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 |
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