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

https://doi.org/10.1016/0891-5849(94)90190-2Get rights and content

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.

References (48)

  • J.M. McCord et al.

    The utility of superoxide dismutase in studying free radical reactions, II. The mechanism of the mediation of cytochrome c reduction by a variety of electron carriers

    J. Biol. Chem.

    (1970)
  • Y. Sawada et al.

    One-electron transfer reactions in biochemical systems VIII. Kinetic study of superoxide dismutase

    Biochim. Biophys. Acta

    (1973)
  • B.F. Van Gelder et al.

    The extinction coefficient of cytochrome c

    Biochim. Biophys. Acta

    (1962)
  • W.F. Beyer et al.

    Phosphate, not superoxide dismutase, facilitates electron transfer from ferrous salts to cytochrome c

    Arch. Biochem. Biophys.

    (1991)
  • S. Belkin et al.

    Reduction and destruction rates of nitroxide spin probes

    Arch. Biochem. Biophys.

    (1987)
  • E. Finkelstein et al.

    Spin trapping of superoxide and hydroxyl radical: Practical aspects

    Arch. Biochem. Biophys.

    (1980)
  • E.G. Janzen

    A critical review of spin trapping in biological systems

  • G.M. Rosen et al.

    Use of spin traps in biological systems

    Adv. Free Radic. Biol. Med.

    (1985)
  • G.R. Buettner

    Spin trapping: ESR parameters of spin adducts

    Free Radic. Biol. Med.

    (1987)
  • K. Chen et al.

    Oxidation of hydroxylamines to nitroxide spin labels in living cells

    Biochim. Biophys. Acta

    (1988)
  • J.L. Zweier et al.

    Electron paramagnetic resonance evidence that cellular oxygen toxicity is caused by the generation of superoxide and hydroxyl free radicals

    FEBS Lett.

    (1989)
  • A. Samuni et al.

    The cellular-induced decay of DMPO spin adducts of ·OH and ·O2

    Free Radic. Biol. Med.

    (1989)
  • B.E. Britigan et al.

    Insight into the nature and site of oxygen-centered free radical generation by endothelial cell monolayers using a novel spin trapping technique

    Blood

    (1992)
  • B.E. Britigan et al.

    Do human neutrophils make hydroxyl radical?

    J. Biol. Chem.

    (1986)
  • Cited by (46)

    • Enhanced ozonation degradation of atrazine in the presence of nano-ZnO: Performance, kinetics and effects

      2017, Journal of Environmental Sciences (China)
      Citation Excerpt :

      This is probably because the facile disproportionation reaction of superoxide in water precludes the slow reactions between O2− and DMPO (k = 10 × 103 mol/(L·sec)) (Chen et al., 2002). Besides, the DMPO-O2− adduct is unstable in aqueous solution and tends to convert into DMPO-OH by combination with H+ (Sanders et al., 1994), which could enhance the DMPO-OH adduct signal. Fig. 9a also reveals that nZnO had a positive effect on the formation of H2O2.

    • Efficient degradation of tetrabromobisphenol A by synergistic integration of Fe/Ni bimetallic catalysis and microbial acclimation

      2017, Water Research
      Citation Excerpt :

      In order to determine the role of the reactive oxygen species (ROSs) played in the TBBPA degradation process, the amount of generated H2O2 and O2− were photometrically analyzed via N, N-diethyl-p-phenylenediamine (DPD)/peroxidase and reduction of cytochrome c, respectively. OH was detected by HPLC using 2,5-dihydroxybenzoic acid (2,5-DHBA) as OH-trapping agent (Bader et al., 1988; Sanders et al., 1994). To further ascertain the production of O2− and OH, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) spin-trapping electron spin resonance (ESR) spectra was recorded in the mixed system.

    • Tetrathiatriarylmethyl radical with a single aromatic hydrogen as a highly sensitive and specific superoxide probe

      2012, Free Radical Biology and Medicine
      Citation Excerpt :

      This technique utilizes the reaction of nitrone spin traps with O2•− resulting in the production of relatively stable spin adducts [15]. Our previous studies showed that the EPR spin trapping technique is more sensitive than the cytochrome c reduction method in quantifying O2•− [44,48]. However, the application of the EPR spin trapping method for very low O2•− fluxes is still limited because of slow spin trap reactivity with O2•−, and short adduct half-life, as well as decreased EPR analytical sensitivity due to the complex multiline EPR spectra of the spin adduct.

    View all citing articles on Scopus
    View full text