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Photodegradation mechanism and reaction kinetics of recombinant human interferon-α2a

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Abstract

The photodegradation mechanism of recombinant human interferon-α2a (IFNα2a) has been investigated using absorption, fluorescence, and circular dichroism (CD) spectroscopies, and fluorescence photobleaching kinetics measurements under various conditions. After photobleaching, the absorption profile of aromatic amino acid residues in IFNα2a was almost absent, and an absorption profile showing a monotonic increase toward short wavelengths was observed. According to the CD spectrum analysis, partial unfolding of IFNα2a was accompanied by a complete loss of fluorescence. This unfolding was attributed to tryptophan-mediated photoinduced disulfide bond cleavage. Photooxygenation and photoionization of tryptophan (Trp) residues followed by subsequent radical reactions were the main photodegradation pathways of IFNα2a. Photobleaching kinetics was faster in acidic solution (pH 2.5) than in neutral solution (pH 7.4). The variation of photobleaching kinetics seemed to be caused by the structural differences in IFNα2a according to the solution pH. The relationship between the protein conformation and photobleaching rate could be explained based on the competition between excited state energy transfer and the photoionization process in Trp residues.

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References

  1. C. R. Caldwell, Ultraviolet-induced photodegradation of cucumber (Cucumis sativus L.) microsomal and soluble protein tryptophanyl residues in vitro, Plant Physiol., 1993, 101, 947–953.

    Article  CAS  Google Scholar 

  2. J. A. Schauerte, A. Gafni, Photodegradation of Tryptophan residues and attenuation of molecular chaperone activity in α-crystallin are correlated, Biochem. Biophys. Res. Commun., 1995, 212, 900–905.

    Article  CAS  Google Scholar 

  3. R. F. Borkman, A. Douhal, K. Yoshihara, Picosecond fluorescence decay in photolyzed lens protein α-crystallin, Biochemistry, 1993, 32, 4787–4792.

    Article  CAS  Google Scholar 

  4. B. L. Miller, M. J. Hageman, T. J. Thamann, L. B. Barròn, C. Schöneich, Solid-state photodegradation of bovine somatotropin (bovine growth hormone): Evidence for tryptophan-mediated photooxidation of disulfide bonds, J. Pharm. Sci., 2003, 92, 1698–1709.

    Article  CAS  Google Scholar 

  5. J. J. Prompers, C. W. Hilbers, H. A. M. Pepermans, Tryptophan mediated photoreduction of disulfide bond causes unusual fluorescence behavior of Fusarium solani pisi cutinase, FEBS Lett., 1999, 456, 409–416.

    Article  CAS  Google Scholar 

  6. M. T. Neves-Petersen, Z. Gryczynski, J. Lakowicz, P. Fojan, S. Pedersen, E. Petersen, S. B. Petersen, High probability of disrupting a disulfide bridge mediated by an endogenous excited Tryptophan residue, Protein Sci., 2002, 11, 588–600.

    Article  CAS  Google Scholar 

  7. A. Vanhooren, B. Devreese, K. Vanhee, J. V. Beeumen, I. Hanssens, Photoexcitation of tryptophan groups induces reduction of two disulfide bonds in goat α-lactalbumin, Biochemistry, 2002, 41, 11035–11043.

    Article  CAS  Google Scholar 

  8. S. Pestka and S. Baron, Definition and classification of the interferons, in Methods in Enzymology; Interferons Part A, ed. S. Pestka, Academic Press, London, 1981, vol. 78, pp. 3–14.

  9. V. K. Sharma, D. S. Kalonia, Temperature- and pH-induced multiple partially unfolded states of recombinant human interferon-α2a: possible implications in protein stability, Pharm. Res., 2003, 20, 1721–1729.

    Article  CAS  Google Scholar 

  10. W. Klaus, B. Gsell, A. M. Labhardt, B. Wipf, H. Senn, The three-dimensional high resolution structure of human interferon-α2a determined by heteronuclear NMR spectroscopy in solution, J. Mol. Biol., 1997, 274, 661–675.

    Article  CAS  Google Scholar 

  11. O. H. Lowry, N. J. Rosebrough, A. L. Farr, R. J. Randall, Protein measurement with the Folin-Phenol reagents, J. Biol. Chem., 1951, 193, 265–275.

    Article  CAS  Google Scholar 

  12. P. K. Smith, R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson, D. C. Klenk, Measurement of Protein Using Bicinchoninic Acid, Anal. Biochem., 1985, 150, 76–85.

    Article  CAS  Google Scholar 

  13. G. L. Ellman, Tissue sulfhydryl group, Arch. Biochem. Biophys., 1959, 82, 70–77.

    Article  CAS  Google Scholar 

  14. J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum Press, New York and London, 1983, pp. 341–381.

    Book  Google Scholar 

  15. C. H. I. Ramos, Laboratory Exercises. A spectroscopic-based laboratory experiment for protein conformational studies, BAMBED, 2004, 32, 31–34.

