Identification of the myoglobin tyrosyl radical by immuno-spin trapping and its dimerization

5,5-Dimethyl-1-pyrroline N-oxide (DMPO) spin trapping in conjunction with antibodies specific for the DMPO nitrone epitope was used on hydrogen peroxide-treated sperm whale and horse heart myoglobins to determine the site of protein nitrone adduct formation. The present study demonstrates that the sperm whale myoglobin tyrosyl radical, formed by hydrogen peroxide-dependent self-peroxidation, can either react with another tyrosyl radical, resulting in a dityrosine cross-linkage, or react with the spin trap DMPO to form a diamagnetic nitrone adduct. The reaction of sperm whale myoglobin with equimolar hydrogen peroxide resulted in the formation of a myoglobin dimer detectable by electrophoresis/protein staining. Addition of DMPO resulted in the trapping of the globin radical, which was detected by Western blot. The location of this adduct was demonstrated to be at tyrosine-103 by MS/MS and site-specific mutagenicity. Interestingly, formation of the myoglobin dimer, which is known to be formed primarily by cross-linkage of tyrosine-151, was inhibited by the addition of DMPO.     

Probing the free radicals formed in the metmyoglobin-hydrogen peroxide reaction

The reaction between metmyoglobin and hydrogen peroxide results in the two-electron reduction of H2O2 by the protein, with concomitant formation of a ferryl-oxo heme and a protein-centered free radical. Sperm whale metmyoglobin, which contains three tyrosine residues (Tyr-103, Tyr-146, and Tyr-151) and two tryptophan residues (Trp-7 and Trp-14), forms a tryptophanyl radical at residue 14 that reacts with O2 to form a peroxyl radical and also forms distinct tyrosyl radicals at Tyr-103 and Tyr-151. Horse metmyoglobin, which lacks Tyr-151 of the sperm whale protein, forms an oxygen-reactive tryptophanyl radical and also a phenoxyl radical at Tyr-103. Human metmyoglobin, in addition to the tyrosine and tryptophan radicals formed on horse metmyoglobin, also forms a Cys-110-centered thiyl radical that can also form a peroxyl radical. The tryptophanyl radicals react both with molecular oxygen and with the spin trap 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS). The spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) traps the Tyr-103 radicals and the Cys-110 thiyl radical of human myoglobin, and 2-methyl-2-nitrosopropane (MNP) traps all of the tyrosyl radicals. When excess H2O2 is used, DBNBS traps only a tyrosyl radical on horse myoglobin, but the detection of peroxyl radicals and the loss of tryptophan fluorescence support tryptophan oxidation under those conditions. Kinetic analysis of the formation of the various free radicals suggests that tryptophanyl radical and tyrosyl radical formation are independent events, and that formation of the Cys-110 thiyl radical on human myoglobin occurs via oxidation of the thiol group by the Tyr-103 phenoxyl radical. Peptide mapping studies of the radical adducts and direct EPR studies at low temperature and room temperature support the conclusions of the EPR spin trapping studies.     

Reassignment of organic peroxyl radical adducts.

