PBN Spin Adducts Research Articles

Vitamin E Forms Domains with Pufa in Membranes: An EPR Spin Trapping Study of Lipid Peroxidation in Model Membranes

Spin trapping in conjunction with EPR spectroscopy is a widely used technique for measurement of short-lived free radical species of biological interest due to its high sensitivity and specificity. In spin trapping a molecule (the spin trap) reacts with the free radical producing a spin adduct that is sufficiently stable to be detected by EPR. In this study, we employ lipid-soluble N-tert-butyl-α-phenylnitrone (PBN) as our spin trap. PBN spin adducts detected by EPR are believed to be lipid hydroperoxides that are secondary species resulting from free radical attack on cell membranes. Thus the presence of the spin-adducts is reflective of ROS levels. Lipid auto oxidation will be monitored in model membranes containing 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PDPC) with various concentrations of α-tocopherol, the major lipid soluble antioxidant in membranes and a primary component of vitamin E. Preliminary data of PDPC without α-tocopherol show auto oxidation that leads to a free radical cascade. By measuring the reduction in lipid peroxidation due to the presence of α-tocopherol, we test the hypothesis that the vitamin co-localizes with polyunsaturated lipids in membrane domains to ensure its close proximity to the most vulnerable lipid species.

EPR spin trapping and DFT studies on structure of active antioxidants in biogycerol

Spin trapping EPR spectroscopy and DFT methods were used to investigate the structure of antioxidants present in bioglycerols [glycerol fractions (GF) derived from biodiesel production]. For this purpose the reactions of GF components and their pure reference chemicals with PBN and H2O2 were examined via EPR measurements. The EPR parameters of formed PBN spin adducts indicated the α-tocopherol origin of the trapped radicals. The comparative analysis of experimental and theoretically calculated hyperfine constants for the spin adducts strongly suggests the carbon-centered nature of the radicals generated and trapped by PBN in the bioglycerol systems.

Free radicals and oxidative stress: targeted ESR measurement of free radicals

The detection of free radicals generated within the body may contribute to clarifying the pathophysiological role of free radicals in disease processes. As an appropriate procedure to examine the generation of free radicals in a biological system, electron spin resonance (ESR) has emerged as a powerful tool for detection and identification. A method for determination of oxygen radical scavenging activity using ESR and the spin trapping technique was developed. Oxygen radicals were trapped by 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) or alpha-phenyl-N-t-butylnitrone (PBN), and the DMPO or PBN spin adduct signal was measured quantitatively by an ESR spectrometer. The spin trapping method using ESR has also been reported for not only in vitro and ex vivo measurements but also in vivo measurements. In in vivo ESR, nitroxyl radical is being used as a spin trap well. ESR signal intensities of nitroxyl radical are measured after administration to animals and the signal decay rates of nitroxyl radical have reported to be influenced by various types of oxidative stress. With this method, it is possible to specify the type of radical or the location at which the free radicals are produced. The spin trapping method by in vivo ESR is an effective procedure for giving non-invasive measurements in animals. ESR imaging in the organs of live animals can also be obtained after injection of nitroxyl radicals as an imaging agent using ESR-computed tomography. In vivo ESR imaging has been established as a powerful technique for determining the spatial distribution of free radicals in living organs and tissues.

Generation of free radicals and the damage done to the sarcoplasmic reticulum during reperfusion injury following brief ischemia in the canine heart.

Free radical generation was studied by the electron spin resonance (ESR) technique using alpha-phenyl N tert butyl nitrone (PBN) in a brief ischemia-reperfusion model of the canine heart, and correlated with biochemical changes of the sarcoplasmic reticulum (SR). ESR spectra (aH=0.3-0.4mT, aN=1.43-1.58mT) were observed as PBN spin adducts, which peaked at levels 5-fold above the control levels at 5 min after reperfusion. The simulated coupling constants of PBN spin adducts suggested that the sample should contain at least 2 carbon-centered radicals at 5 min after reperfusion (radical A: aH=0.350mT, aN=1.485mT; radical B: aH=0.370mT, aN=1.615 mT). At this time point, a significant reduction in Ca-ATPase activity of the SR was found without degradation of the major ATPase protein. Superoxide dismutase (SOD) significantly reduced the intensity of the PBN spin adduct signals and preserved the Ca-ATPase activity of the SR to 80% of the control level. Reperfusion injury after brief ischemia may be the result of inactivation of intracellular Ca-ATPase by free radicals generated during reperfusion, and SOD contributes to the protective effect by scavenging the radicals.

