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Mass spectral analysis of protein-based radicals using DBNBS. Nonradical adduct formation versus spin trapping.

Author(s): Filosa A, English AM

J Biol Chem. 2001 Jun 15;276(24):21022-7 Authors: Filosa A, English AM

Article GUID: 11262405
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J Biol Chem. 2003 May 16;278(20):17895-900 Authors: Gidda SK, Miersch O, Levitin A, Schmidt J, Wasternack C, Varin L

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Author(s): Mallick J, Whiteway M

J Biol Chem. 2013 Jun 14;288(24):17508-19 Authors: Mallick J, Whiteway M

Article GUID: 23625919
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Author(s): Jones DR, Uddin MS, Gruninger RJ, Pham TTM, Thomas D, Boraston AB, Briggs J, Pluvinage B, McAllister TA, Forster RJ, Tsang A, Selinger LB, Abbott DW

J Biol Chem. 2017 07 28;292(30):12606-12620 Authors: Jones DR, Uddin MS, Gruninger RJ, Pham TTM, Thomas D, Boraston AB, Briggs J, Pluvinage B, McAllister TA, Forster RJ, Tsang A, Selinger LB, Abbott DW

Article GUID: 28588026
Title:Mass spectral analysis of protein-based radicals using DBNBS. Nonradical adduct formation versus spin trapping.
Authors:Filosa A,  English AM
Link:https://www.ncbi.nlm.nih.gov/pubmed/11262405?dopt=Abstract
DOI:10.1074/jbc.M100644200
Category:J Biol Chem
PMID:11262405
Dept Affiliation: CHEMBIOCHEM
1 Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec H3G 1M8, Canada.

Description:

Mass spectral analysis of protein-based radicals using DBNBS. Nonradical adduct formation versus spin trapping.

J Biol Chem. 2001 Jun 15;276(24):21022-7

Authors: Filosa A, English AM

Abstract

Protein-based radicals generated in the reaction of ferricytochrome c (cyt c) with H(2)O(2) 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 H(2)O(2) and substituting cyt c 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 alpha-lactalbumin, were substituted for cyt c in the cyt c/H(2)O(2)/DBNBS reaction. A maximum of two DBNBS adducts were observed in the mass spectra of the products of the Mb/H(2)O(2)/DBNBS reactions, whereas no adducts were detected following alpha-lactalbumin/H(2)O(2)/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.

PMID: 11262405 [PubMed - indexed for MEDLINE]