.Yu B.P. Free Radicals in Aging. Boca Raton, FL: CRC Press; 1993. [Google Scholar]
  • 3.Yu B.P., Suescun E.A., Yang S.Y. Effect of age-related lipid peroxidation on membrane fluidity and phospholipase A2, modulation by dietary restriction. Mech. Ageing Dev. 1992;65:17–33. doi: 10.1016/0047-6374(92)90123-U. [DOI] [PubMed] [Google Scholar]
  • 4.Choi J.H., Yu B.P. Brain synaptosomal aging. Free radicals and membrane fluidity. Free Rad. Biol. Med. 1995;18:133–139. doi: 10.1016/0891-5849(94)00106-T. [DOI] [PubMed] [Google Scholar]
  • 5.Miyazawa T., Suzuki T., Fujimoto K. Age-dependent accumulation of phosphatidylcholine hydroperoxide in the brain and liver of the rat. Lipids. 1993;28:789–793. doi: 10.1007/BF02536232. [DOI] [PubMed] [Google Scholar]
  • 6.Ames, B.N., Shigenaga, M.K., and Hagan, T.M.: Oxidant, antioxidants and the degenerative diseases of aging. Proc. Natl. Acad. Sci. USA, 90: 79515–7922, 1993. [DOI] [PMC free article] [PubMed]
  • 7.Kaneko T., Tahara S., Matsuo M. Non-linear accumulation of 8-hydroxyguanosine, a marker of oxidized DNA damage, during aging. Mut. Res. 1996;316:277–285. doi: 10.1016/s0921-8734(96)90010-7. [DOI] [PubMed] [Google Scholar]
  • 8.Shigenaga, N.K., Hagen, T.M., and Ames, B.N.: Oxidative damage and mitochondrial decay in aging. Proc. Natl. Acad. Sci. USA, 91: 10771–10778, 1994. [DOI] [PMC free article] [PubMed]
  • 9.Stadtman E.R. Protein oxidation and aging. Science. 1992;257:1220–1224. doi: 10.1126/science.1355616. [DOI] [PubMed] [Google Scholar]
  • 10.Sohal R.S., Weindruch R. Oxidative stress, caloric restriction, and aging. Science. 1996;273:59–63. doi: 10.1126/science.273.5271.59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Martin G.M., Austad S.N., Johnson T.E. Genetic analysis of ageing: role of oxidative damage and environmental stresses. Nature Genetics. 1996;13:25–34. doi: 10.1038/ng0596-25. [DOI] [PubMed] [Google Scholar]
  • 12.Stadtman E.R. Minireview: Protein modification in aging. J. Gerontol. 1988;43:B112–120. doi: 10.1093/geronj/43.5.b112. [DOI] [PubMed] [Google Scholar]
  • 13.Stadman E.R. Covalent modification reactions are marking steps in protein turnover. Biochemistry. 1990;29:6323–6331. doi: 10.1021/bi00479a001. [DOI] [PubMed] [Google Scholar]
  • 14.Selkloe, D.J.: Amyloid b-protein and the genetics of Alzheimer’s disease. J. Biol Chem., 271: 18295–18298, 1996 [DOI] [PubMed]
  • 15.Stadtman E.R. Role of oxidized amino acids in protein breakdown and stability. Methods Enzymol. 1995;258:379–393. doi: 10.1016/0076-6879(95)58057-3. [DOI] [PubMed] [Google Scholar]
  • 16.Gafni A., Noy N. Age-related effects in enzyme catalysis. Mol. Cell. Biochem. 1984;59:113–129. doi: 10.1007/BF00231308. [DOI] [PubMed] [Google Scholar]
  • 17.Farber J.M., Levine R.L. Sequence of a peptide susceptible to mixed-function oxidation. Probable cation binding site in glutamine synthetase. J.Biol.Chem. 1986;261:4574–4578. [PubMed] [Google Scholar]
  • 18.Stadtman E.R., Starke-Reed P.E., Oliver C.N., Carney J.M., Floyd R.A. Protein modification in aging. In: Emeritt I., Chance B., editors. Free Radical and Aging. Basel, Switzerland: Birkhauser Verlag; 1992. pp. 64–72. [Google Scholar]
  • 19.Uchida K., Fukuda A., Kawashima S., Hirai H., Toyokuni S. A renal carcinogen ferric nitrilotriacetate mediates a temporary accumulation of aldehyde-modified proteins within cytoplasmic compartment of rat kidney. Arch. Biochem. Biophys. 1995;317:405–411. doi: 10.1006/abbi.1995.1181. [DOI] [PubMed] [Google Scholar]
  • 20.Amici A., Levine R.L., Tsai L., Stadtman E.R. Conversion of amino acid residues in proteins and amino acid homopolymers to carbonyl derivatives by metal-catalyzed oxidation. J. Biol. Chem. 1989;264:3341–3346. [PubMed] [Google Scholar]
