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Spin traps inhibit formation of hydrogen peroxide via the dismutation of superoxide: implications for spin trapping the hydroxyl free radical.
Biochim Biophys Acta 1991; 1075(3):213-22BB

Abstract

To enhance the sensitivity of EPR spin trapping for radicals of limited reactivity, high concentrations (10-100 mM) of spin traps are routinely used. We noted that in contrast to results with other hydroxyl radical detection systems, superoxide dismutase (SOD) often increased the amount of hydroxyl radical-derived spin adducts of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) produced by the reaction of hypoxanthine, xanthine oxidase and iron. One possible explanation for these results is that high DMPO concentrations (approximately 100 mM) inhibit dismutation of superoxide (O2.-) to hydrogen peroxide (H2O2). Therefore, we examined the effect of DMPO on O2.- dismutation to H2O2. Lumazine +/- 100 mM DMPO was placed in a Clark oxygen electrode following which xanthine oxidase was added. The amount of H2O2 formed in this reaction was determined by introducing catalase and measuring the amount of generated via O2.- dismutation as compared to direct divalent O2 reduction. In the presence of 100 mM DMPO, H2O2 generation decreased 43%. DMPO did not scavenge H2O2 nor alter the rate of O2.- production. The effect of DMPO was concentration-dependent with inhibition of H2O2 production observed at [DMPO] greater than 10 mM. Inhibition of H2O2 production by DMPO was not observed if SOD was present or if the rate of O2.- formation increased. The spin trap 2-methyl-2-nitroso-propane (MNP, 10 mM) also inhibited H2O2 formation (81%). However, alpha-phenyl-N-tert-butylnitrone (PBN, 10 mM), 3,3,5,5 tetramethyl-1-pyrroline N-oxide (M4PO, 100 mM), alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN, 100 mM) had no effect. These data suggest that in experimental systems in which the rate of O2.- generation is low, formation of H2O2 and thus other H2O2-derived species (e.g., OH) may be inhibited by commonly used concentrations of some spin traps. Thus, under some experimental conditions spin traps may potentially prevent production of the very free radical species they are being used to detect.

Authors+Show Affiliations

Department of Internal Medicine, VA Medical Center, Iowa City, IA.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

1659450

Citation

Britigan, B E., et al. "Spin Traps Inhibit Formation of Hydrogen Peroxide Via the Dismutation of Superoxide: Implications for Spin Trapping the Hydroxyl Free Radical." Biochimica Et Biophysica Acta, vol. 1075, no. 3, 1991, pp. 213-22.
Britigan BE, Roeder TL, Buettner GR. Spin traps inhibit formation of hydrogen peroxide via the dismutation of superoxide: implications for spin trapping the hydroxyl free radical. Biochim Biophys Acta. 1991;1075(3):213-22.
Britigan, B. E., Roeder, T. L., & Buettner, G. R. (1991). Spin traps inhibit formation of hydrogen peroxide via the dismutation of superoxide: implications for spin trapping the hydroxyl free radical. Biochimica Et Biophysica Acta, 1075(3), pp. 213-22.
Britigan BE, Roeder TL, Buettner GR. Spin Traps Inhibit Formation of Hydrogen Peroxide Via the Dismutation of Superoxide: Implications for Spin Trapping the Hydroxyl Free Radical. Biochim Biophys Acta. 1991 Oct 31;1075(3):213-22. PubMed PMID: 1659450.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Spin traps inhibit formation of hydrogen peroxide via the dismutation of superoxide: implications for spin trapping the hydroxyl free radical. AU - Britigan,B E, AU - Roeder,T L, AU - Buettner,G R, PY - 1991/10/31/pubmed PY - 1991/10/31/medline PY - 1991/10/31/entrez SP - 213 EP - 22 JF - Biochimica et biophysica acta JO - Biochim. Biophys. Acta VL - 1075 IS - 3 N2 - To enhance the sensitivity of EPR spin trapping for radicals of limited reactivity, high concentrations (10-100 mM) of spin traps are routinely used. We noted that in contrast to results with other hydroxyl radical detection systems, superoxide dismutase (SOD) often increased the amount of hydroxyl radical-derived spin adducts of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) produced by the reaction of hypoxanthine, xanthine oxidase and iron. One possible explanation for these results is that high DMPO concentrations (approximately 100 mM) inhibit dismutation of superoxide (O2.-) to hydrogen peroxide (H2O2). Therefore, we examined the effect of DMPO on O2.- dismutation to H2O2. Lumazine +/- 100 mM DMPO was placed in a Clark oxygen electrode following which xanthine oxidase was added. The amount of H2O2 formed in this reaction was determined by introducing catalase and measuring the amount of generated via O2.- dismutation as compared to direct divalent O2 reduction. In the presence of 100 mM DMPO, H2O2 generation decreased 43%. DMPO did not scavenge H2O2 nor alter the rate of O2.- production. The effect of DMPO was concentration-dependent with inhibition of H2O2 production observed at [DMPO] greater than 10 mM. Inhibition of H2O2 production by DMPO was not observed if SOD was present or if the rate of O2.- formation increased. The spin trap 2-methyl-2-nitroso-propane (MNP, 10 mM) also inhibited H2O2 formation (81%). However, alpha-phenyl-N-tert-butylnitrone (PBN, 10 mM), 3,3,5,5 tetramethyl-1-pyrroline N-oxide (M4PO, 100 mM), alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN, 100 mM) had no effect. These data suggest that in experimental systems in which the rate of O2.- generation is low, formation of H2O2 and thus other H2O2-derived species (e.g., OH) may be inhibited by commonly used concentrations of some spin traps. Thus, under some experimental conditions spin traps may potentially prevent production of the very free radical species they are being used to detect. SN - 0006-3002 UR - https://www.unboundmedicine.com/medline/citation/1659450/Spin_traps_inhibit_formation_of_hydrogen_peroxide_via_the_dismutation_of_superoxide:_implications_for_spin_trapping_the_hydroxyl_free_radical_ L2 - https://linkinghub.elsevier.com/retrieve/pii/0304-4165(91)90269-M DB - PRIME DP - Unbound Medicine ER -