Pharmaceutical Research

, Volume 21, Issue 12, pp 2377–2383 | Cite as

Effects of Antioxidants on the Hydrogen Peroxide-Mediated Oxidation of Methionine Residues in Granulocyte Colony-Stimulating Factor and Human Parathyroid Hormone Fragment 13-34

  • Jin Yin
  • Jhih-Wei Chu
  • Margaret Speed Ricci
  • David N. Brems
  • Daniel I. C. Wang
  • Bernhardt L. Trout

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The effects and mechanisms of different antioxidants, methionine, glutathione, acetylcysteine, and ascorbic acid (AscH2), on the oxidation of methionine residues in granulocyte colony-stimulating factor (G-CSF) and human parathyroid hormone fragment 13-34 (hPTH 13-34) by hydrogen peroxide (H2O2) were quantified and analyzed.


The rates of oxidation of methionine residues in G-CSF were determined by peptide mapping analyses, and the oxidation of methionine residue in hPTH 13-34 was quantified by reverse-phase HPLC.


At pH 4.5, free methionine reduces, glutathione and acetylcysteine have no obvious effect on, and AscH2 promotes the rates of oxidation of methionine residues in G-CSF. The H2O2-induced oxidation rate constants for free methionine, acetylcysteine, and glutathione at pH 4.5 were measured to be 32.07, 1.00, and 1.63 M-1h-1, respectively, while the oxidation rate constant for Met1, the most readily oxidizable methionine residue in G-CSF, is 13.95 M−1h−1. Therefore, the different effects of free methionine, acetylcysteine, and glutathione on the rates of oxidation of methionine residues in G-CSF are consistent with their different reactivity toward oxidation by H2O2. By using hPTH 13-34, the effect of AscH2 on the H2O2-induced oxidation of methionine residue was quantified, and the mechanisms involved were proposed. Because of the presence of trace transition metal ions in solution, at low concentrations, AscH2 is prone to be a prooxidant, increasing the hydroxyl radical (⋅OH) production rate via Fenton-type reactions. In addition to peroxide oxidation, these radicals lead to the degradation of hPTH 13-34 to smaller peptide fragments. At high concentrations, AscH2 tends to act as an ⋅OH scavenger. EDTA inhibits ⋅OH production and thus eliminates the degradation of hPTH 13-34 by forming complexes with transition metal ions. However, the rate of oxidation of the methionine residue in hPTH 13-34 increases as the concentration of AscH2 is increased from 0 to 200 mM, and the reason for this is still not clear.


Our results demonstrate that free methionine is an effective antioxidant to protect G-CSF against methionine oxidation at pH 4.5. Acetylcysteine and glutathione are not effective antioxidants at pH 4.5. Their oxidation rates at different pH values imply that they would be much more effective antioxidants than free methionine at alkaline conditions. AscH2 is a powerful electron donor. It acts as a prooxidant in the conditions in this study and is unlikely to prevent oxidation by H2O2 in protein formulation, whether or not EDTA is present.

Key words:

antioxidants ascorbic acid granulocyte colony-stimulating factor (G-CSF) human parathyroid hormone (hPTH) methionine oxidation 