  • 21.Huggins, T.G., Well-Knecht, M.C., Detorie, N.A., Baynes, J.W., and Thorpe, S.R.: Formation of othyrosine and dityrosine in proteins during radiolytic and metal-catalyzed oxidation. J. Biol. Chem., 268: 12341–12347, 1993. [PubMed]
  • 22.Well-Knecht, M.C., Huggins, T.G., Dyer, D.G., Thorpe, S.R., and Baynes, J.W.: Oxidized amino acids in lens proteins with age. Measurement of othyrosine and dityrosine in the aging human lens. J. Biol. Chem., 268: 12348–12352, 1993. [PubMed]
  • 23.Gieseg S.P., Simpson J.A., Charlton T.S., Duncan M.K., Dean R.T. Protein-bound 3,4-dihydroxyphenylalanine is a major reductant formed during hydroxyl radical damage to proteins. Biochemistry. 1993;32:4780–4786. doi: 10.1021/bi00069a012. [DOI] [PubMed] [Google Scholar]
  • 24.Maier K.L., Matejkova E., Hinze H., Leuschel L., Weber H., Beck-Speir I. Different selectivities of oxidants during oxidation of methionine residues in the a-1-proteinase inhibitor. FEBS Lett. 1989;250:221–226. doi: 10.1016/0014-5793(89)80725-2. [DOI] [PubMed] [Google Scholar]
  • 25.Moskovitz J., Rahman M.A., Strassman J., Yancey S.O., Kushner S.R., Brot N., Weissbach H. Eschenchia coli peptide methionine sulfoxide reductase gene: Regulation of expression and role in protecting against oxidative damage. J. Bact. 1995;177:502–507. doi: 10.1128/jb.177.3.502-507.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Moskovitz J., Weissbach H., Brot N. Cloning and expression of a mammalian gene involving reduction of methionine sulfoxide residues in proteins. Proc. Natl. Acad. Sci. USA. 1996;93:2095–2099. doi: 10.1073/pnas.93.5.2095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Rothstein M. Biochemical Approaches to Aging. New York: Academic Press; 1982. Enzyme and altered proteins; pp. 213–255. [Google Scholar]
  • 28.Oliver C.N., Ahn B., Moerman E.J., Goldstein S., Stadtman E.R. Age-related changes in oxidized proteins. J. Biol. Chem. 1987;262:5488–5491. [PubMed] [Google Scholar]
  • 29.Takahashi R., Goto S. Alteration of aminoacyl-tRNA synthetase with age: Heat-labilization of the enzyme by oxidative damage. Arch. Biochem. Biophys. 1990;277:228–233. doi: 10.1016/0003-9861(90)90573-H. [DOI] [PubMed] [Google Scholar]
  • 30.Zhou J.Q., Gafni A. Exposure of rat muscle phosphoglycerate kinase to enzymatic MFO system generate the old form of the enzyme. J. Gerontol. 1991;465:B217–121. doi: 10.1093/geronj/46.6.b217. [DOI] [PubMed] [Google Scholar]
  • 31.Stadtman, E.R.: Ascorbic acid and oxidative inactivation of proteins. Am. J. Clin. Nutr., 54: 1125S–1128S, 1991. [DOI] [PubMed]
  • 32.Levine R.L., Garland D., Oliver C.N., Amici A., Climent I., Lenz A.G., Ahn B.W., Shaltiel S., Stadtman E.R. Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol. 1990;186:464–478. doi: 10.1016/0076-6879(90)86141-h. [DOI] [PubMed] [Google Scholar]
  • 33.Levine R.L., Williams J.A., Stadtman E.R., Shacter E. Carbonyl assays for determination of oxidatively modified proteins. Methods Enzymol. 1994;233:346–357. doi: 10.1016/s0076-6879(94)33040-9. [DOI] [PubMed] [Google Scholar]
  • 34.Reznick A.Z., Packer L. Oxidative damage to proteins: Spectrophotometric method for carbonyl assay. Methods Enzymol. 1994;233:357–363. doi: 10.1016/s0076-6879(94)33041-7. [DOI] [PubMed] [Google Scholar]
  • 35.Starke-Reed P.E., Oliver C.N. Protein oxidation and proteolysis during aging and oxidative stress. Arch. Biochem. Biophys. 1989;275:559–567. doi: 10.1016/0003-9861(89)90402-5. [DOI] [PubMed] [Google Scholar]