Direct measurement of oxygen free radicals during in utero hypoxia in the fetal guinea pig brain

The present study tested the hypothesis that maternal hypoxia induces oxygen free radical generation in the fetal guinea pig brain utilizing techniques of electron spin resonance spectroscopy and alpha-phenyl- tert-butyl nitrone (PBN) spin trapping. Pregnant guinea pigs of 60 days gestation were divided into normoxic and hypoxic groups and exposed to 21% or 7% oxygen for 60 min. Free radical generation was documented by measuring the signal of PBN spin adducts. Fluorescent compounds were determined as an index of lipid peroxidation and the activity of Na +,K +-ATPase was determined as an index of brain cell membrane function. Hypoxic fetal cerebral cortical tissue showed a significant increase in spin adducts (normoxic: 33.8±9.3 units/g tissue vs. hypoxic: 57.9±9.2 units/g tissue, p<0.01) and fluorescent compounds (normoxic: 0.639±0.054 μg quinine sulfate/g brain vs. 0.810±0.102 μg quinine sulfate/g brain, p<0.01) and a decrease in Na +,K +-ATPase activity (normoxic: 43.04±2.50 μmol Pi/mg protein/h vs. hypoxic: 33.80±3.51 μmol Pi/mg protein/h, p<0.001). These results demonstrate an increased free radical generation during hypoxia in the fetal guinea pig brain. The spectral characteristics of the radicals were consistent with those of alkoxyl radicals. The increased level of fluorescent compounds and decreased activity of Na +,K +-ATPase indicated hypoxia induced brain cell membrane lipid peroxidation and dysfunction, respectively. These results directly demonstrate an increased oxygen free radical generation during hypoxia and suggest that hypoxia-induced increase in lipid peroxidation and decrease in membrane function, as indicated by a decrease in Na +,K +-ATPase activity, are consequences of increased free radicals. The nature of predominantly present alkoxyl radical indicates ongoing lipid peroxidation during hypoxia. The direct demonstration of oxygen free radical generation during hypoxia is the critical missing link in the mechanism of hypoxia-induced brain cell membrane dysfunction and damage.

Oxidative stress following traumatic brain injury in rats

Free radicals may be involved in the pathophysiology of traumatic brain injury (TBI) through oxidative damage of neurovascular structures. Endogenous antioxidants, such as ascorbate and alpha-tocopherol, may play a critical role in combating these oxidative reactions and their oxidized products can serve as an important index of oxidative stress. We used electron spin resonance (ESR) spectroscopy and in vivo spin trapping (reaction of an organic compound with free radical species) to detect the possible generation of free radicals after TBI. Injury was inflicted by a weight drop technique over the head (5.7 kg-cm). Rats were intravenously infused with either 1 mL, 0.1 M of the spin trap, alpha-phenyl-N-tert-butyl nitrone (PBN), or an equivalent volume of saline immediately before TBI or sham-injury. Animals were divided into four groups: (1) Group I: PBN-infused sham-injured, (2) Group II: PBN-infused injured, (3) Group III: saline-infused sham-injured, and (4) Group IV: saline-infused injured. Additional groups of saline-infused uninjured, saline-infused, and PBN-infused injured animals were used for histopathology. Sixty minutes after TBI or sham-injury, rats were again anesthetized and decapitated. The brains were removed within 1 minute, homogenized, and extracted for lipids. The extracts were analyzed by ESR spectroscopy. Brain ascorbic acid (AA) concentration was determined spectrophotometrically, using the ascorbate oxidase assay. No PBN spin adduct signals (indicating trapped free radical species) were visible 60 minutes after TBI. All groups of rats showed an ascorbyl free radical signal. The ascorbyl signal intensity (AI) was, however, significantly higher in the injured rats, while the brain (AA) was significantly reduced. In addition, the ratio of AI/AA, which eliminates the effect of variable ascorbate concentrations in the brain, was also significantly higher in the injured animals. We conclude that 60 minutes following TBI there was a significantly increased level of oxidative stress in the brain. This may reflect formation of free radical species with subsequent interaction with ascorbate (antioxidant) during the 60 minute period. The lack of PBN spin adduct signals 1 hour after TBI may indicate that free radical generation is time dependent and might be detectable earlier or later than the 60 minute period.