The study of the important role of peroxyl radicals in biological systems is limited by their difficult detection with direct electron spin resonance (ESR). Many ESR spectra were assigned to 5,5-dimethyl-1-pyrroline N-oxide (DMPO)/peroxyl radical adducts based only on the close similarity of their ESR spectra to that of DMPO/superoxide radical adduct in conjunction with their insensitivity to superoxide dismutase, which distinguishes the radical adduct from DMPO/superoxide radical adduct. Later, the spin-trapping literature reported that DMPO/peroxyl radical adducts have virtually the same hyperfine coupling constants as synthesized alkoxyl radical adducts, raising the issue of the correct assignment of peroxyl radical adducts. However, using 17O-isotope labelling, the methylperoxyl and methoxyl radical adducts should be distinguishable. We have reinvestigated the spin trapping of the methylperoxyl radical. The methylperoxyl radical was generated in aerobic solution with 17O-molecular oxygen either in a Fenton system with dimethylsulfoxide or in a chloroperoxidase system with tert-butyl hydroperoxide. Two different spin traps, DMPO and 2,2,4-trimethyl-2H-imidazole-1-oxide (TMIO), were used to trap methylperoxyl radical. 17O-labelled methanol was used to synthesize methoxyl radical adducts by nucleophylic addition. It was shown that the 17O hyperfine coupling constants of radical adducts formed in methylperoxyl radical-generating systems are identical to that of the methoxyl radical adduct. Therefore, methylperoxyl radical-producing systems form detectable methoxyl radical adduct, but not detectable methylperoxyl radical adducts at room temperature. One of the possible mechanisms is the decomposition of peroxyl radical adduct with the formation of secondary alkoxyl radical adduct. These results allow us to reinterpret previously published data reporting detection of peroxyl radical adducts. We suggest that detection of 17O-alkoxyl radical adduct from 17O-labelled molecular oxygen can be used as indirect evidence for peroxyl radical generation.     

Spin trapping of lipid radicals with DEPMPO-derived spin traps: detection of superoxide, alkyl and alkoxyl radicals in aqueous and lipid phase

The spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) forms a superoxide adduct with a half-life of almost 15 min. DEPMPO is very hydrophilic and its use for the detection of radicals in the lipid phase (lipid-derived radicals and superoxide generated in the lipid phase) is therefore limited due to its very low concentration in the lipid phase. For the detection of lipid-derived radicals, three derivatives of DEPMPO with increasing degree of lipid solubility have been investigated: 5-(di-n-propoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DPPMPO), 5-(di-n-butoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DBPMPO), and 5-(bis-(2-ethylhexyloxy)phosphoryl)-5-methyl-1-pyrroline N-oxide (DEHPMPO). As compared with the spin trap DMPO, the half-lives of the respective superoxide adducts were clearly higher in aqueous solutions of the spin traps, which facilitates qualitative ESR measurements. The stability of the superoxide spin adducts formed with the various lipophilic spin traps in aqueous buffer were similar to those observed with DEPMPO (half-life: 7-11 min.). In model experiments using Fe(3+)-catalyzed nucleophilic addition of methanol or tert-butanol to the respective spin trap the respective alkoxyl radical adducts were formed in aqueous solution as transient species in the presence of high concentrations of the alcohol. Upon dilution with water the alkoxyl group was substituted by water, giving the respective hydroxyl adduct of the spin trap. Care must therefore be taken when Fenton-type reactions are used for the generation of radicals such as the use of Fe(2+) complexes with phosphate or DTPA or inactivation of iron by addition of "Desferal" (Novarti's Pharma GmbH, Vienna, Austria) after a short incubation time. Addition of Fe(2+) under anaerobic conditions to an aqueous suspension of linoleic acid hydroperoxide and the spin trap resulted in the detection of three different species: a carbon-centered radical adduct, an acyl radical adduct, and the hydroxyl adduct. In the presence of oxygen a different species was observed with DEPMPO, DPPMPO, and DBPMPO, which was only slightly suppressed upon the addition of SOD, possibly the respective spin adduct of either the alkylperoxyl radical or, in analogy to DMPO, a secondary alkoxyl radical.     

Stabilities of hydroxyl radical spin adducts of PBN-type spin traps.

The stability of the hydroxyl spin adduct of nine different PBN-type spin traps has been examined in phosphate buffer solutions of various pH. The hydroxyl adduct is produced by short illumination of hydrogen peroxide with UV light in the presence of spin trap and the decay of its EPR signal followed. The stability measured by the half life of the first-order decay is strongly dependent on the pH of the solution and the structure of the aromatic ring used in the trap. All hydroxyl adducts are more stable in acidic media. tert-Butyl hydroaminoxyl is detected as a degradation product of the hydroxyl adduct from all spin traps.     

