Comparative investigation of superoxide trapping by cyclic nitrone spin traps: the use of singular value decomposition and multiple linear regression analysis.
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 superoxide radical adduct, as has been previously suggested [correction]. 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 20 degrees C.
Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Agnes
A
Keszler
B
B
Kalyanaraman
Neil
N
Hogg
D003497:Cyclic N-Oxides / Q000032:analysis; D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016014:Linear Models; D009589:Nitrogen Oxides / Q000032:analysis* / Q000737:chemistry*; D019097:Spin Trapping / Q000379:methods*; D013481:Superoxides / Q000032:analysis*; D014969:Xanthine Oxidase / Q000378:metabolism
C441196:5-tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide; D003497:Cyclic N-Oxides; D009589:Nitrogen Oxides; C015388:nitrones; D013481:Superoxides; C098505:5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D014969:Xanthine Oxidase