Amino Acids

, Volume 3, Issue 2, pp 183–194 | Cite as

Oxygen radical induced fluorescence in proteins; identification of the fluorescent tryptophan metabolite, N-formyl kynurenine, as a biological index of radical damage

  • H. R. Griffiths
  • J. Lunec
  • D. R. Blake
Article

Summary

The effect of oxygen derived free radicals (OFR) on aromatic and sulphur containing amino acids has been investigated, both in their free form and within protein backbones. Aerated amino acids and proteins in solution were exposed to three discrete OFR generating systems; (1) gamma radiation in the presence or absence of formate (2) photolysis by UV light at 254 and 366 nm, and (3) site specific modification by H2O2 in the presence of CuII ions.

A sensitive reverse-phase HPLC technique with dual detection systems (UV absorbance and fluorescence monitoring) was developed to analyse the products of amino acid oxidation. OFR denatured amino acids were chromatographed by this procedure, and all radical species generated, with the exception of the superoxide anion, resulted in the formation of identifiable fluorescent metabolites of tryptophan, kynurenines. The identity of peaks was confimed by spiking with authentic material and scanning absorption spectroscopy. After complete proteolytic hydrolysis, OFR treated proteins were also analysed by this technique; again the dose dependent production of kynurenines was detected in IgG,γ lens crystallins and albumin. Bityrosine was not detected in any of the proteins studied using this procedure, however, several novel unidentified fluorophores were detected in proteolytic hydrolysates, possibly the product of two different amino acid radicals.

Immunoglobulin G isolated from the sera of normals and rheumatoid arthritis (RA) patients was examined for the presence of one specific tryptophan metabolite, N-formyl kynurenine. Significantly elevated levels of this metabolite were detected in rheumatoid sera, suggesting increased OFR activity in RA.

These results have demonstrated firstly, that specific oxidised products of amino acids are retained in the protein backbone after exposure to OFR generating systems. Secondly, in aerated solution, oxidised tryptophan residues confer the major new visible fluorescence in non-haem proteins, not tyrosine products. In addition, this work has demonstrated that the measurement of a specific product of an oxidised amino acid can be applied to biological macromolecules, and may be important in implicating free radical reactions in certain disease processes.

Keywords

Amino Acids Protein fluorescence Kynurenines Oxygen free radicals 

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References

  1. 1.
    Halliwell B, Gutteridge JMC (1984) Biochem J 219: 1–4Google Scholar
  2. 2.
    Weiss SJ (1986) Acta Physiol Scand [Suppl] 548: 9–37Google Scholar
  3. 3.
    Fantone JL, Ward PA (1982) A J Pathol 107: 397–418Google Scholar
  4. 4.
    Halliwell B, Gutteridge JMC, Blake, DR (1985) Phil Trans R Soc Lond M B311: 659–671Google Scholar
  5. 5.
    Tate RM, Repine JE (1984) In: Pryor WA (ed) Free radicals in biology, vol 6. Academic Press, Florida, pp 199–207Google Scholar
  6. 6.
    Garner MM, Spector A (1980) Proc Natl Acad Sci USA 77: 1274–1277Google Scholar
  7. 7.
    Griffiths HR, Lunec J, Gee CA, Willson RL (1988) FEBS Lett 230: 155–158Google Scholar
  8. 8.
    Lunec J, Blake DR, McCleary SJ, Brailsford S, Bacon PA (1985) J Clin Invest 76: 2084–2090Google Scholar
  9. 9.
    Carp H, Janoff A (1979) J Clin Invest 63: 793–797Google Scholar
  10. 10.
    Wolff SP, Garner A, Dean RT (1986) Trends Biochem Sci 11: 27–31Google Scholar
  11. 11.
    Pryor WA (1986) Ann Rev Physiol 48: 657–667Google Scholar
  12. 12.
    Frankel EN (1980) Prog Lip Res 19: 1–22Google Scholar
  13. 13.
    Lunec J, Griffiths HR, Blake DR (1987) In: Rice-Evans C (ed) Free radicals, cell damage and disease. Richelieu Press, London, pp 151–167Google Scholar
  14. 14.
    Davies KJA (1987) J Biol Chem 236: 397–400Google Scholar
  15. 15.
    Jones AF, Lunec J (1987) Br J Cancer 55 [Suppl]: 60–65Google Scholar
  16. 16.
    Marx G, Chevion M (1985) Biochem J 236: 397–400Google Scholar
  17. 17.
    Wolff SP, Dean RT (1986) Biochem J 234: 399–403Google Scholar
  18. 18.
    Ahn B, Rhee, SG, Stadtman ER (1987) Analyt Biochem 161: 245–257Google Scholar
  19. 19.
    Lunec J (1980) Biochem Soc Proc 10: 21Google Scholar
  20. 20.
    Aloedees HJ, Bloemendal H (1981) In: Bloemendal H (ed) Molecular and cellular biology of the eye lens. John Wiley, New YorkGoogle Scholar
  21. 21.
    Creed D (1984) Photochem Photobiol 39: 577–583Google Scholar
  22. 22.
    Creed D (1984) Photochem Photobiol 39: 537–562Google Scholar
  23. 23.
    Singh A, Singh H, Kremers W, Koroll GW (1981) Bull Europ Physiopath Resp 17: 31–41Google Scholar
  24. 24.
    Prutz WA, Butler J, Land EJ (1983) Int J Radiat Biol 44: 183–196Google Scholar
  25. 25.
    Bodaness RS, Leclair M, Zigler JS (1984) Arch Biochem Biophys 231: 461–469Google Scholar
  26. 26.
    Pirie A (1971) Biochem J 125: 203–208Google Scholar
  27. 27.
    Susho P, Pulcini D, Ragone R, Miele L, Della Pietra G, Colonna G (1988) Arch Biochem Biophys 266: 61–71Google Scholar
  28. 28.
    McKinney MM, Parkinson A (1987) J Immunol Methods 96: 291–298Google Scholar
  29. 29.
    Lowry OH, Rosenborough NJ, Farr AL, Randall KJ (1951) Protein measurement with the protein-phenol reagent. J Biol Chem 193: 265–275Google Scholar
  30. 30.
    Tsan MF, Chen JW (1980) J Clin Invest 65: 1041–1050Google Scholar
  31. 31.
    Levine RL (1983) J Biol Chem 258: 11823–11827Google Scholar
  32. 32.
    Sahlin M, Petersson L, Graslund A, Ehrenberg A, Sjoberg BM, Nelander L (1987) Biochemistry 26: 5541–5548Google Scholar
  33. 33.
    Tew D, Cortiz de Montellano, PR (1988) J Biol Chem 263: 17880–17886Google Scholar
  34. 34.
    Freeman BA, J Crapo JD (1982) Lab Invest 47/5: 412–426Google Scholar
  35. 35.
    Jasin HE (1983) J Immunol 130: 1918–1923Google Scholar
  36. 36.
    Lunec J, Blake DR (1985) Free Radic Res Commun 1: 131–139Google Scholar

Copyright information

© Springer-Verlag 1992

Authors and Affiliations

  • H. R. Griffiths
    • 1
  • J. Lunec
    • 1
  • D. R. Blake
    • 2
  1. 1.Molecular Toxicity GroupUniversity of Leicester, Glenfield HospitalLeicesterEngland
  2. 2.Bone and Joint Research UnitLondon Hospital Medical CollegeLondenEngland

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