Spin Trapping of Phosphorus-Centered Radicals Produced by the Reactions of Dibenzoyl Peroxide with Organophosphorus Compounds

Abstract The reactions of dibenzoyl peroxide (BPO) with organophosphorus compounds have been studied by means of an ESR technique combined with spin trapping. The intermediate phosphorus-centered radicals were trapped by N-benzylidene-t-butylamine N-oxide (PBN) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The spin adducts with a characteristic hyperfine coupling constant (hfcc) due to a phosphorus atom were observed. From identification of trapped radicals, possible mechanisms of the reactions have been proposed. The hfccs due to β-hydrogen and phosphorus atoms exhibited a large temperature dependence for phosphoranyl–PBN spin adducts, while for phosphinyl–PBN spin adduct the dependence was small. Based on the P and H hfccs, the conformational positions of the adducts trapped by PBN and DMPO are discussed.

Photosensitization by Harmine: An ESR Spin Trapping Study on the Generation of the Superoxide Anion Radical

Harmine was in situ UV-irradiated in the presence of 5,5-dimethyl-1-pyrroline- N-oxide (DMPO), α-phenyl- N-tert-butyl nitrone (PBN), or α-(4-pyridyl-1-oxide) N-tert-butyl nitrone (4-POBN) as spin traps and observed by electron spin resonance (ESR) spectroscopy. The superoxide radical (O2·−) was detected as the corresponding DMPO, PBN, or 4-POBN spin adduct in dimethylsulfoxide (DMSO) or acetonitrile solution. The detection of these spin adducts was prevented by the addition of superoxide dismutase (SOD) to the solution. Also, the O2·− adduct formation was inhibited by the addition of antioxidants as cysteine methyl ester or ascorbic acid, in a dose-dependent manner. The studies carried out in aqueous buffered solution did not allow superoxide radical adduct detection. In the presence of DMPO as a spin trap, the DMPO–OH spin adduct was detected. On the other hand, classical scavengers of the hydroxyl radical such as mannitol or glycerol abolished DMPO–OH spin adduct detection. Other scavengers such as ethanol, DMSO, or sodium azide inhibited the trapping of hydroxyl radicals and resulted in the formation of new radical adducts. No PBN or 4-POBN spin adducts were detected in aqueous solution. The origin of the hydroxyl radical is discussed.