The Effect of Myoglobin on the Stability of the Hydroxyl-Radical Adducts of 5, 5 Dimethyl-1-Pyrolline-N-Oxide (DMPO), 3, 3,5, 5 Tetramethyl-1-pyrolline-N-Oxide(TMPO) and 1-Alpha-Phenyl-Tert-Butyl Nitrone (PBN) in the Presence of Hydrogen Peroxide

The hydroxyl radical adducts of 5, 5 dimethyl-1-pyrolline-N-oxide (DMPO) and 3, 3,5, 5 tetramethyl-1-pyrolline-N-oxide (TMPO) formed in the presence of hydrogen peroxide and Fe are normally quite stable, but in the presence of 5-20 micromolar myoglobin their ESR signals decay rapidly. This decay probably reflects further oxidation of the adduct to nonparamgnetic products.

The ESR signal of the hydroxyl radical adduct of 1-alpha-phenyl-tert-butyl nitrone (PBN) formed under similar conditions is subject to non-heme dependent attenuation, possibly via hydroxyl radical scavenging, but not to heme dependent decay. Hydrogen peroxide readily converts myoglobin to its ferryl (Fe^IV) derivative, and this centre may be responsible for the oxidation of the DMPO and TMPO adducts. The different behaviour of PBN may be due to differences in susceptibility to ferrylmyoglobin mediated oxidation, or to steric differences controlling access to the heme pocket of myoglobin, and is relevant to the choice of spin trap for biological experiments aimed at detecting hydroxyl radicals in the presence of myoglobin or other heme proteins.     

Detection of nucleic acid-derived radicals formed by alloxan

Pyrimidine base-derived radical spin adducts were detected in reaction mixtures containing pyrimidine bases, glutathione, and alloxan by the ESR spin trapping technique with a spin trap, alpha-phenyl-N-tert-butyl nitrone (PBN). Pyrimidine nucleoside- and nucleotide-, and ribose- and deoxyribose-derived radical spin adducts of PBN were also observed. However, purine base- and nucleoside-derived radical spin adducts of PBN were not detected. A cytosine-derived radical spin adduct of PBN was not generated under anaerobic conditions. Catalase and mannitol inhibited the formation of the cytosine-derived radical spin adduct of PBN but superoxide dismutase (SOD) did not. EDTA stimulated it and desferrioxamine suppressed it nearly completely. From these results it is presumed that the hydroxyl radical is involved in the formation of the cytosine-derived radical spin adduct of PBN generated by alloxan.     

Differential additions to the myoglobin prosthetic heme group. Oxidative gamma-meso substitution by alkylhydrazines.

The oxidative reaction of equine myoglobin with alkylhydrazines results primarily in introduction of the alkyl group at the sterically hindered gamma-meso position. The gamma-meso adducts formed with ethyl- and n-butylhydrazine have been isolated and unambiguously identified. With high pressure liquid chromatography, evidence for the formation of similar adducts with methyl- and n-propylhydrazine but not tert-butyl-, 2,2,2-trifluoroethyl-, or 2-phenylethylhydrazine has also been obtained. The gamma regiospecificity of the reaction of myoglobin with alkylhydrazines contrasts with the delta meso regiospecificity in the alkylation of peroxidases. Addition to the porphyrin vinyl groups is not detected, but N-alkylheme adducts appear to be formed in very low yield. Cofactor studies establish that H2O2 is absolutely required for meso heme alkylation and EPR/spin trapping studies show that alkyl free radicals are the probable alkylating species. In contrast, the reductive reaction of sperm whale myoglobin with CBrCl3 results in addition of the CCl3.radical to the 2-vinyl moiety of the heme group (Osawa, Y., Highet, R. J., Murphy, C. M., Cotter. R.J., and Pohl, L.R. (1989) J. Am. Chem. Soc. 111, 4462-4467). Carbon radicals thus apparently add to different sites of the myoglobin prosthetic group under reductive and oxidative conditions, presumably because of differences in the oxidation state of the heme and/or the intrinsic reactivities of alkyl and polyhaloalkyl radicals.     