  • 36.Sohal R.S., Ku H., Agawal S., Forster M.J., Lal H. Oxidative damage, mitochondrial oxidant generation and antioxidant defenses during aging and in response to food restriction in the mouse. Mech. Ageing Dev. 1994;74:121–133. doi: 10.1016/0047-6374(94)90104-X. [DOI] [PubMed] [Google Scholar]
  • 37.Sohal R.S., Agawal S., Sohal B.H. Oxidative stress and aging in the Mongolian gerbil (Meriones unguiculantus) Mech. Ageing Dev. 1995;81:15–25. doi: 10.1016/0047-6374(94)01578-A. [DOI] [PubMed] [Google Scholar]
  • 38.Sohal R.S., Agarwal S., Dubey A., Orr W.C. Protein oxidative damage is associated with life expectancy of houseflies. Proc. Natl. Acad. Sci. USA. 1993;90:7255–7259. doi: 10.1073/pnas.90.15.7255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Cao G., Cutler R.G. Protein oxidation and aging. I. Difficulties in measuring reactive protein carbonyls in tissue using 2,4-dinitrophenyl-hydrazine. Arch. Biochem. Biophys. 1995;320:106–114. doi: 10.1006/abbi.1995.1347. [DOI] [PubMed] [Google Scholar]
  • 40.Ayene I.S., Dodia C., Fisher A.B. Role of oxygen in oxidation of lipid and protein during ischemia/reperfusion in isolated perfused rat lung. Arch. Biochem. Biophys. 1992;296:183–189. doi: 10.1016/0003-9861(92)90561-A. [DOI] [PubMed] [Google Scholar]
  • 41.Nakamura A., Goto S. Analysis of protein carbonyls with 2,4-dinitrophenylhydrazine and its antibodies by immunoblot in two-dimensional gel electrophoresis. J. Biochem. 1996;119:768–774. doi: 10.1093/oxfordjournals.jbchem.a021306. [DOI] [PubMed] [Google Scholar]
  • 42.Lee Y.-J., Shacter E. Role of carbohydrates in oxidative modification of fibrinogen and other plasma proteins. Arch. Biochem. Biophys. 1995;321:175–181. doi: 10.1006/abbi.1995.1383. [DOI] [PubMed] [Google Scholar]
  • 43.Esterbauer H., Zollner H. Methods for determination of aldehydic lipid peroxidation products. Free Rad. Biol. Med. 1995;7:197–203. doi: 10.1016/0891-5849(89)90015-4. [DOI] [PubMed] [Google Scholar]
  • 44.Blakeman D.P., Ryan T.P., Jolly R.A., Petry T.W. Diquat-dependent protein carbonyl formation. Identification of lipid-dependent and lipid-independent pathways. Biochemical. Pharmacol. 1995;50:929–935. doi: 10.1016/0006-2952(95)00214-K. [DOI] [PubMed] [Google Scholar]
  • 45.Burchan P.C., Kuhan Y.T. Introduction of carbonyl groups into proteins by lipid peroxidation products, malondialdehydes. Biochem. Biophys. Res. Comm. 1996;220:996–1001. doi: 10.1006/bbrc.1996.0521. [DOI] [PubMed] [Google Scholar]
  • 46.Janes S.M., Klinman P. Isolation of 2,4,5-trihydroxyphenylalanine quinone (topa quinone) from copper amine oxidase. Methods Enzymol. 1995;258:20–34. doi: 10.1016/0076-6879(95)58034-4. [DOI] [PubMed] [Google Scholar]
  • 47.Shacter E., Williams J.A., Lim M., Levine R.L. Differential susceptibility of plasma proteins to oxidative modification: Examination by Western blot immunoassay. Free Rad. Biol. Med. 1994;17:429–437. doi: 10.1016/0891-5849(94)90169-4. [DOI] [PubMed] [Google Scholar]
  • 48.Cabiscol, E., and Levine, R.L.: Carbonic anhydrase. III. Oxidative modification in vivo and loss of phosphatase activity during aging. J. Biol. Chem., 270: 14742–14747, 1995. [DOI] [PubMed]
  • 49.Radak Z., Asano K., Lee K.C., Ohno H., Nakamura A., Nakamoto H., Goto S. High altitude training increases the reactive carbonyl derivatives but not lipid peroxidation in skeletal muscle of rats. Free Rad. Biol. Med. 1997;22:1109–1114. doi: 10.1016/S0891-5849(96)00350-4. [DOI] [PubMed] [Google Scholar]
  • 50.Van Remmen, H., Ward, W.F., Sabia, R.V., and Richardson, A.: Gene expression and protein degradation, in Handbook of Physiology. A critical, comprehensive presentation of physiological knowledge and concepts. Section 11: Aging, edited by Masoro, E.J., Oxford Univ. Press, 1995, pp. 171–234.
