Radicals in the reaction between peroxynitrite and uric acid identified by electron spin resonance spectroscopy and liquid chromatography mass spectrometry.
Peroxynitrite is a reactive oxidant produced in vivo in response to oxidative and other stress by the diffusion-limited reaction of nitric oxide and superoxide. This article is focused on the identification of free radical intermediates of uric acid formed during its reaction with peroxynitrite. The experimental approach included the ESR spin trapping of the radical generated from the reaction between uric acid and peroxynitrite at pH 7.4 and mass spectrometry studies of the trapped radicals. Using PBN (N-tert-butyl-alpha-phenylnitrone) as the spin trapping agent, a six-line ESR spectrum was obtained and its hyperfine coupling constants, a(N)=15.6 G and a(H)=4.4 G, revealed the presence of carbon-based radicals. Further structural identification of the PBN-radical adducts was carried out using liquid chromatography-mass spectrometry. After comparison with the control reactions, two species were identified that correspond to the protonated molecules (M+1) at m/z 352 and 223, respectively. The ions of m/z 352 were characterized as the PBN-triuretcarbonyl radical adduct and the m/z 223 ion was identified as the PBN-aminocarbonyl radical adduct. Their mechanism of formation is discussed.
Imaram
Witcha
W
Department of Chemistry, University of Florida, Gainesville, FL 32611, USA. wimaram@chem.ufl.edu
Gersch
Christine
C
Kim
Kyung Mee
KM
Johnson
Richard J
RJ
Henderson
George N
GN
Angerhofer
Alexander
A
D055598:Chemical Phenomena; D002853:Chromatography, Liquid; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000737:chemistry*; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D008956:Models, Chemical; D010084:Oxidation-Reduction; D030421:Peroxynitrous Acid / Q000737:chemistry*; D050050:Protein Carbonylation; D014527:Uric Acid / Q000031:analogs & derivatives / Q000737:chemistry* / Q000302:isolation & purification
D005609:Free Radicals; D030421:Peroxynitrous Acid; D014527:Uric Acid
Nat Rev Drug Discov. 2007 Aug;6(8):662-80
17667957
Nucleosides Nucleotides Nucleic Acids. 2009 Feb;28(2):118-49
19219741
Arch Biochem Biophys. 2004 Mar 1;423(1):213-7
14871483
Nucleosides Nucleotides Nucleic Acids. 2008 Jun;27(6):608-19
18600514
ACS Chem Biol. 2009 Mar 20;4(3):161-77
19267456
Chem Res Toxicol. 1997 Jul;10(7):786-94
9250413
Free Radic Res. 1996 Sep;25(3):275-83
8889493
Biochem Pharmacol. 2005 Aug 1;70(3):343-54
15963955
J Biol Chem. 1988 Feb 5;263(4):1709-12
2828349
Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):11173-7
7972029
Anal Chem. 1989 Aug 1;61(15):1709-17
2774199
Anal Biochem. 1996 May 1;236(2):242-9
8660500
Hypertension. 2001 Nov;38(5):1101-6
11711505
Free Radic Biol Med. 1987;3(4):259-303
2826304
Proc Natl Acad Sci U S A. 1981 Nov;78(11):6858-62
6947260
Free Radic Biol Med. 2009 Nov 15;47(10):1401-7
19686842
Arch Biochem Biophys. 1999 Dec 15;372(2):285-94
10600166
Chem Res Toxicol. 1999 Oct;12(10):1010-8
10525279
Nitric Oxide. 2006 Mar;14(2):109-21
16352449
Arch Biochem Biophys. 2000 Apr 15;376(2):333-7
10775420
Free Radic Biol Med. 2002 May 1;32(9):841-59
11978486
Chem Res Toxicol. 2008 Apr;21(4):879-87
18361509
Chem Res Toxicol. 1997 Nov;10(11):1285-92
9403183
J Chromatogr B Analyt Technol Biomed Life Sci. 2009 Jan 1;877(1-2):65-70
19081307
Hypertension. 2003 Jun;41(6):1183-90
12707287
Biochem J. 1996 Mar 15;314 ( Pt 3):869-76
8615782
Chem Res Toxicol. 1992 Nov-Dec;5(6):834-42
1336991
J Mol Evol. 1992 Jan;34(1):78-84
1556746