Carolyn Mottley

Sulfate Anion Free Radical Formation by the Peroxidation of (Bi)sulfite and Its Reaction with Hydroxyl Radical Scavengers

The one-electron oxidation of (bi)sulfite is catalyzed by peroxidases to yield the sulfur trioxide radical anion (SO3-), a predominantly sulfur-centered radical as shown by studies with 33S-labeled (bi)sulfite. This radical reacts with molecular oxygen to form a peroxyl radical. The subsequent reaction of this peroxyl radical with (bi)sulfite has been proposed to form the sulfate anion radical, which is nearly as strong an oxidant as the hydroxyl radical. We used the spin trapping electron spin resonance technique to provide for the first time direct evidence for sulfate anion radical formation during (bi)sulfite peroxidation. The sulfate anion radical is known to react with many compounds more commonly thought of as hydroxyl radical scavengers such as formate and ethanol. Free radicals derived from these scavengers are trapped in systems where (bi)sulfite peroxidation has been inhibited by these scavengers.

[17O]Oxygen hyperfine structure for the hydroxyl and superoxide radical adducts of the spin traps DMPO, PBN and 4-POBN

[17O]oxygen hyperfine coupling constants are reported for the superoxide and hydroxyl radical adducts with the spin traps 5,5-dimethyl-1-pyrroline N-oxide, N-t-butyl-alpha-phenylnitrone and alpha-(4-pyridyl 1-oxide)-N-t-butylnitrone. These couplings provide spectroscopic evidence that the spin adducts have been correctly identified.

On the use of organic extraction in the spin-trapping technique as applied to biological systems

A back-extraction methodology is presented which involves extraction of a spin adduct from an organic medium into an aqueous medium where its spectral parameters are well established. This technique should prove very useful in properly identifying spin adducts formed in organic media. Some of the hazards of extracting spin adducts into organic solvents for study are pointed out.

SPIN TRAPPING ARTIFACTS DUE TO THE REDUCTION OF NITROSO SPIN TRAPS

The use of nitroso compounds as spin traps in chemical and biological systems has become widespread, and many interesting, unstable radicals have been trapped in this manner [l-4]. However, as we will show, extreme caution must be taken when trapping radicals in a reducing medium. The technique of spin trapping involves producing the unstable free radical of interest and allowing it to react with a diamagnetic compound (the spin trap, usually a nitroso compound or a nitrone) to form a relatively stable free radical (the spin adduct) which can be observed by electron spin resonance (ESR). Observation of a stable free radical, however, is no guarantee that the radical of interest has been trapped. Spectral artifacts can arise due to nitroxide impurities or nucleophilic addition to nitroso compounds followed by oxidation to the nitroxide [5]. We report another way in which spin trapping artifacts may arise, that of direct reduction of anitroso spin trap to a nitroxide free radical. Though nitrose spin traps have been used extensively [l-4], apparently not much attention has been given to the possibility of this reduction, though reduction of the spin adduct has been proposed as a ‘decay process [3,6]. The reduction of the spin trap itself is particularly important in biological systems because of the presence of endogenous reducing agents such as ascorbate.

Free radical formation in the oxidation of malondialdehyde and acetylacetone by peroxidase enzymes

Malondialdehyde, a product of lipid peroxidation, and acetylacetone undergo one-electron oxidation by peroxidase enzymes to form free radical metabolites, which were detected with ESR using the spin-trapping technique. The structures of the radical adducts were assigned using isotope substitution. With both malondialdehyde and acetylacetone and the enzymes myeloperoxidase and chloroperoxidase, carbon-centered radicals were detected. With horseradish peroxidase, a carbon-centered radical was initially trapped and then disappeared with the concomitant appearance of an iminoxyl radical.

Thiyl Free Radical Metabolites of Thiol Drugs and Glutathione

Lactoperoxidase, a prototypical mammalian peroxidase, is part of an antimicrobial system found in secreted fluids.1 It is believed to act by oxidation of thiocyanate.2 Not surprisingly, it will also metabolize thiol moieties, oxidizing them to free radical intermediates. Thiol compounds are important in pharmacology and toxicology, and we have examined the production of highly reactive thiyl free radicals by this mechanism.

An ESR Study of the Oxidation and Reduction of Bisulfite (Hydrated Sulfur Dioxide) in Biological Systems

Sulfur dioxide is recognized as a major air pollutant, particularly near large cities (Rall, 1974), while the ionized forms, bisulfite and sulfite, are found as preservatives in food and wine. In the lung, sulfur dioxide is hydrated rapidly according to the following equation,

A Direct Electron Spin Resonance and Spin-trapping Investigation of Peroxyl Free Radical Formation by Hematin/Hydroperoxide Systems*

{H_2}O + S{O_2} \rightleftharpoons HSO_3^ - + {H^ + }