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. 1998 Nov;7(11):2391–2397. doi: 10.1002/pro.5560071116

Identification of tryptophan oxidation products in bovine alpha-crystallin.

E L Finley 1, J Dillon 1, R K Crouch 1, K L Schey 1
PMCID: PMC2143850  PMID: 9828005

Abstract

Oxidation is known to affect the structure, activity, and rate of degradation of proteins, and is believed to contribute to a variety of pathological conditions. Metal-catalyzed oxidation (MCO) is a primary oxidizing system in many cell types. In this study, the oxidative effects of a MCO system (the Fenton reaction) on the structure of the tryptophan residues of alpha-crystallin were determined. Tandem mass spectrometry (MS/MS) was utilized to identify specific tryptophan and methionine oxidation products in the bovine alpha-crystallin sequence. After oxidative exposure, alpha-crystallin was digested with trypsin, and the resulting peptides were fractionated by reverse-phase HPLC. Structural analysis by mass spectrometry revealed that tryptophan 9 of alphaA- and tryptophan 60 of alphaB-crystallin were each converted into hydroxytryptophans (HTRP), N-formylkynurenine (NFK), and kynurenine (KYN). However, only HTRP and KYN formation were detected at residue 9 of alphaB-crystallin. Oxidation of methionine 1 of alphaA- and methionine 1 and 68 of alphaB-crystallin was also detected. The products NFK and KYN are of particular importance in the lens, as they themselves are photosensitizers that can generate reactive oxygen species (ROS) upon UV light absorption. The unambiguous identification of HTRP, NFK, and KYN in intact alpha-crystallin represents the first structural proof of the formation of these products in an intact protein, and provides a basis for detailed structural analysis of oxidized proteins generated in numerous pathological conditions.

