hexacyanoferrate-iii and 5-5-dimethyl-1-pyrroline-1-oxide

Nitrone spin traps such as 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) are commonly used for free radical detection. Though proven examples are rare, artifact formation must be considered. For example, the Forrester-Hepburn mechanism yields the same radical adduct as that formed by genuine radical trapping. A hydroxylamine is formed by nucleophilic attack of the substrate on DMPO and subsequently oxidized to the respective nitroxide radical. One potential candidate for this artifact is the sulfur trioxide radical adduct (DMPO/(•)SO(3)(-)), as detected in spin-trapping experiments with horseradish peroxidase and sulfite. It has previously been shown by NMR experiments that the hydroxylamine intermediate does indeed form, but no direct proof for the ESR artifact has been provided. Here, we used isotopically labeled DMPO with horseradish peroxidase and ferricyanide to test for the Forrester-Hepburn artifact directly in a spin-trapping experiment. Besides sulfite, we investigated other nucleophiles such as cyanide, cysteine, and glutathione. Neither sulfite nor biological thiols produced detectable spin-trapping artifacts, but with cyanide the relatively weak signal originated entirely from the nucleophilic reaction. The hydroxylamine intermediate, which is more abundant with cyanide than with sulfite, was identified as cyano-hydroxylamine by means of 2D NMR experiments. Although our study found that spin trapping provided authentic free radical signals with most of the substrates, the occurrence of the Forrester-Hepburn mechanism artifact with cyanide emphasizes the importance of isotope measurements with nucleophile substrates.

Topics: Cyclic N-Oxides; Cysteine; Electron Spin Resonance Spectroscopy; Ferricyanides; Glutathione; Horseradish Peroxidase; Hydrogen Peroxide; Hydroxylamine; Nitric Oxide; Nitrogen Oxides; Oxidation-Reduction; Spin Trapping; Sulfites

It is well known that oxygen enhances the relaxation of free radical EPR probes through spin lattice and Heisenberg spin-spin interactions with consequent effect on the line height and width. The two relaxation processes have opposing effects on the signal heights and depend on the concentration of oxygen, the incident microwave power, and the presence of other paramagnetic species. During EPR studies of chemical, biochemical, and cellular processes involving free radicals, molecular oxygen has significant magnetic influence on the EPR signal intensity of the free radical species under investigation in addition to affecting the rates of production of the primary species and the stability of the spin adduct nitroxides. These effects are often overlooked and can cause artifacts and lead to erroneous interpretation. In the present study, the effects of oxygen and ferricyanide on the EPR signal height of stable and persistent spin adduct nitroxides at commonly employed microwave powers were examined. The results show that under commonly adopted EPR spectrometer instrumental conditions, artifactual changes in the EPR signal of spin adducts occur and the best way to avoid them is by keeping the oxygen level constant using a gas-permeable cell.

Topics: Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Ferricyanides; Free Radicals; Microwaves; Oxygen; Spin Labels; Triacetoneamine-N-Oxyl