    CAS  PubMed  Google Scholar 

  16. C. Eggeling, J. Widengren, R. Rigler, C. A. M. Seidel, Photobleaching of fluorescent dyes under conditions used for single-molecule detection: Evidence of two-step photolysis, Anal. Chem., 1998, 70, 2651–2659.

    Article  CAS  Google Scholar 

  17. J. Mertz, Molecular photodynamics involved in multi-photon excitation fluorescence microscopy, Eur. Phys. J. D, 1998, 3, 53–66.

    Article  CAS  Google Scholar 

  18. M. Lippitz, W. Erker, H. Decker, K. E. van Holde, T. Basché, Two-photon excitation microscopy of tryptophan-containing proteins, PNAS, 2002, 99, 2772–2777.

    Article  CAS  Google Scholar 

  19. E. Fujimori, Crosslinking and blue-fluorescence of photo-oxidized calf-lens α-crystallin, Exp. Eye Res., 1982, 34, 381–388.

    Article  CAS  Google Scholar 

  20. A. Pirie, Fluorescence of N′-formylkynurenine and of proteins exposed to sunlight, Biochem. J., 1972, 128, 1365–1367.

    Article  CAS  Google Scholar 

  21. E. L. Finley, J. Dillon, R. K. Crouch, K. L. Schey, Identification of tryptophan oxidation products in bovine α-crystallin, Protein Sci., 1998, 7, 2391–2397.

    Article  CAS  Google Scholar 

  22. R. Santus, A. Hélène, C. Hélène, M. Ptak, Reactions of electrons photoejected from aromatic amino acids in frozen aqueous solutions of divalent metal salts, J. Phys. Chem., 1970, 74, 550–561.

    Article  CAS  Google Scholar 

  23. K. R. Millington, G. Maurdev, The generation of superoxide and hydrogen peroxide by exposure of fluorescent whitening agents to UVA radiation and its relevance to the rapid photoyellowing of whitened wool, J. Photochem. Photobiol., A, 2004, 165, 177–185.

    Article  CAS  Google Scholar 

  24. D. V. Bent, E. Hayon, Excited state chemistry of aromatic amino acids and related peptides. III. tryptophan, J. Am. Chem. Soc., 1975, 97, 2612–2619.

    Article  CAS  Google Scholar 

  25. E. P. Melo, T. Q. Faria, L. O. Martins, A. M. Gonçalves, J. M. S. Cabral, Cutinase unfolding and stabilization by trehalose and mannosylglycerate, Proteins, 2001, 42, 542–552.

    Article  CAS  Google Scholar 

  26. K. Ado, N. Takeda, M. Kikuchi, Y. Taniguchi, The pressure effect on the structure and functions of protein disulfide isomerase, Biochim. Biophys. Acta, 2006, 1764, 586–592.

    Article  CAS  Google Scholar 

  27. P. C. M. Weisenborn, H. Meder, M. R. Egmond, T. J. W. G. Visser, A. van Hoek, Photophysics of the single tryptophan residue in fusarium solani cutinase: evidence for the occurrence of conformational substates with unusual fluorescence behavior, Biophys. Chem., 1996, 58, 281–288.

    Article  CAS  Google Scholar 

  28. T. B. Truong, Charge transfer to a solvent state. 5. Effect of solute-solvent interaction on the ionization potential of the solute. Mechanism for photoionization, J. Phys. Chem., 1980, 84, 964–970.

    Article  CAS  Google Scholar 

  29. M. Bazin, L. K. Patterson, R. Santus, Direct observation of monophotonic photoionization in tryptophan excited by 300-nm radiation. A laser photolysis study, J. Phys. Chem., 1983, 87, 189–190.

    Article  CAS  Google Scholar 

  30. J. F. Baugher, L. I. Grossweiner, Photolysis mechanism of aqueous tryptophan, J. Phys. Chem., 1977, 81, 1349–1354.

    Article  CAS  Google Scholar 

  31. C. Aubert, M. H. Vos, P. Mathis, A. P. M. Eker, K. Brettel, Intraprotein radical transfer during photoactivation of DNA photolyase, Nature, 2000, 405, 586–590.

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Health Metrology Group, Division of Metrology for Quality Life, Korea Research Institute of Standards & Science, P.O. Box 102, Yuseong, Daejeon, 305-600, Korea

    Hyong-Ha Kim

  2. Nanobio Fusion Research Center, Division of Advanced Technology, Korea Research Institute of Standards & Science, P.O. Box 102, Yuseong, Daejeon, 305-600, Korea

    Yun Mi Lee & Nam Woong Song

  3. Korean German Institute of Technology Biotech Research Center, KGIT Mokdong Center, 905-27, Mok-dong, Yangcheon-gu, Seoul, 158-050, Korea

    Jung-Keun Suh

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  1. Hyong-Ha Kim
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  2. Yun Mi Lee
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Correspondence to Hyong-Ha Kim or Nam Woong Song.

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Kim, HH., Lee, Y.M., Suh, JK. et al. Photodegradation mechanism and reaction kinetics of recombinant human interferon-α2a. Photochem Photobiol Sci 6, 171–180 (2007). https://doi.org/10.1039/b614971e

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