DOSE-DEPENDENT EFFECT OF ALLOPURINOL ON FREE RADICAL PRODUCTION DURING CEREBRAL HYPOXIA IN NEWBORN PIGLETS. ▴ 413

Previous studies have shown that allopurinol, a xanthine oxidase inhibitor and free radical scavenger, has a neuroprotective effect during hypoxia-ischemia. The present study tests the hypothesis that allopurinol has a dose-dependent effect on the hypoxia-induced generation of free radicals in the cerebral cortex of newborn piglets. Studies were performed on 15 piglets divided into 6 groups. Group 1 (control n=5) pretreated with saline and Groups 2-6 (n=2 each) pretreated with allopurinol at doses of 10, 20, 50, 100 and 200 mg/kg IV respectively. Hypoxia was induced by lowering the FiO2 to 5-7% for 1 hr to achieve a PaO2 of 18-20 mmHg. Cortical biopsies were obtained via cranial windows and tissue homogenized in 100 mMα-phenyl-N-tert-butyl-nitrone (PBN). PBN spin adducts were extracted in a 2:1 mixture of chloroform:methanol and electron spin resonance spectroscopy performed. Signal height (intensity) of spectrum divided by tissue weight was expressed as mm/g tissue. Brain tissue ATP and phosphocreatine (PCr) concentrations were used to determine cellular energy metabolism. Tissue levels of allopurinol were measured by HPLC and increased linearly with drug dose. Free radical intensity in Group 1 was 387±16 mm/g tissue(mean±S.E.). Groups 2-6 showed a linear decrease in free radical intensity with values approaching zero in the groups receiving 100 and 200 mg/kg of allopurinol (p<0.01). ATP concentrations (μmole/g tissue) increased linearly from 1.09±0.55 in Group 1 to 5.02±0.45 in the 200 mg/kg group (p<0.01). PCr concentrations (μmol/g brain) increased linearly from 0.19±0.03 in Group 1 to 1.10±0.29 in the 200 mg/kg group (p<0.01). The data show that administration of allopurinol decreases free radical production and preserves high energy phosphate compounds during hypoxia in a dose-dependent menner. We speculate that allopurinol reduces free radical production by inhibiting xanthine oxidase and scavenging free radicals and may play a direct neuromodulatory role in reducing excitotoxicity during hypoxia. (NIH-HD-20337, MOD-#6-FY94-0135).

DIRECT DETECTION OF OXYGEN FREE RADICAL GENERATION DURING INUTERO HYPOXIA IN THE FETAL GUINEA PIG BRAIN. ▴ 364

Previous studies have shown that hypoxia is associated with lipid peroxidation in the brain of guinea pig fetuses. The present study tests the hypothesis that the hypoxia-induced increase lipid peroxidation is related to increase of oxygen free radical generation. Free radical generation was documented by measuring the signal of spin adducts using electron spin resonance spectroscopy (ESR). Fourteen pregnant guinea pigs of 16 days gestation (term=63 days) were divided into normoxic (n=7) and hypoxic (n=7) groups and exposed to 21% or 7% oxygen for 60 min., respectively. Fetal brain hypoxia was documented biochemically by decreased levels of ATP and phosphocreatine. Following anesthesia, fetal cerebral cortical tissue was excised and homogenized in a N2 bubbled ice-cold 100 mMα-phenyl-N-tert-butylnitrone (PBN) to trap oxygen free radicals generated in brain tissue. Trapped PBN spin adducts were extracted in toluene and ESR measurements were performed at -20°C. Peak height of the spin adduct signal spectrum was used as an index of the intensity of free radical formation and expressed as units/g dry tissues. Cortical tissue from hypoxic fetuses showed a significant increase in spin adducts (normoxic: 33.8±9.3 units/g tissue vs. hypoxic: 57.9±9.2 units/g tissue, 71% increase. p<0.01). The results demonstrate an increased production of oxygen free radicals in the guinea pig fetal brain during hypoxia, suggesting that hypoxia-induced oxidation of cell components, such as cell membrane lipids, proteins, (enzymes and receptors) and nucleic acids follows increased generation of oxygen free radicals, therefore it is a primary mechanism of hypoxic cellular dysfunction. (This study was supported by NIH HD-20337; MOD#94-135 and UCP#R-506-93;WUH).