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  1. 1.
    1. S. H. Li, C. Schöneich, and R. T. Borchardt. Chemical-instability of protein pharmaceuticals - mechanisms of oxidation and strategies for stabilization. Biotechnol. Bioeng. 48:490–500 (1995).Google Scholar
  2. 2.
    2. W. Wang. Instability, stabilization, and formulation of liquid protein pharmaceuticals. Int. J. Pharm. 185:129–188 (1999).Google Scholar
  3. 3.
    3. Y. Nabuchi, E. Fujiwara, K. Ueno, H. Kuboniwa, Y. Asoh, and H. Ushio. Oxidation of recombinant human parathyroid hormone: Effect of oxidized position on the biological activity. Pharm. Res. 12:2049–2052 (1995).Google Scholar
  4. 4.
    4. S. W. Griffiths and C. L. Cooney. Relationship between protein structure and methionine oxidation in recombinant human alpha 1-antitrypsin. Biochemistry 41:6245–6252 (2002).Google Scholar
  5. 5.
    5. S. M. Van Patten, E. Hanson, R. Bernasconi, K. Zhang, P. Manavalan, E. S. Cole, J. M. McPherson, and T. Edmunds. Oxidation of methionine residues in antithrombin - Effects on biological activity and heparin binding. J. Biol. Chem. 274:10268–10276 (1999).Google Scholar
  6. 6.
    6. E. T. Duenas, R. Keck, A. De Vos, A. J. S. Jones, and J. L. Cleland. Comparison between light induced and chemically induced oxidation of rhVEGF. Pharm. Res. 18:1455–1460 (2001).Google Scholar
  7. 7.
    7. J. L. Liu, K. V. Lu, T. Eris, V. Katta, K. R. Westcott, L. O. Narhi, and H. S. Lu. In vitro methionine oxidation of recombinant human leptin. Pharm. Res. 15:632–640 (1998). Google Scholar
  8. 8.
    8. H. S. Lu, P. R. Fausset, L. O. Narhi, T. Horan, K. Shinagawa, G. Shimamoto, and T. C. Boone. Chemical modification and site-directed mutagenesis of methionine residues in recombinant human granulocyte colony-stimulating factor: effect on stability and biological activity. Arch. Biochem. Biophys. 362:1–11 (1999).Google Scholar
  9. 9.
    9. J. L. Cleland and R. Langer. Formulation and delivery of proteins and peptides - design and development strategies. ACS Sym. Ser 567:1–19 (1994).Google Scholar
  10. 10.
    10. T. Osterberg and A. Fatouros. Oxygen-reduced aqueous solution of factor VIII. U.S. Patent No. 5,962,650 (1999)Google Scholar
  11. 11.
    11. T. Osterberg and A. Fatouros. Protein formulation comprising coagulation factor VIII or factor IX in an aqueous solution. U.S. Patent No. 5,919,908 (1999)Google Scholar
  12. 12.
    12. S. H. Li, C. Schöneich, and R. T. Borchardt. Chemical pathways of peptide degradation. VIII. Oxidation of methionine in small model peptides by prooxidant/transition metal ion systems: influence of selective scavengers for reactive oxygen intermediates. Pharm. Res. 12:348–355 (1995).Google Scholar
  13. 13.
    13. V. M. Knepp, J. L. Whatley, A. Muchnik, and T. S. Calderwood. Identification of antioxidants for prevention of peroxide-mediated oxidation of recombinant human ciliary neurotrophic factor and recombinant human nerve growth factor. PDA J. Pharm. Sci. Technol. 50(3):163–171 (1996).Google Scholar
  14. 14.
    14. X. M. Lam, J. Y. Yang, and J. L. Cleland. Antioxidants for prevention of methionine oxidation in recombinant monoclonal antibody HER2. J. Pharm. Sci. 86:1250–1255 (1997).Google Scholar
  15. 15.
    15. J. A. Cadee, M. J. van Steenbergen, C. Versluis, A. J. R. Heck, W. J. M. Underberg, W. den Otter, W. Jiskoot, and W. E. Hennink. Oxidation of recombinant human interleukin-2 by potassium peroxodisulfate. Pharm. Res. 18:1461–1467 (2001).Google Scholar
  16. 16.
    16. S. H. Li, C. Schöneich, G. S. Wilson, and R. T. Borchardt. Chemical pathways of peptide degradation. V. ascorbic-acid promotes rather than inhibits the oxidation of methionine to methionine sulfoxide in small model peptides. Pharm. Res. 10:1572–1579 (1993).Google Scholar
  17. 17.
    17. J. C. Deutsh. Ascorbic acid oxidation of hydrogen peroxide. Anal. Biochem. 255:1–7 (1998).Google Scholar
  18. 18.
    18. B. Bishop, D. C. Koay, A. C. Sartorelli, and L. Regan. Reengineering granulocyte colony-stimulating factor for enhanced stability. J. Biol. Chem. 276:33465–33470 (2001).Google Scholar