Immunochemical detection of hemoglobin-derived radicals formed by reaction with hydrogen peroxide: involvement of a protein-tyrosyl radical

To investigate the involvement of a hemoglobin radical in the human oxyhemoglobin (oxyHb) or metHb/H2O2 system, we have used a new approach called "immuno-spin trapping," which combines the specificity and sensitivity of both spin trapping and antigen:antibody interactions. Previously, a novel rabbit polyclonal anti-DMPO nitrone adduct antiserum, which specifically recognizes protein radical-derived nitrone adducts, was developed and validated in our laboratory. In the present study, the formation of nitrone adducts on hemoglobin was shown to depend on the oxidation state of the iron heme, the concentrations of H2O2 and DMPO, and time as determined by enzyme-linked immunosorbent assay (ELISA) and by Western blotting. The presence of reduced glutathione or L-ascorbate significantly decreased the level of nitrone adducts on metHb in a dose-dependent manner. To confirm the ELISA results, Western blotting analysis showed that only the complete system (oxy- or metHb/DMPO/H2O2) generates epitopes recognized by the antiserum. The specific modification of tyrosine residues on metHb by iodination nearly abolished antibody binding, while the thiylation of cysteine residues caused a small but reproducible decrease in the amount of nitrone adducts. These findings strongly suggest that tyrosine residues are the site of formation of the immunochemically detectable hemoglobin radical-derived nitrone adducts. In addition, we were able to demonstrate the presence of hemoglobin radical-derived nitrone adducts inside red blood cells exposed to H2O2 and DMPO. In conclusion, our new approach showed several advantages over EPR spin trapping with the anti-DMPO nitrone adduct antiserum by demonstrating the formation of tyrosyl radical-derived nitrone adduct(s) in human oxyHb/metHb at much lower concentrations than was possible with EPR and detecting radicals inside RBC exposed to H2O2.     

Detection of superoxide production in stimulated and unstimulated living cells using new cyclic nitrone spin traps

Reactive oxygen species (ROS), including superoxide anion and hydrogen peroxide (H₂O₂), have a diverse array of physiological and pathological effects within living cells depending on the extent, timing, and location of their production. For measuring ROS production in cells, the ESR spin trapping technique using cyclic nitrones distinguishes itself from other methods by its specificity for superoxide and hydroxyl radical. However, several drawbacks, such as the low spin trapping rate and the spontaneous and cell-enhanced decomposition of the spin adducts to ESR-silent products, limit the application of this method to biological systems. Recently, new cyclic nitrones bearing a triphenylphosphonium (Mito-DIPPMPO) or a permethylated β-cyclodextrin moiety (CD-DIPPMPO) have been synthesized and their spin adducts demonstrated increased stability in buffer. In this study, a comparison of the spin trapping efficiency of these new compounds with commonly used cyclic nitrone spin traps, i.e., 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and analogs BMPO, DEPMPO, and DIPPMPO, was performed on RAW 264.7 macrophages stimulated with phorbol 12-myristate 13-acetate. Our results show that Mito-DIPPMPO and CD-DIPPMPO enable a higher detection of superoxide adduct, with a low (if any) amount of hydroxyl adduct. CD-DIPPMPO, especially, appears to be a superior spin trap for extracellular superoxide detection in living macrophages, allowing measurement of superoxide production in unstimulated cells for the first time. The main rationale put forward for this extreme sensitivity is that the extracellular localization of the spin trap prevents the reduction of the spin adducts by ascorbic acid and glutathione within cells.     

