Direct Electron Spin Resonance Research Articles

55] Assay of in situ radicals by electron spin resonance

Most biological Electron-Spin Resonance (ESR) studies of enzymes and their free-radical metabolites have used one of two techniques, continuous rapid flow or freeze-quench. Unfortunately, the freeze-quench technique is of very limited utility in the study of organic-free-radical metabolites. Many classes of free radicals can be detected under physiological steady-state conditions for periods of several minutes to over an hour at room temperature with the use of very simple procedures. In the steady-state condition, the rate of radical formation is equal to the rate of radical decay. Any strategy that will increase the rate of free-radical formation or decrease the rate of radical decay will help achieve the necessary 10 -8 -10 -7 M steady-state radical concentration. The spin-trapping technique is a very effective approach for extending the lifetime of the paramagnetic species. Spin traps scavenge many reactive free radicals, even under physiological conditions, to form relatively stable nitroxide adducts. The formation of these secondary nitroxides enables the study of superoxide and other free-radical metabolites that would otherwise be impossible to study with direct ESR under steady-state conditions. A more convenient technique, known as stationary flat cell, explained in detail, is to use aspiration (controlled by a stopcock) or a modified Gilford rapid sampler to fill the flat cell. This approach has various advantages that are discussed in the chapter.

Application of stable radical-containing reagents to the ESR determination of metals

The emergence of complexing reagents whose molecules contain a stable free-radical group affords new opportunities to considerably expand the potential of electron spin resonance (ESR) as an analytical tool. These reagents make ESR determinations of practically every metal possible instead of only those possessing paramagnetic ions. Furthermore, the use of complexing reagents may improve the detection limits characteristic of direct ESR determinations.

A direct electron spin resonance and spin-trapping investigation of peroxyl free radical formation by hematin/hydroperoxide systems.

Direct electron spin resonance was used to detect tert-alkylperoxyl radicals generated by hematin and the corresponding hydroperoxides at near-physiological pH values. The spin-trapping method was necessary to detect the less persistent primary ethylperoxyl radical. Under a nitrogen atmosphere, the electron spin resonance signal of the tert-alkylperoxyl radicals decreased, and the ethylperoxyl spin-adduct concentration did not change. Concomitant studies, using a Clark oxygen electrode, show that oxygen was consumed by the hematin-tert-alkyl hydroperoxide systems, but was released by the hematin-ethyl hydroperoxide reaction. Thus, molecular oxygen seems to play a subsidiary role in the hematin-catalyzed decomposition of hydroperoxides. Based on the electron spin resonance and oxygen electrode results, a mechanism for the continuous production of the peroxyl free radicals is proposed for hematin/hydroperoxide systems. The present spectroscopic methodology can be used to search for peroxyl free radical formation by hemoprotein/hydroperoxide systems.

SPECTROSCOPIC STUDIES OF CUTANEOUS PHOTOSENSITIZING AGENTS—II. SPIN TRAPPING OF PHOTOLYSIS PRODUCTS FROM SULFANILAMIDE AND 4-AMINOBENZOIC ACID USING 5,5-DIMETHYL-1-PYRROLINE-1-OXIDE

— The photodecomposition of sulfanilamide, 4-aminobenzoic acid and related analogs in aqueous solution has been studied with the aid of spin traps 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and CH3NO2 as well as by direct electron spin resonance techniques. The NH2 radical was trapped by DMPO during the photolysis of aqueous solutions of sulfanilamide with a Xe arc lamp. Studies with [15N1]-sulfanilamide indicated that the NH2 radical was generated by homolytic fission of the sulfur-nitrogen bond. Under the same conditions DMPO trapped the H and SO−3 radicals during photolysis of sulfanic acid. Direct photolysis of sulfanilamide, sulfanilic acid and Na2SO3 in the absence of any spin trap yielded the SO−3 radical. Photolysis of 4-aminobenzoic acid at pH 7 gave the H radical which was trapped by DMPO. At low pH values OH and C6H4COOH radicals were generated during the photolysis of 4-aminobenzoic acid. No e−aq were trapped by CH3NO2 when acid (pH 4) and neutral aqueous solutions of sulfanilamide or 4-aminobenzoic acid were photoirradiated. The mechanism of formation of known photoproducts from the free radicals generated by sulfanilamide and 4-aminobenzoic acid during irradiation are discussed. The free radicals generated by these agents may play an important role in their phototoxic and photoallergic effects.

Spin-trapping and direct electron spin resonance investigations of the redox metabolism of quinone anticancer drugs

The superoxide free radical has been spin trapped in microsomal incubations containing adriamycin, daunorubicin, and mitomycin C. The time sequence of the appearance of the spin-trapped superoxide and the semiquinone radical metabolite of these quinone-containing anticancer drugs indicates that air oxidation of the semiquinone is responsible for the superoxide formation. Superoxide dismutase prevents the formation of the superoxide spin adducts. Microsomal incubations containing anthracyclines intercalated in DNA produce much less superoxide than incubations free of DNA. The first unambiguous ESR evidence for the semiquinone metabolite of mitomycin C in a biological system is also presented.

Spin trapping of radicals formed during radiolysis of aqueous solutions. Direct electron spin resonance observations

It is shown that esolv, H•, and OH formed by the radiolysis of water by 3 MeV electrons are trapped by nitroso and nitrone compounds to give nitroxides with well defined esr spectra. Three spin trapping agents were used, nitroso-t-butane, phenyl-t-butyl nitrone, and 5,5′-dimethyl pyrroline-1-oxide. The latter was shown to be an excellent compound for these studies. Complications due to the instability of some of the nitroxides were overcome by coupling the electron accelerator to the esr spectrometer to permit direct observation of the spectra.

Determination of human liver cytochrome P-450 level by electron paramagnetic resonance of liver biopsies

Human liver cytochrome P-450 level has been determined by direct electron paramagnetic resonance spectroscopy at 77° K of liver specimens obtained by surgical biopsy. A preliminary experiment with rat livers was performed in order to calibrate the intensity of the cytochrome P-450 EPR signal as a function of the cytochrome concentration obtained by the classical spectrophotometric method. 44 subjects have been examined. We found a cytochrome P-450 value of 21.5 ± 11.2 nmoles per gram of liver in normal subjects. Variations of this level in some pathological liver states are discussed.

Direct electron spin resonance detection of free radicals produced in electron-irradiated liquid alcohols

Direct electron spin resonance detection of free radicals produced in electron-irradiated liquid alcohols

Radical cations. I. Direct electron paramagnetic resonance observation of the chlorine molecule cation Cl2+, a stable diatomic radical cation, in SbF5, FSO3H-SbF5, and HF-SbF5 solutions of ClF

Radical cations. I. Direct electron paramagnetic resonance observation of the chlorine molecule cation Cl2+, a stable diatomic radical cation, in SbF5, FSO3H-SbF5, and HF-SbF5 solutions of ClF