Spin trapping chemistry of acyl radicals

AbstractThe EPR spectra of a variety of different acyl radical adducts of PBN (C‐phenyl N‐tert‐butyl nitrone) and MNP (2‐methyl‐2‐nitrosopropane) were measured and their hyperfine splitting constants determined. Included are benzoyl, acetyl and other alkylacyl, alkoxyacyl, aminoacyl and alkylaminoacyl adducts. All examples gave relatively large β‐hydrogen hyperfine splittings ranging from 2.71 to 5.93 G in the PBN spin adducts. Long‐range hyperfine splitting was found to both nitrogen and amino‐hydrogen atoms in the aminoacyl adducts, indicating specific preferred conformations of the spin adducts. The MNP adducts have the well known 7‐8 G nitrogen hyperfine splitting with little else. The existence of hydrogen atom abstraction from benzaldehyde by nitroxides was established, indicating that nitroxides cannot be considered as inert reagents when sent to probe systems containing aldehydes. For example, the EPR signal due to a nitronyl nitroxide was rapidly quenched in the presence of benzaldehyde in benzene. Also, a surprising increase in intensity of benzoyl PBN adduct was noted after photolysis when the light was turned off. Double acyl adduct formation is proposed.

Tandem mass spectrometry study of C-phenyl- N-tert-butyl nitrone spin adducts from in vitro rat liver microsomal metabolism of bromotrichloromethane and carbon tetrachloride

Electron ionization and thermospray were used in conjunction with tandem mass spectrometry methods to identify trichloromethyl/C-phenyl-N-tert-butyl nitrone (PBN) spin adducts produced in rat liver microsomal dispersions that had been treated with reduced nicotinamide adenine dinucleotide phosphate (NADPH)-generating system and BrCCl3 (or CCl4). In the identification of PBN spin adducts, a scan of precursors of m / z 57 was utilized to confirm the presence of PBN spin adducts, because PBN spin adducts produce m / z 57 from tert-butyl as a characteristic fragment. Use of deuterated PBN (PBN-d9 deuterated at tert-butyl; PBN-d 14 deuterated at both phenyl and tert-butyl) improved the recognition of PBN adducts in mixtures by precursor ion scans, because m / z 66 (which corresponds to the deuterated tert-butyl group) is characteristic and, unlike m / z 57, it is not a common fragment for any other compounds. Two new PBN spin adducts that were not detected before by electron paramagnetic resonance or mass spectrometry were identified by these methods for the first time.

Studies of PBN Spin Adducts by the Combined Technique of Quartz Capillary Column GC/MS/ESR

A new technique, high resolution quartz capillary column GC/MS/ESR combined with the silanization approach of free radical adduct was successfully used in the analysis and determination of 6 types of PBN spin adducts. Satisfactory mass spectra of spin adducts were obtained. The results show that it is an efficient method for separating and identifying mixed free radicals.

Free radical generation in the brain precedes hyperbaric oxygen-induced convulsions

We tested the hypothesis that hyperbaric oxygenation (HBO) generates free radicals in the brain before the onset of neurological manifestations of central nervous system (CNS) oxygen poisoning. Chronically cannulated, conscious rats were individually placed in a transparent pressure chamber and exposed to (1) 5 atmospheres absolute (ATA) oxygen for 15 min ( n = 4); (2) 5 ATA oxygen for 30 min ( n = 5), during which no visible convulsions occurred; (3) 5 ATA oxygen for 30 min with recurrent convulsions ( n = 6); (4) 5 ATA oxygen until the appearance of the first visible convulsions ( n = 5); (5) 4 ATA oxygen for 60 min during which no convulsions occured ( n = 5); and (6) 5 ATA air for 30 min ( n = 5, controls). Immediately before compression, 1 mL of 0.1 M of alpha-phenyl- N-tert-butyl nitrone (PBN) was administered intravenously (iv) for spin trapping. At the termination of each experiment,, rats were euthanized by pentobarbital iv and decompressed within 1 min. Brains were rapidly removed for preparation of lipid extracts (Folch). The presence of PBN spin adducts in the lipid extracts was examined by electron spin resonance (ESR) spectroscopy. ESR spectra from unconvulsed 5 ATA oxygen for 30 min revealed both oxygen-centered and carbon-centered PBN spin adducts in three of the brains. One of the five rats in this group showed an ascorbyl signal in the ESR spectrum. In contrast, only one of the six rats with recurrent convulsions and one of five which were sacrified immediately after the first visible convulsions showed a low-intensity ESR signal due to PBN spin adducts. Unconvulsed rats exposed to 5 ATA oxygen for 15 min or 4 ATA oxygen for 60 min showed no PBN spin adducts but had an ascorbyl signal in two of four and three of five ESR spectra, respectively. Lipid extracts from control rats exposed to 5 ATA air showed no ascorbyl ESR signal. The preconvulsive appearance of PBN spin adducts and ascorbyl radical ESR signals is consistent with the free radical theory of the CNS oxygen toxicity. However, the reason for diminution or disappearance of PBN spin adducts after HBO-induced convulsions remains to be investigated.