Spin trapping of polyunsaturated fatty acid-derived peroxyl radicals: reassignment to alkoxyl radical adducts

Polyunsaturated fatty acid (PUFA) peroxyl radicals play a crucial role in lipid oxidation. ESR spectroscopy with the spin-trapping technique is one of the most direct methods for radical detection. There are many reports of the detection of PUFA peroxyl radical adducts; however, it has recently been reported that attempted spin trapping of organic peroxyl radicals at room temperature formed only alkoxyl radical adducts in detectable amounts. Therefore, we have reinvestigated spin trapping of the linoleic, arachidonic, and linolenic acid-derived PUFA peroxyl radicals. The slow-flow technique allowed us to obtain well-resolved ESR spectra of PUFA-derived radical adducts in a mixture of soybean lipoxygenase, PUFA, and the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). However, interpretation of the ESR spectra was complicated by the overlapping of the PUFA-derived alkoxyl radical adduct spectra. In order to understand these spectra, PUFA-derived alkoxyl radical adducts were modeled by various alkoxyl radical adducts. For the first time, we synthesized a wide range of DMPO adducts with primary and secondary alkoxyl radicals. It was found that many ESR spectra previously assigned as DMPO/peroxyl radical adducts based on their close similarity to the ESR spectrum of the DMPO/superoxide radical adduct, in conjunction with their insensitivity to superoxide dismutase, are indeed alkoxyl radical adducts. We have reassigned the PUFA alkylperoxyl radical adducts to their corresponding alkoxyl radical adducts. Using hyperfine coupling constants of model DMPO/alkoxyl radical adducts, the computer simulation of DMPO/PUFA alkoxyl radical adducts was performed. It was found that the trapped, oxygen-centered PUFA-derived radical is a secondary, chiral alkoxyl radical. The presence of a chiral carbon atom leads to the formation of two diastereomers of the DMPO/PUFA alkoxyl radical adduct. Therefore, attempted spin trapping of the PUFA peroxyl radical by DMPO at room temperature leads to the formation of the PUFA alkoxyl radical adduct.     

Spin Trapping of Free Radicals Formed During Peroxidation of Sarcolemmal Membranes (SLM)

The generation of free radicals in a superoxide (O₂-)driven Fe⁺³ catalysed reactions with isolated myocytic sarcolemma using electron spin resonance was investigated. Incubation of highly purified canine myocytic sarcolemma in the presence of the spin trap, 2-methyl-2-nitrosopropane (MNP). followed by the addition of dihydroxyfurmarate (DHF) and Fe⁺³-ADP resulted in the generation and detection of radical adducts of this spin trap. Spin trapping of the alkyl radicals with 2-methyl-2-nitrosopropane led to the identification of methyl radical adduct following exposure to DHF/Fe⁺³-ADP. With sarcolemma and the alkyl nitroso compound, the only radical product trapped was the methyl radical formed by β-scission of alkoxyl radical. The participation of hydroperoxide-derived radicals in this system verified that the decomposition of unsaturated hydroperoxy fatty acid does proceed via a free radical mechanism.     

Immunological identification of the heart myoglobin radical formed by hydrogen peroxide

This study reports the detection of protein free radicals using the specific free radical reactivity of nitrone spin traps in conjunction with nitrone-antibody specificity. Polyclonal antibodies were developed that bind to protein adducts of the nitrone spin-trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The antibodies were used to detect DMPO protein adducts produced on horse myoglobin resulting from self-peroxidation. Western blot analysis demonstrates that myoglobin forms the predominant radical-derived nitrone adduct in rat heart supernatant.     

Using anti-5,5-dimethyl-1-pyrroline N-oxide (anti-DMPO) to detect protein radicals in time and space with immuno-spin trapping

The detection of protein free radicals using the specific free radical reactivity of nitrone spin traps in conjunction with nitrone-antibody sensitivity and specificity greatly expands the utility of the spin trapping technique, which is no longer dependent on the quantum mechanical electron spin resonance (ESR). The specificity of the reactions of nitrone spin traps with free radicals has already made spin trapping with ESR detection the most universal, specific tool for the detection of free radicals in biological systems. Now the development of an immunoassay for the nitrone adducts of protein radicals brings the power of immunological techniques to bear on free radical biology. Polyclonal antibodies have now been developed that bind to protein adducts of the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). In initial studies, anti-DMPO was used to detect DMPO protein adducts produced on myoglobin and hemoglobin resulting from self-peroxidation by H2O2. These investigations demonstrated that myoglobin forms the predominant detectable protein radical in rat heart supernatant, and hemoglobin radicals form inside red blood cells. In time, all of the immunological techniques based on antibody-nitrone binding should become available for free radical detection in a wide variety of biological systems.     