  19. 19.
    19. J.-W. Chu, J. Yin, D. I. C. Wang, and B. L. Trout. Molecular dynamics and oxidation rates of methionine residues of granulocyte colony-stimulating factor (G-CSF) at different pH values. Biochemistry 43:1019–1029 (2004).Google Scholar
  20. 20.
    20. A. L. Frelinger and J. E. Zull. Oxidized forms of parathyroid-hormone with biological-activity - separation and characterization of hormone forms oxidized at methionine-8 and methionine-18. J. Biol. Chem. 259:5507–5513 (1984).Google Scholar
  21. 21.
    21. Available at: (accessed 4/27/2004).Google Scholar
  22. 22.
    22. S. P. Wolff. Ferrous ion oxidation in presence of ferric ion indicator xylenol orange for measurement of hydroperoxides. Method. Enzymol. 233:182–189 (1994).Google Scholar
  23. 23.
    23. C. C. Winterbourn and D. Metodiewa. Reactivity of biologically important thiol compounds with superoxide and hydrogen peroxide. Free Radical Bio. Med. 27:322–328 (1999).Google Scholar
  24. 24.
    24. J. C. Deutsh. Oxygen-accepting antioxidants which arise during ascorbate oxidation. Anal. Biochem. 265:238–245 (1998).Google Scholar
  25. 25.
    25. J. C. Deutsh. Dehydroascorbic acid. J. Chromatogr. A. 881:299–307 (2000).Google Scholar
  26. 26.
    26. P. Wardman and L. P. Candeias. Fenton chemistry: an introduction. Radiat. Res. 145:523–531 (1996).Google Scholar
  27. 27.
    27. M. J. Burkitt and B. C. Gilbert. Model studies of the iron-catalysed Haber-Weiss cycle and the ascorbate-driven Fenton reation. Free Radical Res. Com. 10:265–280 (1990). Google Scholar
  28. 28.
    28. M. J. Zhao and L. Jung. Kinetics of the competitive degradation of deoxyribose and other molecules by hydroxyl radicals produced by the Fenton reaction in the presence of ascorbic-acid. Free Radic. Res. 23:229–243 (1995).Google Scholar
  29. 29.
    29. G. R. Buettner and B. A. Jurkiewicz. Catalytic metals, ascorbate and free radicals: combinations to avoid. Radiat. Res. 145:532–541 (1996).Google Scholar
  30. 30.
    30. J. E. Biaglow, Y Manevich, F. Uckun, and K.D. Held. Quantitation of hydroxyl radicals produced by radiation and copper-linked oxidation of ascorbate by 2-deoxy-D-ribose method. Free Radical Bio. Med. 22:1129–1138 (1997).Google Scholar
  31. 31.
    31. A. Rees and T. F. Slater. Ascorbic acid and lipid peroxidation: the cross-over effect. Acta Biochim. Biophys. 22:241–249 (1987).Google Scholar
  32. 32.
    32. L. Packer and J. Fuchs. Health and Disease, Marcel Dekker Inc., New York, 1997, pp. 75–94Google Scholar
  33. 33.
    33. G. R. Buettner. In the absence of catalytic metals ascorbate does not autoxidize at pH 7: ascorbate as a test for catalytic metals. J. Biochem. Biophys. Methods 16:27–40 (1988).Google Scholar
  34. 34.
    34. G. R. Buettner, T. P. Doherty, and L. K. Patterson. The kinetics of the reaction of superoxide radical with Fe(III) complexes of EDTA, DETAPAC and HEDTA. FEBS Lett. 158:143–146 (1983).Google Scholar
  35. 35.
    35. J. Butler and B. Halliwell. Reaction of iron EDTA chelates with the superoxide radical. Arch. Biochem. Biophys. 218:174–178 (1982).Google Scholar
  36. 36.
    36. K. D. Welch, T. Z. Davis, and S. D. Aust. Iron autoxidation and free radical generation: effects of buffers, ligands, and chelators. Arch. Biochem. Biophys. 397:360–369 (2002).Google Scholar
  37. 37.
    37. J. Van der Zee and P. J. A. Van den Broek. Determination of the ascorbate free radical concentration in mixtures of ascorbate and dehydroascorbate. Free Radical Bio. Med. 25:282–286 (1998).Google Scholar

Copyright information

© Springer Science+Business Media, Inc. 2004

Authors and Affiliations

  • Jin Yin
    • 1
  • Jhih-Wei Chu
    • 1
  • Margaret Speed Ricci
    • 2
  • David N. Brems
    • 2
  • Daniel I. C. Wang
    • 1
  • Bernhardt L. Trout
    • 1
  1. 1.Department of Chemical EngineeringMassachusetts Institute of TechnologyCambridgeUSA
  2. 2.Pharmaceutics DepartmentAmgen, Inc.Thousand OaksUSA

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