A COMPARATIVE-STUDY OF EPR SPIN-TRAPPING AND CYTOCHROME-C REDUCTION TECHNIQUES FOR THE MEASUREMENT OF SUPEROXIDE ANIONS

Superoxide anions (O-2(.-)) generated by the reaction of xanthine with xanthine oxidase were measured by the reduction of cytochrome c and b y electron paramagnetic resonance (EPR) spectroscopy using the spin tr ap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Studies were performed to determine the relative sensitivities of these two techniques for the m easurement 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 (SO D), demonstrating they originated from O2(.-), and were also significa ntly decreased when the experiments were performed using unchelated bu ffers, suggesting that metal ion impurities in unchelated buffers alte r the formation or degradation of DMPO-adducts. O2(.-), generated by c oncentrations of xanthine as low as 0.05 mu M, were detectable using E PR spin trapping. In contrast, mixtures of xanthine, xanthine oxidase, and cytochrome c measured spectrophotometrically at 550 nm demonstrat ed that concentrations of xanthine above 1 mu M were required to produ ce measurable levels of reduced cytochrome c. These studies demonstrat e that spin trapping using DMPO was at least 20-fold more sensitive th an the reduction of cytochrome c for the measurement of superoxide ani ons. However, at levels of superoxide generation where cytochrome c pr ovides a linear measurement of production, EPR spin trapping may under estimate radical production, probably due to degradation of DMPO radic al adducts.