The Effects of Myoglobin and Apomyoglobin on the Formation and Stability of the Hydroxyl Radical Adduct of 5,5'-Dimethyl-1-Pyrroline-N-Oxide

When aqueous solutions of the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) are treated with hydrogen peroxide in the presence of either Fe or light, the hydroxyl radical adduct DMPO-OH is formed, with a characteristic 4 line ESR spectrum. When oxy- or metmyoglobin is added to such a system the initial yield and the halife of DMPO-OH are reduced, and at high myoglobin concentrations (about 0.1 mmol dm ^-l3) DMPO-OH becomes undetectable. Using the stable nitroxide 2,2,6,6-tetramethyl-1-piperidinyloxy-N-oxyl (TMPO) for comparison it was found that neither hydrogen peroxide nor myoglobin alone caused a loss of signal, but together a marked loss of signal was induced. From the evidence of these and other experiments it was concluded that the DMPO-OH adduct reacts with hydrogen peroxide and myoglobin to give non-paramagnetic products, and hence that the use of the DMPO spin trap to detect hydroxyl or other active radicals in systems containing physiological concentrations of myoglobin may give misleading results.     

Fatty acid radical formation in rats administered oxidized fatty acids: in vivo spin trapping investigation.

We report in vivo evidence for fatty acid-derived free radical metabolite formation in bile of rats dosed with spin traps and oxidized polyunsaturated fatty acids (PUFA). When rats were dosed with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and oxidized PUFA, the DMPO thiyl radical adduct was formed due to a reaction between oxidized PUFA and/or its metabolites with biliary glutathione. In vitro experiments were performed to determine the conditions necessary for the elimination of radical adduct formation by ex vivo reactions. Fatty acid-derived radical adducts of alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) were detected in vivo in bile samples collected into a mixture of iodoacetamide, desferrioxamine, and glutathione peroxidase. Upon the administration of oxidized 13C-algal fatty acids and 4-POBN, the EPR spectrum of the radical adducts present in the bile exhibited hyperfine couplings due to 13C. Our data demonstrate that the carbon-centered radical adducts observed in in vivo experiments are unequivocally derived from oxidized PUFA. This in vivo evidence for PUFA-derived free radical formation supports the proposal that processes involving free radicals may be the molecular basis for the previously described cytotoxicity of dietary oxidized PUFA.     

Free radical intermediates in the reaction of pyruvate:ferredoxin oxidoreductase in Tritrichomonas foetus hydrogenosomes

Aerobic incubations of the Tritrichomonas foetus hydrogenosomal fraction containing pyruvate, CoA, and the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) gave spectra of two radical adducts. One was a carbon-centered radical adduct of DMPO. This radical was centered at C-3 of pyruvate as determined in experiments using [13C]pyruvate. The other radical detected was identified as the CoA radical adduct of DMPO by comparison with an adduct obtained by incubating CoA with DMPO, H2O2 and horseradish peroxidase. Deletion of CoA led to an increased stability of the carbon-centered radical adduct of DMPO, disappearance of the thiyl radical adduct of DMPO, and appearance of a hydroxyl radical adduct of DMPO. Superoxide dismutase suppressed the appearance of the DMPO-hydroxyl radical adduct but did not have any inhibitory effect on the appearance of the other adducts. Catalase had no significant effect on any of the adducts. Addition of pyruvate to these hydrogenosomal preparations stimulated oxygen consumption. Addition of CoA led to a further increase in the rate of O2 uptake but had no effect in the absence of pyruvate. The formation of two substrate free radicals as intermediates in the generation of acetyl-CoA represents a novel mechanism for this enzymatic reaction and indicates that the pyruvate:ferredoxin oxidoreductase from T. foetus differs significantly from the pyridine nucleotide-dependent pyruvate dehydrogenase complex of other eukaryotic cells in its catalytic mechanism.     