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Selected References

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  1. 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 reactions. J Biol Chem. 1989 Feb 25;264(6):3341–3346. [PubMed] [Google Scholar]
  2. Andley U. P., Clark B. A. Generation of oxidants in the near-UV photooxidation of human lens alpha-crystallin. Invest Ophthalmol Vis Sci. 1989 Apr;30(4):706–713. [PubMed] [Google Scholar]
  3. Andley U. P., Clark B. A. Photoreactions of human lens monomeric crystallins. Biochim Biophys Acta. 1989 Aug 31;997(3):284–291. doi: 10.1016/0167-4838(89)90199-4. [DOI] [PubMed] [Google Scholar]
  4. Andley U. P., Mathur S., Griest T. A., Petrash J. M. Cloning, expression, and chaperone-like activity of human alphaA-crystallin. J Biol Chem. 1996 Dec 13;271(50):31973–31980. doi: 10.1074/jbc.271.50.31973. [DOI] [PubMed] [Google Scholar]
  5. Aruoma O. I., Halliwell B. Inactivation of alpha 1-antiproteinase by hydroxyl radicals. The effect of uric acid. FEBS Lett. 1989 Feb 13;244(1):76–80. doi: 10.1016/0014-5793(89)81166-4. [DOI] [PubMed] [Google Scholar]
  6. Bando M., Nakajima A., Satoh K. Coloration of human lens protein. Exp Eye Res. 1975 May;20(5):489–492. doi: 10.1016/0014-4835(75)90089-5. [DOI] [PubMed] [Google Scholar]
  7. Berlett B. S., Stadtman E. R. Protein oxidation in aging, disease, and oxidative stress. J Biol Chem. 1997 Aug 15;272(33):20313–20316. doi: 10.1074/jbc.272.33.20313. [DOI] [PubMed] [Google Scholar]
  8. Bhat S. P., Nagineni C. N. alpha B subunit of lens-specific protein alpha-crystallin is present in other ocular and non-ocular tissues. Biochem Biophys Res Commun. 1989 Jan 16;158(1):319–325. doi: 10.1016/s0006-291x(89)80215-3. [DOI] [PubMed] [Google Scholar]
  9. Biemann K., Scoble H. A. Characterization by tandem mass spectrometry of structural modifications in proteins. Science. 1987 Aug 28;237(4818):992–998. doi: 10.1126/science.3303336. [DOI] [PubMed] [Google Scholar]
  10. Chapman M. L., Rubin B. R., Gracy R. W. Increased carbonyl content of proteins in synovial fluid from patients with rheumatoid arthritis. J Rheumatol. 1989 Jan;16(1):15–18. [PubMed] [Google Scholar]
  11. Dillon J., Garcia Castineiras S., Santiago M. A., Spector A. The endopeptidase-resistant protein fraction from human cataractous lenses. Exp Eye Res. 1984 Jul;39(1):95–106. doi: 10.1016/0014-4835(84)90118-0. [DOI] [PubMed] [Google Scholar]
  12. Finley E. L., Busman M., Dillon J., Crouch R. K., Schey K. L. Identification of photooxidation sites in bovine alpha-crystallin. Photochem Photobiol. 1997 Nov;66(5):635–641. doi: 10.1111/j.1751-1097.1997.tb03200.x. [DOI] [PubMed] [Google Scholar]
  13. Fujimori E. Crosslinking and photoreaction of ozone-oxidized calf-lens alpha-crystallin. Invest Ophthalmol Vis Sci. 1982 Mar;22(3):402–405. [PubMed] [Google Scholar]
  14. Garland D. Role of site-specific, metal-catalyzed oxidation in lens aging and cataract: a hypothesis. Exp Eye Res. 1990 Jun;50(6):677–682. doi: 10.1016/0014-4835(90)90113-9. [DOI] [PubMed] [Google Scholar]
  15. Garland D., Russell P., Zigler J. S., Jr The oxidative modification of lens proteins. Basic Life Sci. 1988;49:347–352. doi: 10.1007/978-1-4684-5568-7_52. [DOI] [PubMed] [Google Scholar]
  16. Gershon H., Gershon D. Detection of inactive enzyme molecules in ageing organisms. Nature. 1970 Sep 19;227(5264):1214–1217. doi: 10.1038/2271214a0. [DOI] [PubMed] [Google Scholar]
  17. Grossweiner L. I. Photochemistry of proteins: a review. Curr Eye Res. 1984 Jan;3(1):137–144. doi: 10.3109/02713688408997195. [DOI] [PubMed] [Google Scholar]
  18. Holt L. A., Milligan B., Rivett D. E., Stewart F. H. The photodecomposition of tryptophan peptides. Biochim Biophys Acta. 1977 Aug 25;499(1):131–138. doi: 10.1016/0304-4165(77)90235-5. [DOI] [PubMed] [Google Scholar]
  19. Imlay J. A., Linn S. DNA damage and oxygen radical toxicity. Science. 1988 Jun 3;240(4857):1302–1309. doi: 10.1126/science.3287616. [DOI] [PubMed] [Google Scholar]
  20. Kitano H., Maeda Y., Furukawa K., Yamamoto T., Izumida R., Matsugo S. Modification of alpha-chymotrypsin using a water-soluble photo-Fenton reagent. Photochem Photobiol. 1995 Nov;62(5):809–812. doi: 10.1111/j.1751-1097.1995.tb09140.x. [DOI] [PubMed] [Google Scholar]
  21. Lagerwerf F. M., van de Weert M., Heerma W., Haverkamp J. Identification of oxidized methionine in peptides. Rapid Commun Mass Spectrom. 1996;10(15):1905–1910. doi: 10.1002/(SICI)1097-0231(199612)10:15<1905::AID-RCM755>3.0.CO;2-9. [DOI] [PubMed] [Google Scholar]
  22. Maskos Z., Rush J. D., Koppenol W. H. The hydroxylation of tryptophan. Arch Biochem Biophys. 1992 Aug 1;296(2):514–520. doi: 10.1016/0003-9861(92)90605-v. [DOI] [PubMed] [Google Scholar]
  23. Murphy M. E., Kehrer J. P. Oxidation state of tissue thiol groups and content of protein carbonyl groups in chickens with inherited muscular dystrophy. Biochem J. 1989 Jun 1;260(2):359–364. doi: 10.1042/bj2600359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Okajima T., Kawata Y., Hamaguchi K. Chemical modification of tryptophan residues and stability changes in proteins. Biochemistry. 1990 Oct 2;29(39):9168–9175. doi: 10.1021/bi00491a010. [DOI] [PubMed] [Google Scholar]