Direct detection of free radical generation in an in vivo model of acute lung injury.

Electron spin resonance (ESR) spectroscopy has been used to provide direct evidence that free radical production occurs in an in vivo model of acute lung injury. Two experimental groups of rabbits were given the spin trap alpha-phenyl N-tert.-butyl nitrone (PBN), together with endotoxin in the test group, and saline in the control group. Both groups were subsequently briefly ventilated with air containing cigarette smoke. Plasma samples from the endotoxin pretreated group showed a sudden burst of radical formation, detected as PBN spin adduct, which peaked in the first ten minutes after smoke exposure. No signals were detected in the control group. Permeability of the alveolar capillary barrier of the lung, measured by the clearance of 99mTc-DTPA, demonstrated significantly greater damage following smoke in the endotoxin primed animals than in the controls. Temporal studies suggest that this increase in permeability occurred after a burst of radical production. These studies provide supportive evidence for the hypothesis that endotoxin promotes the accumulation of a population of primed white cells within the lung, which when triggered by cigarette smoke, are able to generate a burst of free radicals which produce tissue damage and acute lung injury.

Recombinant human extracellular superoxide dismutase reduces concentration of oxygen free radicals in the reperfused rat heart

The aim of the study was to determine whether recombinant human extracellular superoxide dismutase type C (rh-EC-SOD C) has antioxidant effects similar to those of the previously studied Cu-Zn superoxide dismutase. Electron paramagnetic resonance spectroscopy was used for quantification of free radical generation during reperfusion of postischaemic isolated rat hearts. The hearts were perfused with Krebs-Henseleit buffer and then subjected to 15 min global ischaemia. N-tert-butyl-alpha-phenylnitrone (PBN) was added as a spin trapping agent to the perfusate. 18 hearts from Sprague-Dawley rats (280-400 g) were used. Reperfusion resulted in a burst of radical formation. In presence of rh-EC-SOD C plus catalase, the PBN spin adduct concentration was only 18% (p less than 0.05) of the value in the control group. With bovine Cu-Zn superoxide dismutase plus catalase added to the buffer the corresponding spin adduct formation was 44% of control (NS = v rh-EC-SOD C). In isolated rat heart, rh-EC-SOD C reduces free radical concentrations at least to the same extent as Cu-Zn superoxide dismutase.

Mass Spectroscopy and Chromatography of the Trichloromethyl Radical Adduct of PhenylTer T-Butyl Nitrone

Positive structural identification of the PBN-trichloromethyl spin adduct in vitro was accomplished with the use of high pressure liquid chromatography and/or gas chromatography coupled with mass spectrometry. Both thin layer and liquid chromatography were used to separate a complex mixture of compounds from rat liver extracts treated with CCl4 in vitro and in vivo. Deuterated PBN's (PBN-d9; tert-butyl deuteration, or PBN-d14; both phenyl and tert-butyl deuteration) were also used to aid in the mass spectral analysis of spin adducts from liver extracts of CCl4 exposed rat livers, since the tert-butyl group fragment ion. C4D9+ (m/z = 66) is always present for PBN and PBN spin adducts. In addition, the masses of the ion peaks increase by the amount of deuteration, i.e. an increase of 9 for PBN-d9 or PBN-d14 in comparison to normally synthesized PBN.

