Protein Oxidation Society: Primer and Definition

1. Protein Oxidation Society For Free Radical Biology and Medicine Shacter 1 Protein Oxidation: A primer on characterization, detection, and consequences Emily Shacter, Ph.D. Chief, Laboratory of Biochemistry Division of Therapeutic Proteins Center for Drug Evaluation and Research Food and Drug Administration Bethesda, MD 20892 Ph: 301-827-1833 Fax: 301-480-3256 Email: emily.shacter@fda.hhs.gov Virtual Free Radical School

2. Protein Oxidation Society For Free Radical Biology and Medicine Shacter 2 What is protein oxidation? Covalent modification of a protein induced by reactive oxygen intermediates or by-products of oxidative stress.

3. Protein Oxidation Society For Free Radical Biology and Medicine Shacter 3 Agents that lead to protein oxidation Chemical Reagents (H 2 O 2 , Fe 2+ , Cu 1+ , glutathione, HOCl, HOBr, 1 O 2 , ONOO - ) Activated phagocytes (oxidative burst activity) -irradiation in the presence of O 2 UV light, ozone Lipid peroxides (HNE, MDA, acrolein) Mitochondria (electron transport chain leakage) Oxidoreductase enzymes (xanthine oxidase, myeloperoxidase, P-450 enzymes) Drugs and their metabolites

4. Protein Oxidation Society For Free Radical Biology and Medicine Shacter 4 General types of protein oxidative modification Sulfur oxidation (Cys disulfides, S-thiolation; Met sulfoxide) Protein carbonyls (side chain aldehydes, ketones) Tyrosine crosslinks, chlorination, nitrosation, hydroxylation Tryptophanyl modifications Hydro(pero)xy derivatives of aliphatic amino acids Chloramines, deamination Amino acid interconversions ( e.g ., His to Asn; Pro to OH-Pro) Lipid peroxidation adducts (MDA, HNE, acrolein) Amino acid oxidation adducts ( e.g ., p -hydroxyphenylacetaldehyde) Glycoxidation adducts ( e.g ., carboxymethyllysine) Cross-links, aggregation, peptide bond cleavage

5. Amino acids most susceptible to oxidation and their main reaction products Amino Acid Physiological oxidation products Cysteine Disulfides, mixed disulfides ( e.g ., glutathiolation), HNE-Cys Methionine Methionine sulfoxide Tyrosine Dityrosine, nitrotyrosine, chlorotyrosines, dopa Tryptophan Hydroxy- and nitro-tryptophans, kynurenines Phenylalanine Hydroxyphenylalanines Valine, Leucine Hydro(pero)xides Histidine 2-Oxohistidine, asparagine, aspartate, HNE-His Glutamyl Oxalic acid, pyruvic acid Proline Hydroxyproline, pyrrolidone, glutamic semialdehyde Threonine 2-Amino-3-ketobutyric acid Arginine Glutamic semialdehyde, chloramines Lysine a -Aminoadipic semialdehyde, chloramines, MDA-Lys, HNE-Lys, acrolein-Lys, carboxymethyllysine, pHA-Lys Protein Oxidation Society For Free Radical Biology and Medicine Shacter 5

6. Reaction scheme showing how metal-catalyzed protein oxidation is a site-specific process Stadtman, E.R. and Levine, R.L. (2000) Ann. N.Y. Acad. Sci. 899 , 191-208 Oxidized & Dysfunctional + Fe (II) Peroxide Reactive Species Fe (III) or O 2 etc Protein Oxidation Society For Free Radical Biology and Medicine Shacter 6

7. Protein Oxidation Society For Free Radical Biology and Medicine Shacter 7 Biochemical consequences of protein oxidative modification Loss or gain of enzyme activity Loss of protein function ( e.g ., fibrinogen/fibrin clotting) Loss of protease inhibitor activity ( e.g. , -1-antitrypsin, 2-macroglobulin) Protein aggregation ( e.g ., IgG, LDL, a-synuclein, amyloid protein, prion protein) Enhanced susceptibility to proteolysis ( e.g ., IRP-2, HIF-1 , glutamine synthetase) Diminished susceptibility to proteolysis Abnormal cellular uptake ( e.g ., LDL) Modified gene transcription ( e.g ., SoxR, IkB) Increased immunogenicity ( e.g ., ovalbumin; HNE- or acrolein-LDL)

