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Mass Spectral Analysis of Protein-based Radicals Using DBNBS: Nonradical adduct formation versus spin trapping

Title:

Mass Spectral Analysis of Protein-based Radicals Using DBNBS: Nonradical adduct formation versus spin trapping

Filosa, Angelo and English, Ann M. ORCID: https://orcid.org/0000-0002-3696-7710 (2001) Mass Spectral Analysis of Protein-based Radicals Using DBNBS: Nonradical adduct formation versus spin trapping. Journal of Biological Chemistry, 276 (24). pp. 21022-21027. ISSN 0021-9258

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Official URL: https://doi.org/10.1074/jbc.m100644200

Abstract

Protein-based radicals generated in the reaction of ferricytochrome c (cyt c) with H2O2 were investigated by electrospray mass spectrometry (ESI-MS) using 3,5-dibromo-4-nitrosobenzenesulfonate (DBNBS). Up to four DBNBS-cyt c adducts were observed in the mass spectra. However, by varying the reaction conditions (0–5 molar equivalents of H2O2 and substituting cytc with its cyanide adduct which is resistant to peroxidation), noncovalent DBNBS adduct formation was inferred. Nonetheless, optical difference spectra revealed the presence of a small fraction of covalently trapped DBNBS. To probe the nature of the noncovalent DBNBS adducts, the less basic proteins, metmyoglobin (Mb) and α-lactalbumin, were substituted for cyt c in the cytc/H2O2/DBNBS reaction. A maximum of two DBNBS adducts were observed in the mass spectra of the products of the Mb/H2O2/DBNBS reactions, whereas no adducts were detected following α-lactalbumin/H2O2/DBNBS incubation, which is consistent with adduct formation via spin trapping only. Titration with DBNBS at pH 2.0 yielded noncovalent DBNBS-cyt c adducts and induced folding of acid-denatured cyt c, as monitored by ESI-MS and optical spectroscopy, respectively. Thus, the noncovalent DBNBS-cyt c mass adducts observed are assigned to ion pair formation occurring between the negatively charged sulfonate group on DBNBS and positively charged surface residues on cyt c. The results reveal the pitfalls inherent in using mass spectral data with negatively charged spin traps such as DBNBS to identify sites of radical formation on basic proteins such as cyt c.

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Article
Refereed:Yes
Authors:Filosa, Angelo and English, Ann M.
Journal or Publication:Journal of Biological Chemistry
Date:15 June 2001
Digital Object Identifier (DOI):10.1074/jbc.m100644200
ID Code:985175
Deposited By: MIA MASSICOTTE
Deposited On:28 May 2019 14:07
Last Modified:29 May 2019 12:42

References:

1. Radi, R., Turrens, J. F., and Freeman, B. A. (1991) Arch. Biochem. Biophys. 288, 118–125
2. Radi, R., Bush, K. M., and Freeman, B. A. (1993) Arch. Biochem. Biophys. 300, 409–415
3. Perkins, M. J. (1980) Adv. Phys. Org. Chem. 17, 1–61
4. Iwahashi, H., Parker, C. E., Mason, R. P., and Tomer, K. B. (1990) Rapid Commun. Mass. Spectrom. 4, 352–354
5. Iwahashi, H., Parker, C. E., Mason, R. P., and Tomer, K. B. (1991) Biochem. J. 276, 447–453
6. Janzen, E. G., Towner, R. A., Krygsman, P. H., Lai, E. K., Poyer, J. L., Brueggemann, G., and McCay, P. B. (1990) Free Radical. Res. Commun. 9, 353–360
7. Filosa, A., and English, A. M. (1999) Proceedings of the 47th ASMS Conference on Mass Spectrometry and Allied Topics, June 12–17, Dallas, TX
8. Tsapraillis, G. (1997) Probing the Redox-active Residues in Cytochrome Peroxidase. Ph.D. thesis, Concordia University, Montreal, Canada
9. Fenwick, C. W. (1997) Study of the Highly Oxidizing Centers in the Metmyoglobin- H2O2 Reaction. Ph.D. thesis, Concordia University, Montreal, Canada
10. Fenwick, C. W., and English, A. M. (1996) J. Am. Chem. Soc. 118, 12236–12237
11. Barr, D. P., Gunther, M. R., Deterding, L. J., and Tomer, K. B. (1996) J. Biol. Chem. 271, 15498–15508
12. Kaur, H., Leung, K. H. W., and Perkins, M. J. (1981) J. Chem. Soc. Chem. Commun. 142–143
13. Kalyanaraman, B., Joseph, J., Kondratenko, N., and Parthasarathy, S. (1992) Biochim. Biophys. Acta 1126, 309–313
14. Hiramoto, K., Hasegawa, Y., and Kikugawa, K. (1994) Free Radic. Res. 21, 341–349
15. Deterding, L. J., Barr, D. P., Mason, R. P., and Tomer, K. B. (1998) J. Biol. Chem. 273, 12863–12869
16. Shidoji, Y., Hayashi, K., Komura, S., Ohishi, N., and Yagi, K. (1999) Biochem. Biophys. Res. Commun. 264, 343–347
17. Dyer, C., Schubert, A., Timkovich, R., and Feinberg, B. A. (1979) Biochim. Biophys. Acta 579, 253–268
18. Goto, Y., Takahashi, N., and Fink, A. L. (1990) Biochemistry 29, 3480–3488
19. Ali, V., Prakash, K., Kulkarni, S., Ahmad, A., Madhusudan, K. P., and Bhakuni, V. (1999) Biochemistry 38, 13635–13642
20. Blumenthal, D. C., and Kassner, R. J. (1980) J. Biol. Chem. 255, 5859–5863
21. Kim, Y. M., Jeong, S. H., Yamazaki, I., Piette, L. H., Han, S., and Hong, S. J.(1995) Free Radic. Res. 22, 11–21
22. Mason, R. P., Kalyanaraman, B., Tainer, B. E., and Eling, T. E. (1980) J. Biol. Chem. 255, 5019–5022
23. Eberson, L. (1998) Adv. Phys. Org. Chem. 31, 91–141
24. Nazhat, N. B., Saadalla-Nazhat, R. A., Fairburn, K., Jones, P., Blake, D. R., Nielsen, B. R., Symons, M. C., and Winyard, P. G. (1999) Biochim. Biophys. Acta 1427, 276–286
25. Chen, Y. R., Gunther, M. R., and Mason, R. (1999) J. Biol. Chem. 274, 3308–3314
26. Gunther, M. R., Kelman, D. J., Corbett, J. T., and Mason, R. P. (1995) J. Biol. Chem. 270, 16075–16081
27. Kohno, M., Yamada, M., Mitsuta, K., Mizuta, Y., and Yoshikawa, T. (1991) Bull. Chem. Soc. Jpn. 64, 1447–1453
28. Ozawa, T., and Hanaki, A. (1991) Bull. Chem. Soc. Jpn. 64, 1976–1978
29. Righetti, P. G., and Caravaggio, T. (1976) J. Chromatogr. 127, 1–28
30. Filosa, A., and English, A. M. (2000) Proceedings of the 48th ASMS Conference on Mass Spectrometry and Allied Topics, June 11–15, Long Beach, CA
31. Witting, P. K., Douglas, D. J., and Mauk, A. G. (2000) J. Biol. Chem. 275, 20391–20398
32. Gunther, M. R., Tschirret-Guth, R. A., Witkowska, H. E., Fann, Y. C., Barr, D. P., Ortiz De Montellano, P. R., and Mason, R. P. (1998) Biochem. J. 330, 1293–1299
33. Catalano, C. E., Choe, Y. S., and Ortiz de Montellano, P. R. (1989) J. Biol. Chem. 264, 10534–10541
34. George, P., and Irvine, D. H. (1952) Biochem. J. 52, 511–517
35. Matulis, D., and Lovrien, R. (1998) Biophys. J. 74, 422–429
36. Moore, G. R., and Pettigrew, G. W. (1987) Cytochromes c: Biological Aspects, pp. 83–84, Springer-Verlag, Berlin
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