NMR and EPR studies of the reaction of nucleophilic addition of (bi)sulfite to the nitrone spin trap DMPO

Potapenko, Dmitrii I.; Bagryanskaya, Elena G.; Reznikov, Vladimir V.; Clanton, Thomas L.; Khramtsov, Valery V.

doi:10.1002/mrc.1226

Cited by 19 publications

(53 citation statements)

References 27 publications

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“…The DMPOH/CN hydroxylamine is formed in a reversible equilibrium (Supplemental Figure 2), but the signal in the corresponding ESR experiment was clearly identified as artifact [7]. For sulfite, Potapenko showed indirect kinetic evidence for the Forrester-Hepburn artifact in experiments with DMPO [8]. In that publication, the ferricyanide consumption rates of the sulfite oxidation experiment were investigated by means of UV-Vis spectroscopy in the presence and absence of DMPO.…”

Section: Discussionmentioning

confidence: 99%

“…In that publication, the ferricyanide consumption rates of the sulfite oxidation experiment were investigated by means of UV-Vis spectroscopy in the presence and absence of DMPO. It was found that in the presence of the spin trap, the ferricyanide reduction was much faster; this increased rate was interpreted as evidence of the Forrester-Hepburn mechanism [8]. …”

Section: Discussionmentioning

confidence: 99%

“…The mixed sample was loaded within seconds into the ESR spectrometer using an aspiration technique. As did Potapenko et al [8], we used ferricyanide as a mild oxidant to make genuine radical formation less favorable (see below). With these experimental conditions, we indeed found proof for the Forrester-Hepburn artifact in the ESR preincubation assay: The proportion of the isotope present during preincubation was elevated significantly 30 s after mixing (Figure 2a).…”

Section: Discussionmentioning

confidence: 99%

“…Also, the existence of the Forrester-Hepburn mechanism was proposed because, in a sulfite oxidation experiment, ferricyanide was reduced faster in the presence of DMPO [8]. We have now investigated whether this faster reduction may be due to oxidation of the DMPO/ • SO 3 – adduct as an alternative explanation and have reinvestigated our preincubation experiments with respect to the equilibrium chemistry.…”

Section: Introductionmentioning

confidence: 97%

“…Hydroxylamine equilibrium chemistry has been reported for sulfite and DMPO [8]. Also, the existence of the Forrester-Hepburn mechanism was proposed because, in a sulfite oxidation experiment, ferricyanide was reduced faster in the presence of DMPO [8].…”

Section: Introductionmentioning

confidence: 99%

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Investigation of spin-trapping artifacts formed by the Forrester-Hepburn mechanism

Leinisch¹,

Jiang²,

DeRose³

et al. 2013

Free Radical Biology and Medicine

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Free radical detection with ESR spin trapping relies on the specific addition of the radical to nitrone/nitroso compounds. It has been proposed that spin traps can react also in biological systems to give false-positive results. For nitrone spin traps, the reaction with nucleophiles, first described by Forrester and Hepburn, has been discussed as the most critical source of artifacts. For artifact identification, the ESR preincubation method may be used, which employs isotopically marked spin traps. Here we investigated the influence of fast sulfite-hydroxylamine equilibrium chemistry on the validity of this assay. Using the (faster) aspiration technique, we found that the Forrester-Hepburn mechanism also contributes to DMPO/•SO3– adduct formation during ferricyanide-mediated sulfite oxidation, but no evidence for artifactual DMPO/•SO3– formation was found if the more potent horseradish peroxidase was used. This is ESR evidence that the Forrester-Hepburn mechanism can occur under mild conditions, depending on the experimental details. This technique can also be used to test for other artifact mechanisms. We investigated the known ene reaction of DBNBS and tryptophan in more detail. We found that a strong artifact signal is induced by light; however, with atypically long incubations, we found that the artifact is also formed thermally.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Investigation of spin-trapping artifacts formed by the Forrester-Hepburn mechanism

Leinisch¹,

Jiang²,

DeRose³

et al. 2013

Free Radical Biology and Medicine

Self Cite

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5,5-Dimethyl-1-pyrroline-N -oxide

Cordero

Brandi

2017

Encyclopedia of Reagents for Organic Synthesis

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The fidelity of spin trapping with DMPO in biological systems

Ranguelova

Mason

2011

Magnetic Reson in Chemistry

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Unlike direct ESR, spin trap methodology depends on the absolute fidelity of the spin trap reaction. Two alternative reactions of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) leading to radical adduct artifacts have been discovered and investigated: inverted spin trapping and the Forrester-Hepburn nucleophilic mechanisms. These two alternate pathways to radical adducts are a combination of one-electron oxidation and nucleophilic addition, in either order. In biological systems, serious artifacts have been reported due to the Forrester-Hepburn mechanism, which is initiated by the addition of a nucleophile to DMPO. It has recently been demonstrated that (bi)sulfite (hydrated sulfur dioxide) can react with DMPO via a nonradical, nucleophilic reaction, and it has been further proposed that DMPO/•SO3− formation in biological systems is an artifact and not the result of spin trapping of sulfur trioxide anion radical (•SO3−). The one-electron oxidation of (bi)sulfite catalyzed by horseradish peroxidase (HRP)/hydrogen peroxide (H2O2) has been reinvestigated by ESR spin trapping with DMPO and oxygen uptake studies to obtain further evidence for the radical reaction mechanism. In the absence of DMPO, the initial rate of (bi)sulfite-dependent oxygen and H2O2 consumption was determined to be half of the initial rate of DMPO/•SO3− radical adduct formation as determined by ESR, demonstrating that, under our experimental conditions, DMPO exclusively forms the radical adduct by trapping the •SO3−.

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NMR and EPR studies of the reaction of nucleophilic addition of (bi)sulfite to the nitrone spin trap DMPO

Cited by 19 publications

References 27 publications

Investigation of spin-trapping artifacts formed by the Forrester-Hepburn mechanism

Investigation of spin-trapping artifacts formed by the Forrester-Hepburn mechanism

5,5-Dimethyl-1-pyrroline-N -oxide

The fidelity of spin trapping with DMPO in biological systems

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