  • 51.Goto, S., Hasegawa, A., Nakamoto, H., Nakamura, A., Takahashi, R., and Kurochkin, I.V.: Age-associated changes of oxidative modification and turn-over of proteins, in Oxidative Stress and Aging, edited by Cutler, R., Packer, L., Bertram, J., and Mori, A., Birkhauser Verlag, 1995, pp. 151–158.
  • 52.Lavie L., Reznick A.Z., Gershon D. Decreased protein and puromycinyl-peptide degradation in livers of senescent mice. Biochem.J. 1982;202:47–51. doi: 10.1042/bj2020047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Rivett, A.J.: Purification of a liver alkaline protease which degrades oxidatively modified glutamine synthetase: characterization as a high molecular weight cysteine proteinase. J. Biol. Chem., 260: 12600–1206, 1985. [PubMed]
  • 54.Pacifici R.E., Salo D.C., Davies K.J.A. Macroxyproteinase (M.O.P.) — A 670 kDa proteinase complex that degrades oxidatively denatured proteins in red blood cells. Free Rad. Biol. Med. 1989;7:521–536. doi: 10.1016/0891-5849(89)90028-2. [DOI] [PubMed] [Google Scholar]
  • 55.Fagan, J.M., and Waxman, L.: The ATP-independent pathway in red blood cells that degrades oxidant-damaged hemoglobin. J. Biol. Chem., 267: 23015–23022, 1992. [PubMed]
  • 56.Grune T., Reinheckel T., Joshi M., Davies K.J.A. Proteolysis in cultured liver epitherial cells during oxidative stress. J. Biol. Chem. 1995;270:2344–2351. doi: 10.1074/jbc.270.5.2344. [DOI] [PubMed] [Google Scholar]
  • 57.Grune, T., Reinheckel, T., and Davies, K.J.A.: Degradation of oxidized proteins in K562 human hematopoietic cells by proteasomes. J. Biol. Chem., 271: 15504–15509, 1996. [DOI] [PubMed]
  • 58.Kuo, W.L., Gehm, B.D., Rosner, M.R., Li, W., and Keller, G.: Inducible expression and cellular localization of insulin-degrading enzyme in a stably transfected cell line. J. Biol. Chem., 269: 22599–22606, 1994. [PubMed]
  • 59.Smith, C.D., Carney, J.M., Starke-Reed, P.E., Oliver, C.N., Stadtman, E.R., Floyd, R.A., and Markesbery, W.R.: Excess brain protein oxidation and enzyme dysfunction in normal aging and in Alzheimer disease. Proc. Natl. Acad. Sci. USA, 88: 10540–10543, 1991. [DOI] [PMC free article] [PubMed]
  • 60.Carney J.M., Starke-Reed P.E., Oliver C.N., Landum R.W., Cheng M.S., Wu J.F., Floyd R.A. Reversal of age-related increase in brain protein oxidation, decrease in enzyme activity, and loss in temporal and spatial memory by chronic administration of the spin-tapping com-pound N-tert-butyl-α-phenylnitrone. Proc. Natl. Acad. Sci. USA. 1991;88:3633–3636. doi: 10.1073/pnas.88.9.3633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 61.Cao G., Cutler R.G. Protein oxidation and aging. II. Difficulties in measuring alkaline protease activity in tissues using the fluorescamine procedure. Arch. Biochem. Biophys. 1995;320:195–201. doi: 10.1006/abbi.1995.1359. [DOI] [PubMed] [Google Scholar]
  • 62.Sahakian J.A., Szweda L.I., Friguet B., Kitani K., Levine R.L. Aging of the liver: Proteolysis of oxidatively modified glutamine synthetase. Arch. Biochem. Biophys. 1995;318:411–417. doi: 10.1006/abbi.1995.1248. [DOI] [PubMed] [Google Scholar]