1H NMR resonance assignments in a paramagnetic heme protein by two-dimensional spectroscopy: heme resonances in equine met-azido myoglobin

Specific heme protons for the majority of resonances in the downfield resolved region of equine met-azido myoglobin have been assigned using solely the two-dimensional 1H NMR experiments NOESY and COSY. Metazido myoglobin provides a useful test case for the applicability of these techniques to paramagnetic proteins for the following reasons. First met-azido myoglobin is a mixed spin-state protein, with significantly shorter relaxation times and broadened lines relative to pure low-spin systems (eg., met-cyano myoglobin). Second, met-azido hemoglobin and met-azido myoglobin are important as models for the physiological forms of hemoglobin. Third, a few sperm whale met-azido myoglobin resonances have been previously assigned, which permits a comparison of assignments for these similar proteins, and a check of the method presented here.     

Spin adducts of superoxide,alkoxyl, and lipid-derived radicals with EMPO and its derivatives

The compound 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide (EMPO) is a hydrophilic cyclic nitrone spin trap, which, in contrast to DMPO, forms a relatively stable superoxide adduct (t(1/2)=8.6 min) with an EPR spectrum similar to the respective DMPO adduct. In order to find the optimal degree of lipophilicity of this novel type of spin trap with respect to the detection of radicals formed during lipid peroxidation, the ethoxy group of EMPO was replaced by alkoxy substituents of increasing chain length, leading to the methoxy- (MeMPO), 1-propoxy- (PrMPO), 1-butoxy- (BuMPO), and 1-octyloxy- (OcMPO) derivatives of EMPO. The stability of their superoxide adducts was found to be strongly dependent on the size of the alkoxycarbonyl group. Increasing chain length of the alkoxyl substituent decreased the stability of alkoxyl radical adducts of MeMPO, EMPO, and PrMPO, but increased the stability of OcMPO adducts. The stability of alkoxyl radical adducts of BuMPO, on the other hand, were practically independent of the size of the alkoxyl group. Detection of lipid alkoxyl radicals formed by peroxidizing linoleic acid in a stationary system was therefore only possible with the most lipophilic spin trap, OcMPO. However, with the more hydrophilic spin traps MeMPO, EMPO, PrMPO, and BuMPO optimal EPR signal intensity could be obtained when a slow-flow system was used. Thus, within this series EMPO is the best spin trap for the detection of superoxide; OcMPO, on the other hand, is most suitable for the detection of lipid alkoxyl radicals.     

Evaluation of dibromonitrosobenzene sulfonate as a spin trap in biological systems

In the present study dibromonitrosobenzene sulfonate (DBNBS) was examined for its suitability for spin trapping for ESR detection of superoxide radicals in biological systems. This nitroso spin trap recently has been reported to yield very persistent spin adducts with O2. as well as with various carbon-centered radicals. In the present work the possible toxicity of DBNBS, the partitioning of its spin adducts into cells, and the stability of the adducts and the parent compound inside cells were studied. No significant toxicity was found. In cellular systems, however, DBNBS did not produce detectable adducts with O2.; it also did not detectably trap superoxide generated in the xanthine/xanthine oxidase system. Both DBNBS and a DBNBS adduct performed extracellularly and then added to cell suspensions were rapidly metabolized by cells. Intracellular spin adducts were not detected under any condition. Evidently, in spite of its promising features, DBNBS will not be useful for spin trapping radicals in cellular systems or for detecting superoxide radicals in any biological system.