Structure Identification of Free Radicals by Esr And Gc/Ms of Pbn Spin Adducts From theIn Vitroandin vivoRat Liver Metabolism Of Halothane

Free radicals were detected from the in vitro metabolism of halothane (rat liver microsomes) by the PBN spin trapping method. The detected radical species include the 1-chloro-2,2,2-trifluoro-1-ethyl radical (I), as determined by mass spectral analysis, and lipid-type radicals assigned by high resolution ESR spectroscopy with the use of d14-deuterated PBN. The lipid-derived radicals are a carbon-centred radical with the partially assigned structure CH2R and an oxygen-centred radical of the OR' type. From the mass spectral analysis of the spin adduct mixture there is also evidence for a halocarbon double adduct of PBN of the type I-PBN-I.

An antimycin-insensitive succinate-cytochrome c reductase activity in pure reconstitutively active succinate dehydrogenase

Antimycin-insensitive succinate-cytochrome c reductase activity has been detected in pure, reconstitutively active succinate dehydrogenase. The enzyme catalyzes electron transfer from succinate to cytochrome c at a rate of 0.7 μmole succinate oxidized per min per mg protein, in the presence of 100 μM cytochrome c. This activity, which is about 2% of that of reconstitutive (the ability of succinate dehydrogenase to reconstitute with coenzyme ubiquinone-binding proteins (QPs) to form succinate-ubiquinone reductase) or succinate-phenazine methosulfate activity in the preparation, differs from antimycin-insensitive succinate-cytochrome c reductase activity detected in submitochondrial particles or isolated succinate-cytochrome c reductase. The K m for cytochrome c for the former is too high to be measured. The K m for the latter is about 4.4 μM, similar to that of antimycin-sensitive succinate-cytochrome c activity in isolated succinate-cytochrome c reductase, suggesting that antimycin-insensitive succinate-cytochrome c activity of succinate-cytochrome c reductase probably results from incomplete inhibition by antimycin. Like reconstitutive activity of succinate dehydrogenase, the antimycin-insensitive succinate-cytochrome c activity of succinate dehydrogenase is sensitive to oxygen; the half-life is about 20 min at 0°C at a protein concentration of 23 mg/ml. In the presence of QPs, the antimycin-insensitive succinate-cytochrome c activity of succinate dehydrogenase disappears and at the same time a thenoyltrifluoroacetone-sensitive succinate-ubiquinone reductase activity appears. This suggests that antimycin-insensitive succinate-cytochrome c reductase activity of succinate dehydrogenase appears when succinate dehydrogenase is detached from the membrane or from QPs. Reconstitutively active succinate dehydrogenase oxidizes succinate using succinylated cytochrome c as electron acceptor, suggesting that a low potential intermediate (radical) may be involved. This suggestion is confirmed by the detection of an unknown radical by spin trapping techniques. When a spin trap, α-phenyl- N- tert-butylnitrone (PBN), is added to a succinate oxidizing system containing reconstitutively active succinate dehydrogenase, a PBN spin adduct is generated. Although this PBN spin adduct is identical to that generated by xanthine oxidase, indicating that a perhydroxy radical might be involved, the insensitivity of this antimycin-insensitive succinate-cytochrome c reductase activity to superoxide dismutase and oxygen questions the nature of this observed radical.

Electrochemical Detection of PBN Spin Adduct Aminoxyls (Nitroxides) Separated by High Performance Liquid Chromatography

Abstract Spin adduct aminoxyls (nitroxides) produced from the reaction of shortlived radicals with α-phenyl-tert-butyl nitrone (PBN) could be separated by high performance liquid chromatography (HPLC) and analyzed by both UV and electrochemical detectors. Peak assignments of the spin adduct aminoxyls were confirmed by collecting peak effluents and analyzing by electron spin resonance (ESR) spectroscopy. Mixtures of aminoxyl spin adducts that were unresolvable by ESR methods were separated by HPLC and their individual ESR spectra recorded.