Journal
FREE RADICAL BIOLOGY AND MEDICINE
Volume 35, Issue 9, Pages 1149-1157Publisher
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/S0891-5849(03)00497-0
Keywords
superoxide; spin trapping; EPR; singular value decomposition; free radicals
Funding
- NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING [P41EB001980] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM055792, R29GM055792] Funding Source: NIH RePORTER
- NIBIB NIH HHS [EB001980] Funding Source: Medline
- NIGMS NIH HHS [GM55792] Funding Source: Medline
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The kinetics of the reaction between superoxide and the spin trapping agents 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO), and 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO) were re-examined in the superoxide-generating xanthine/xanthine oxidase system, by competition with spontaneous dismutation. The approach used singular value decomposition (SVD), multiple linear regression, and spectral simulation. The experiments were carried out using a two-syringe mixing arrangement with fast scan acquisition of 100 consecutive EPR spectra. Using SVD analysis, the extraction of both temporal and spectral information could be obtained from in a single run. The superoxide spin adduct was the exclusive EPR active species in the case of DEPMPO and BMPO, and the major component when DMPO was used. In the latter case a very low concentration of hydroxyl adduct was also observed, which did not change during the decay of the DMPO-superoxide adduct. This indicates that the hydroxyl radical adduct is not formed from the spontaneous decay of the hydroxyl radical adduct, as has been previously suggested. It was established that in short-term studies (up to 100 s) DMPO was the superior spin trapping agent, but for reaction times longer than 100 s the other two spin traps were more advantageous. The second order rate constants for the spin trapping reaction were found to be DMPO (2.4 M-1 s(-1)), DEPMPO (0.53 M-1 s(-1)), and BMPO (0.24 M-1 s(-1)) determined through competition with spontaneous dismutation of superoxide, at pH 7.4 and 20degreesC. (C) 2003 Elsevier Inc.
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