title: Investigation of spin-trapping artifacts formed by the Forrester-Hepburn mechanism.
abstract: Free radical detection with ESR spin trapping relies on the specific addition of the radical to nitrone/nitroso compounds. It also has been proposed that spin traps can react 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.
authors.affiliation: Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, 111 TW Alexander Drive, Research Triangle Park, NC 27709, USA. Electronic address: leinischf@niehs.nih.gov.
authors.forename: Fabian
authors.initials: F
authors.lastname: Leinisch
authors.affiliation: Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, 111 TW Alexander Drive, Research Triangle Park, NC 27709, USA.
authors.forename: Jinjie
authors.initials: J
authors.lastname: Jiang
authors.affiliation: Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, 111 TW Alexander Drive, Research Triangle Park, NC 27709, USA.
authors.forename: Eugene F
authors.initials: EF
authors.lastname: DeRose
authors.affiliation: Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, Department of Internal Medicine, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Valery V
authors.initials: VV
authors.lastname: Khramtsov
authors.affiliation: Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, 111 TW Alexander Drive, Research Triangle Park, NC 27709, USA.
authors.forename: Ronald P
authors.initials: RP
authors.lastname: Mason
mesh_terms: D016477:Artifacts; D001557:Benzenesulfonates / Q000032:analysis / Q000737:chemistry*; D003497:Cyclic N-Oxides / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D005189:False Positive Reactions; D005292:Ferricyanides / Q000737:chemistry; D005609:Free Radicals / Q000032:analysis*; D006735:Horseradish Peroxidase / Q000737:chemistry; D019811:Hydroxylamine / Q000737:chemistry; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy; D009589:Nitrogen Oxides / Q000737:chemistry; D009603:Nitroso Compounds / Q000032:analysis / Q000737:chemistry*; D010084:Oxidation-Reduction; D013113:Spin Labels; D019097:Spin Trapping / Q000379:methods*; D013447:Sulfites / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D001557:Benzenesulfonates; D003497:Cyclic N-Oxides; D005292:Ferricyanides; D005609:Free Radicals; D009589:Nitrogen Oxides; D009603:Nitroso Compounds; D013113:Spin Labels; D013447:Sulfites; C015388:nitrones; D019811:Hydroxylamine; C048757:3,5-dibromo-4-nitrosobenzenesulfonate; D014364:Tryptophan; D006735:Horseradish Peroxidase
keywords: 1-hydroxy,2-cyano,5,5-dimethylpyrrolidine; 3,5-dibromo-4-nitrosobenzenesulfonate; 4-POBN; 5,5-dimethyl-1-pyrroline-N-oxide; Artifact; Cys; DBNBS; DMPO; DMPOH/CN; False-positive; Forrester-Hepburn mechanism; GSH; L-cysteine; L-glutathione reduced; N-tert-butyl-α-(4-pyridyl)nitrone-N′-oxide; Nucleophile; Spin trapping