  25. Oliver C. N., Levine R. L., Stadtman E. R. A role of mixed-function oxidation reactions in the accumulation of altered enzyme forms during aging. J Am Geriatr Soc. 1987 Oct;35(10):947–956. doi: 10.1111/j.1532-5415.1987.tb02297.x. [DOI] [PubMed] [Google Scholar]
  26. Oliver C. N., Starke-Reed P. E., Stadtman E. R., Liu G. J., Carney J. M., Floyd R. A. Oxidative damage to brain proteins, loss of glutamine synthetase activity, and production of free radicals during ischemia/reperfusion-induced injury to gerbil brain. Proc Natl Acad Sci U S A. 1990 Jul;87(13):5144–5147. doi: 10.1073/pnas.87.13.5144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pirie A. Color and solubility of the proteins of human cataracts. Invest Ophthalmol. 1968 Dec;7(6):634–650. [PubMed] [Google Scholar]
  28. Pirie A. Formation of N'-formylkynurenine in proteins from lens and other sources by exposure to sunlight. Biochem J. 1971 Nov;125(1):203–208. doi: 10.1042/bj1250203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Reszka K. J., Bilski P., Chignell C. F., Dillon J. Free radical reactions photosensitized by the human lens component, kynurenine: an EPR and spin trapping investigation. Free Radic Biol Med. 1996;20(1):23–34. doi: 10.1016/0891-5849(95)02018-7. [DOI] [PubMed] [Google Scholar]
  30. Roberts J. E., Dillon J. Comparison of endogenous and exogenous photosensitization in the lens using in vitro and photophysical studies. Lens Eye Toxic Res. 1989;6(1-2):309–318. [PubMed] [Google Scholar]
  31. Samuni A., Aronovitch J., Godinger D., Chevion M., Czapski G. On the cytotoxicity of vitamin C and metal ions. A site-specific Fenton mechanism. Eur J Biochem. 1983 Dec 1;137(1-2):119–124. doi: 10.1111/j.1432-1033.1983.tb07804.x. [DOI] [PubMed] [Google Scholar]
  32. Schauerte J. A., Gafni A. Photodegradation of tryptophan residues and attenuation of molecular chaperone activity in alpha-crystallin are correlated. Biochem Biophys Res Commun. 1995 Jul 26;212(3):900–905. doi: 10.1006/bbrc.1995.2054. [DOI] [PubMed] [Google Scholar]
  33. Sen A. C., Ueno N., Chakrabarti B. Studies on human lens: I. Origin and development of fluorescent pigments. Photochem Photobiol. 1992 May;55(5):753–764. [PubMed] [Google Scholar]
  34. Smith C. D., Carney J. M., Starke-Reed P. E., Oliver C. N., Stadtman E. R., Floyd R. A., Markesbery W. R. Excess brain protein oxidation and enzyme dysfunction in normal aging and in Alzheimer disease. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10540–10543. doi: 10.1073/pnas.88.23.10540. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Smith J. B., Jiang X., Abraham E. C. Identification of hydrogen peroxide oxidation sites of alpha A- and alpha B-crystallins. Free Radic Res. 1997 Feb;26(2):103–111. doi: 10.3109/10715769709097789. [DOI] [PubMed] [Google Scholar]
  36. Spector A., Garner W. H. Hydrogen peroxide and human cataract. Exp Eye Res. 1981 Dec;33(6):673–681. doi: 10.1016/s0014-4835(81)80107-8. [DOI] [PubMed] [Google Scholar]
  37. Spector A. Oxidative stress-induced cataract: mechanism of action. FASEB J. 1995 Sep;9(12):1173–1182. [PubMed] [Google Scholar]
  38. Stadtman E. R. Metal ion-catalyzed oxidation of proteins: biochemical mechanism and biological consequences. Free Radic Biol Med. 1990;9(4):315–325. doi: 10.1016/0891-5849(90)90006-5. [DOI] [PubMed] [Google Scholar]
  39. Stadtman E. R. Oxidation of free amino acids and amino acid residues in proteins by radiolysis and by metal-catalyzed reactions. Annu Rev Biochem. 1993;62:797–821. doi: 10.1146/annurev.bi.62.070193.004053. [DOI] [PubMed] [Google Scholar]
  40. Stadtman E. R. Protein oxidation and aging. Science. 1992 Aug 28;257(5074):1220–1224. doi: 10.1126/science.1355616. [DOI] [PubMed] [Google Scholar]
  41. Thomas C. E., Morehouse L. A., Aust S. D. Ferritin and superoxide-dependent lipid peroxidation. J Biol Chem. 1985 Mar 25;260(6):3275–3280. [PubMed] [Google Scholar]
  42. Van Heyningen R. Fluorescent glucoside in the human lens. Nature. 1971 Apr 9;230(5293):393–394. doi: 10.1038/230393a0. [DOI] [PubMed] [Google Scholar]
  43. Walker M. L., Borkman R. F. Light scattering and photocrosslinking in the calf lens crystallins gamma-II, III and IV. Exp Eye Res. 1989 Mar;48(3):375–383. doi: 10.1016/s0014-4835(89)80006-5. [DOI] [PubMed] [Google Scholar]
  44. Walrant P., Santus R. N-formyl-kynurenine, a tryptophan photooxidation product, as a photodynamic sensitizer. Photochem Photobiol. 1974 Jun;19(6):411–417. doi: 10.1111/j.1751-1097.1974.tb06533.x. [DOI] [PubMed] [Google Scholar]
  45. Zigler J. S., Jr, Goosey J. D. Photosensitized oxidation in the ocular lens: evidence for photosensitizers endogenous to the human lens. Photochem Photobiol. 1981 Jun;33(6):869–874. doi: 10.1111/j.1751-1097.1981.tb05505.x. [DOI] [PubMed] [Google Scholar]

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