A comparative study of EPR spin trapping and cytochrome c reduction techniques for the measurement of superoxide anions

Scherer P. Sanders, Stephen J. Harrison, Periannan Kuppusamy, J. T. Sylvester, Jay L. Zweier

Research output: Contribution to journalArticle

Abstract

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, 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 μM, 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 μM 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.

Original languageEnglish (US)
Pages (from-to)753-761
Number of pages9
JournalFree Radical Biology and Medicine
Volume16
Issue number6
DOIs
StatePublished - Jun 1994

Keywords

  • Cytochrome c
  • EPR spectroscopy
  • Free radicals
  • Spin trapping
  • Superoxide anion

ASJC Scopus subject areas

  • Biochemistry
  • Physiology (medical)

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