  • 63.Conconi M., Szweda L.I., Levine R.L., Stadtman E.R., Friguet B. Age-related decline of rat liver multicatalytic proteinase activity and protection from oxidase inactivation by heat-shock protein 90. Arch. Biochem. Biophys. 1996;331:232–240. doi: 10.1006/abbi.1996.0303. [DOI] [PubMed] [Google Scholar]
  • 64.Shibatani T., Ward W.F. Effect of age and food restriction on alkaline protease activity in rat liver. J. Gerontol. 1995;51A:B175–B178. doi: 10.1093/gerona/51a.2.b175. [DOI] [PubMed] [Google Scholar]
  • 65.Agarwal S., Sohal R.S. Aging and proteolysis of oxidized proteins. Arch. Biochem. Biophys. 1994;309:24–28. doi: 10.1006/abbi.1994.1078. [DOI] [PubMed] [Google Scholar]
  • 66.Foster M., Dubey A., Dawson K.M., Stutts W.A., Lai H., Sohal R. Age-related losses of cognitive function and motor skills in mice are associated with oxidative protein damage in the brain. Proc. Natl. Acad. Sci. USA. 1996;93:4765–4769. doi: 10.1073/pnas.93.10.4765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 67.Dubey A., Foster M.J., Sohal R.S. Effect of the spin-trapping compound N-tert-butyl-α-phenylnitrone on protein oxidation and life span. Arch. Biochem. Biophys. 1995;324:249–254. doi: 10.1006/abbi.1995.0037. [DOI] [PubMed] [Google Scholar]
  • 68.Davies K.J.A., Delsignore M.E., Lin S.W. Protein damage and degradation by oxygen radicals II. Modification of amino acids. J. Biol. Chem. 1987;262:9902–9907. [PubMed] [Google Scholar]
  • 69.Levine, R.L.: Oxidative modification of glutamine synthetase II. Characterization of the ascorbate system. J. Biol. Chem., 258: 11828–11833, 1983. [PubMed]
  • 70.Stadtman, E.R., and Berlett, B.S.: Fenton chemistry. Amino acid oxidation. J. Biol. Chem., 266: 17201–17211, 1991. [PubMed]
  • 71.Youngman L.D., Park J.K., Ames B.N. Protein oxidation with aging is reduced by dietary restriction of protein or calories. Proc. Natl. Acad. Sci. USA. 1992;89:9112–9116. doi: 10.1073/pnas.89.19.9112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 72.Orr W.C., Sohal R.S. Extension of life-span by overexpression of superoxide dismutase and catalase in Drosophila melanogaster. Science. 1994;263:1128–1130. doi: 10.1126/science.8108730. [DOI] [PubMed] [Google Scholar]
  • 73.Cini M., Morettie A. Studies on lipid peroxidation and protein oxidation in the aging brain. Neurobiol. Aging. 1995;16:53–57. doi: 10.1016/0197-4580(95)80007-E. [DOI] [PubMed] [Google Scholar]
  • 74.Tian L., Cai Q., Bowen R., Wei H. Effect of caloric restriction on age-related oxidative modifications of macromolecules and lymphocyte proliferation in rats. Free Rad. Biol. Med. 1995;19:859–865. doi: 10.1016/0891-5849(95)00090-K. [DOI] [PubMed] [Google Scholar]
  • 75.Nagy K., Takacs I.E., Pankucsi C. Age-dependence of free radical-induced oxidative damage in ischemic-reperfused rat heart. Arch. Geront. Geriatr. 1996;22:297–309. doi: 10.1016/0167-4943(96)00700-5. [DOI] [PubMed] [Google Scholar]

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