8. Protein Oxidation Society For Free Radical Biology and Medicine Shacter 8 Diseases and conditions in which protein oxidation has been implicated and specific target proteins, if known Atherosclerosis (LDL) Rheumatoid arthritis (IgG, -1-proteinase inhibitor) Ischemia reperfusion injury Emphysema ( -1-proteinase inhibitor, elastase) Neurodegenerative diseases Alzheimers ( -actin, creatine kinase) Parkinsons Sporadic amyotrophic lateral sclerosis Muscular dystrophy Neonates on ventilators; bronchopulmonary dysplasia Adult respiratory distress syndrome Aging (glutamine synthetase, carbonic anhydrase III, aconitase) Progeria Acute pancreatitis Cataractogenesis (alpha-crystallins) Chronic ethanol ingestion Cancer

9. Protein Oxidation Society For Free Radical Biology and Medicine Shacter 9 How can we inhibit protein oxidation? Antioxidants scavengers (probucol, spin traps, methionine) antioxidant enzymes (catalase, SOD, peroxiredoxins) antioxidant enzyme mimics (ebselen, Tempol, TBAPS) augmentation of cellular antioxidant systems N-acetylcysteine ( intracellular GSH) Chelators (DTPA, Desferal ) Depletion of O 2

10. Protein Oxidation Society For Free Radical Biology and Medicine Shacter 10 Advantages and disadvantages of using proteins as markers of oxidative stress There is no single universal marker for protein oxidation. * With so many different potential reaction products, may need to do several different assays if source of oxidants unknown * If source of oxidation is known, the range narrows ( e.g ., metal-catalyzed oxidation does not cause chlorination or nitrosation, and HOCl does not cause lipid peroxidation adducts)

11. Protein Oxidation Society For Free Radical Biology and Medicine Shacter 11 Advantages and disadvantages of using proteins as markers of oxidative stress Products are relatively stable Types of modification reveal nature of oxidizing species chlorotyrosine from HOCl nitrotyrosine from NO + O 2 - or HOCl glutamic and aminoadipic semialdehydes from metal-catalyzed oxidation Have unique physiological consequences due to the specificity of protein functions Sensitive assays are available (detecting <1 pmol of oxidized product)

12. Protein Oxidation Society For Free Radical Biology and Medicine Shacter 12 Advantages and disadvantages of using proteins as markers of oxidative stress Different forms of oxidative modification have different functional consequences * Met is highly susceptible but oxidation often does not affect protein function * Carbonyls are often associated with dysfunction but may require more stringent oxidative conditions

13. Protein Oxidation Society For Free Radical Biology and Medicine Shacter 13 Advantages and disadvantages of using proteins as markers of oxidative stress Proteins, lipids, and DNA are modified by different oxidants to different degrees e.g ., HOCl generated by myeloperoxidase hits protein >> lipids >> DNA e.g ., H 2 O 2 treatment of cells hits DNA lipids >> proteins

14. Methods for detection of oxidative protein modifications # Modification Methods of Detection Disulfides SDS-gel electrophoresis -ME DTNB Reaction with BIAM* & or MPB* SDS-PAGE Glutathiolation RP-HPLC/mass spectrometry S 35 -Cys/Chx SDS-PAGE Biotinylated glutathione ethyl ester $ Methionine sulfoxide CnBr cleavage/amino acid analysis Carbonyls DNPH**-coupled assays: Spectroscopy HPLC Western blotting ELISA Immunohistochemistry Reduction with NaB 3 H 3 # See Table 4 in Shacter, E. (2000) Drug Metab. Rev. 32 , 307-326. & Kim et al . (2000) Anal. Biochem. 283 , 214-221; $ Sullivan et al . (2000) Biochemistry 39 , 11121-11128. * Biotinylated iodoacetamide or maleimido-propionyl biocytin; **, Dinitrophenylhydrazine Protein Oxidation Society For Free Radical Biology and Medicine Shacter 14

