A comparative study of EPR spin trapping and cytochrome c reduction techniques for the measurement of superoxide anions.
Superoxide anions (O2.-) generated by the reaction of xanthine with xanthine oxidase were measured by the reduction of cytochrome c and by electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Studies were performed to determine the relative sensitivities of these two techniques for the measurement of O2.-. Mixtures of xanthine, xanthine oxidase, and DMPO generated two adducts, a transient DMPO-OOH and a smaller but longer-lived DMPO-OH. Both adducts were inhibited by superoxide dismutase (SOD), demonstrating they originated from O2.-, and were also significantly decreased when the experiments were performed using unchelated buffers, suggesting that metal ion impurities in unchelated buffers alter the formation or degradation of DMPO-adducts. O2.-, generated by concentrations of xanthine as low as 0.05 microM, were detectable using EPR spin trapping. In contrast, mixtures of xanthine, xanthine oxidase, and cytochrome c measured spectrophotometrically at 550 nm demonstrated that concentrations of xanthine above 1 microM were required to produce measurable levels of reduced cytochrome c. These studies demonstrate that spin trapping using DMPO was at least 20-fold more sensitive than the reduction of cytochrome c for the measurement of superoxide anions. However, at levels of superoxide generation where cytochrome c provides a linear measurement of production, EPR spin trapping may underestimate radical production, probably due to degradation of DMPO radical adducts.
Sanders
S P
SP
Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, MD.
Harrison
S J
SJ
Kuppusamy
P
P
Sylvester
J T
JT
Zweier
J L
JL
D003497:Cyclic N-Oxides; D003574:Cytochrome c Group / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D007700:Kinetics; D010084:Oxidation-Reduction; D012680:Sensitivity and Specificity; D013053:Spectrophotometry / Q000379:methods*; D013113:Spin Labels; D013481:Superoxides / Q000032:analysis*; D013997:Time Factors; D019820:Xanthine; D014969:Xanthine Oxidase / Q000378:metabolism; D014970:Xanthines / Q000378:metabolism
D003497:Cyclic N-Oxides; D003574:Cytochrome c Group; D013113:Spin Labels; D014970:Xanthines; D013481:Superoxides; D019820:Xanthine; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D014969:Xanthine Oxidase