15. Methods for detection of oxidative protein modifications, con # Modification Methods of Detection 2-oxo-His Amino acid analysis Dityrosine Fluorescence Proteolysis or hydrolysis HPLC Chlorotyrosine Hydrolysis/nitroso-naphthol/HPLC HBr hydrolysis GC/MS Nitrotyrosine Immunoassay Hydrolysis HPLC HPLC/electrochemical detection Tryptophanyl Fluorescence modifications Amino acid analysis (alkaline hydrolysis) Proteolysis/MS Hydroperoxides KI/I 3 - /spectroscopy NaBH 4 /hydrolysis/OPA-HPLC # See Table 4 in Shacter, E. (2000) Drug Metab. Rev . 32 , 307-326. Protein Oxidation Society For Free Radical Biology and Medicine Shacter 15

16. Methods for detection of oxidative protein modifications, con # Modification Methods of Detection Lipid peroxidation adducts Immunoassays DNPH NaBH 4 /hydrolysis/OPA-HPLC Hydrolysis GC/MS Amino acid oxidation adducts NaCNBH 3 reduction/hydrolysis /H 1 -NMR/MS Glycoxidation adducts Derivitization GC/MS Cross-links, aggregates, SDS-gel electrophoresis fragments HPLC Thiyl radicals ESR # See Table 4 in Shacter, E. (2000) Drug Metab. Rev. 32 , 307-326. Protein Oxidation Society For Free Radical Biology and Medicine Shacter 16

17. Protein Oxidation Society For Free Radical Biology and Medicine Shacter 17 A little more about protein carbonyls Carbonyl groups are stable ( aids detection and storage ) Present at low levels in most protein preparations (~1 nmol/mg protein ~ 0.05 mol/mol ~ 1/3000 amino acids) See 2- to 8- fold elevations of protein carbonyls under conditions of oxidative stress in vivo Induced in vitro by almost all types of oxidants (site-specific metal catalyzed oxidation, -irradiation, HOCl, ozone, 1 O 2 , lipid peroxide adducts) Sensitive assays are available ( 1 pmol)

18. Protein Oxidation Society For Free Radical Biology and Medicine Shacter 18 Amino acids that undergo metal-catalyzed oxidation to form carbonyl products Proline ( -glutamylsemialdehyde) Arginine ( -glutamylsemialdehyde) Lysine (amino-adipicsemialdehyde) Threonine (amino-ketobutyrate)

19. Protein Oxidation Society For Free Radical Biology and Medicine Shacter 19 Detection of protein carbonyls Measure total protein carbonyls levels after reaction with DNPH* followed by spectroscopy (A370), ELISA, or immunohistochemistry Measure carbonyl levels in individual proteins within a mixture of proteins (tissue samples, cell extracts) by reaction with DNPH followed by Western blot immunoassay *DNPH, dinitrophenylhydrazine

20. Protein Oxidation Society For Free Radical Biology and Medicine Shacter 20 Measurement of total carbonyls (Spectrophotometric DNPH assay) protein oxidized protein O Fe DNPH Absorbance at 370 nm e.g. arg ---> -glutamylsemialdehyde H 2 O 2 DNP- protein DNP Dinitrophenylhydrazone-protein activated neutrophil

21. Protein Oxidation Society For Free Radical Biology and Medicine Shacter 21 Immunoassays for protein carbonyls e.g. , Western blot, ELISA, immunohistochemistry

22. Protein Oxidation Society For Free Radical Biology and Medicine Shacter 22 Western blot assay for protein carbonyls Detects individual oxidized proteins within a mixture of proteins Requires ~ 50 ng of protein Sensitivity of 1 pmol of protein carbonyl ~50 ng of a 50 kDa protein oxidized @ 0.5 mol/mol Reveals differential susceptibility of individual proteins to oxidative modification* * Shacter et al . (1994) Free Radic. Biol. Med. 17 , 429-437

23. Protein Oxidation Society For Free Radical Biology and Medicine Shacter 23 Notes Carbohydrate groups of glycoproteins do not contribute to carbonyl levels* Free aldehyde groups from lipid peroxidation adducts ( e.g. , MDA) can react with DNPH Adduct needs to be stable if reduction with NaBH 4 is required to stabilize the adduct, DNPH reactivity will not be seen Western blot assay is only semi-quantitative use titration to estimate carbonyl content** *Lee, Y-J. and Shacter, E. (1995) Arch. Biochem. Biophys. 321 , 175-181 ** Shacter, E. et al. (1994) Free Radic. Biol. Med . 17 , 429-437

24. Protein Oxidation Society For Free Radical Biology and Medicine Shacter 24 Reagents and equipment* 20 mM DNPH in 20% trifluoroacetic acid (TFA) 24% SDS in water Neutralizing solution (2M Tris/30% glycerol 20% -ME) Sample protein(s) Oxidized and native protein samples SDS-gel electrophoresis and Western blotting apparatus and conventional solutions Anti-DNP antibody (Sigma D-8406, IgE) Rat anti-mouse IgE, conjugated for immunoassay detection (biotin, HRP) * See Shacter (2000) Meth. Enzymol. 319 , 428-436 or Levine, R.L., Williams, J., Stadtman, E.R., and Shacter, E. (1994) Meth. Enzymol. 233 , 346-357

25. Protein Oxidation Society For Free Radical Biology and Medicine Shacter 25 Technical Pointers Can be used on cell and tissue extracts Dissolve the DNPH in 100% TFA and then dilute with H 2 O Total protein bands can be visualized with Amido black stain after washing the blot Always run positive and negative controls internal standards of oxidized and non-oxidized control protein adjust exposure time if doing chemiluminescence Run controls without DNPH or primary antibody to establish specificity

26. Protein Oxidation Society For Free Radical Biology and Medicine Shacter 26 Other DNPH immunoassays for protein carbonyls ELISA Buss et al . (1997) Free Radic. Biol. Chem. 23 , 361-366 2D gel electrophoresis/immunoblotting Yan et al . (1998) Anal. Biochem. 263 , 67-71 Immunohistochemistry Smith et al. (1998) J. Histochem. Cytochem. 46 , 731-735

27. Protein Oxidation Society For Free Radical Biology and Medicine Shacter 27 A little more about protein sulfur group oxidations In general, Cys and Met are the amino acids that are most susceptible to oxidation Distinguished from other oxidative protein modifications in that cells have mechanisms to reverse the oxidation e.g ., methonine sulfoxide reductase e.g ., glutathione or thioredoxin redox systems Hence may serve a regulatory function Reversible oxidation/reduction of methionine may protect proteins from more damaging forms of oxidative modification ( e.g ., carbonyl formation)* * Stadtman, E. R., Moskovitz, J., Berlett, B. S., and Levine, R. L. (2002) Mol. Cell. Biochem. 234-235 , 3-9

28. Protein Oxidation Society For Free Radical Biology and Medicine Shacter 28 A little more about HOCl-induced protein oxidation Primary products are chloro- and di-tyrosyl residues, amino acyl aldehyde adducts, and chloramines Represent unique products of myeloperoxidase activity, reflecting neutrophil and monocyte activity Serve as markers for oxidants generated as part of the inflammatory response Are elevated in atherosclerotic plaques Can be detected with sensitive and specific assays See Heinecke, J.W. (2002) Free Radic. Biol. Med. 32 , 1090-1101 Winterbourne, C.C. and Kettle, A.J. (2000) Free Radic. Biol. Med. 29 , 403-409 Hazell, L.J. et al. (1996) J. Clin. Invest. 97 , 1535-1544

29. Protein Oxidation Society For Free Radical Biology and Medicine Shacter 29 A little more about lipid peroxidation adducts Indirect oxidative protein modification through attachment of lipid peroxidation breakdown products ( e.g. , hydroxynonenal, malondialdehyde, acrolein) to Lys, Cys, and His residues in proteins Generated by a variety of oxidizing systems, predominantly metal-catalyzed oxidation and -irradiation Elevated in atherosclerosis and neurodegenerative diseases Detected with immunoassays specific for each type of protein adduct See Uchida, K. (2000) Free Radic. Biol. Med. 28 , 1685-1696

30. Some recent review articles on protein oxidation Protein Oxidation Society For Free Radical Biology and Medicine Shacter 30