Mitochondrial aconitase is a source of hydroxyl radical. An electron spin resonance investigation

Mitochondrial aconitase (m-aconitase) contains a [4Fe-4S](2+) cluster in its active site that catalyzes the stereospecific dehydration-rehydration of citrate to isocitrate in the Krebs cycle. It has been proposed that the [4Fe-4S](2+) aconitase is oxidized by superoxide, generating the inactive [3Fe-4S](1+) aconitase. In this reaction, the likely products are iron(II) and hydrogen peroxide. Consequently, the inactivation of m-aconitase by superoxide may increase the formation of hydroxyl radical ((*)OH) through the Fenton reaction in mitochondria. In this work, evidence for the generation of (*)OH from the reaction of m-aconitase with superoxide is provided using ESR spin trapping experiments with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide and alpha-phenyl-N-tert-butylnitrone. Formation of free ( small middle dot)OH was verified with the (*)OH scavenger Me(2)SO, which forms methyl radical upon reacting with (*)OH. The addition of Me(2)SO to incubation mixtures containing m-aconitase and xanthine/xanthine oxidase yielded methyl radical, which was detected by ESR spin trapping. Methyl radical formation was further confirmed using [(13)C]Me(2)SO. Parallel low temperature ESR experiments demonstrated that the generation of the [3Fe-4S](1+) cluster increased with increasing additions of superoxide to m-aconitase. This reaction was reversible, as >90% of the initial aconitase activity was recovered upon treatment with glutathione and iron(II). This mechanism presents a scenario in which (*)OH may be continuously generated in the mitochondria.     

Superoxide anion and hydroxyl radical scavenging activities of vegetable extracts measured using electron spin resonance.

Radical scavenging by reconstituted lyophilized powders of water extracts from 16 common vegetables was measured using electron spin resonance (ESR) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), hydroxyl radicals, (.OH) or superoxide anion radicals (O2.-), as DMPO-OH or DMPO-OOH spin adducts. On a dry weight basis, eggplant, and red, yellow and green bell pepper extracts showed potent superoxide anion radical scavenging activities (SOD-like activities). Ascorbate oxidase- or heat-treatments, decreased SOD-like activities in bell pepper extracts suggesting that ascorbate accounts for much of their free radical scavenging activity. Eggplant epidermis extract exhibited the most potent hydroxyl radical scavenging and SOD-like activities. Eggplant SOD-like activity did not decrease after ascorbate oxidase treatment, but decreased following ultrafiltration demonstrating that SOD-like activity is partially due to high molecular weight substances. Nasunin, an anthocyanin in eggplant epidermis, showed markedly potent superoxide anion radical scavenging activity, while it inhibited hydroxyl radical generation probably by chelating ferrous ion.     

Superoxide anion radical scavenging activities of herbs and pastures in northern Japan determined using electron spin resonance spectrometry

Free radicals are not only destructive to the living cells but also reduce the quality of animal products through oxidation. As a result the superoxide anion radical (O2-), one of the most destructive reactive oxygen species, is a matter of concern for the animal scientists as well as feed manufacturers to ensure the quality of product to reach consumers demand. The superoxide anion radical scavenging activities (SOSA) of water and MeOH extracts of 2 herbs and 9 pasture samples collected from lowland and highland swards were determined against a 5,5-dimethyl-1-pyroline-N-oxide-O2-spin adduct based on a hypoxanthine-xanthine oxidase reaction using electron spin resonance spectrometry. Both the water and MeOH extracted SOSA differed among the herbs and pastures. Species and altitudinal variations were observed between extraction methods. The herbs were higher in both water and MeOH extracted SOSA than the pastures except for water extracts of one pasture, white clover (Trifolium repens L.). Among the pastures, quackgrass (Agrophyron repens L.) showed higher SOSA in both the MeOH and water extracts, and timothy (Phleum pretense L.) showed higher MeOH extracted SOSA. It is apparent that the kind and amount of antioxidants differ among herbs and pastures. Animal health and quality of animal products could be improved by adequate selection and combining of herbs and pastures having higher SOSA.     

An electron spin resonance investigation of the iron-catalyzed reaction of metronidazole with cysteine

Metronidazole is used in the treatment of protozoal and bacterial infections, and has been used as a radiation sensitizer in experimental research and clinical trials. This drug is rapidly decomposed by cysteine in the presence of ferric or ferrous iron. Electron spin resonance spectroscopy demonstrates the formation of two complexes composed of iron, cysteine, and NO. The nitric oxide is probably formed by the reduction of inorganic nitrite formed by the cleavage of the metronidazole nitro group from the imidazole ring. No such reaction occurred with the 2-nitroimidazole drug, misonidazole.     

An electron spin resonance study of o-semiquinones formed during the enzymatic and autoxidation of catechol estrogens

Electron spin resonance spectroscopy has been used to demonstrate production of semiquinone-free radicals from the oxidation of the catechol estrogens 2- and 4-hydroxyestradiol and 2,6- and 4,6-dihydroxyestradiol. Radicals were generated either enzymatically (using horseradish peroxidase-H2O2 or tyrosinase-O2) or by autoxidation, and were detected as their complexes with spin-stabilizing metal ions (Zn2+ and/or Mg2+). In the peroxidase system, radicals are produced by one-electron oxidation of the catechol estrogen and their decay is by a second-order pathway, consistent with their disproportionation to quinone and catechol products. With tyrosinase-O2, radical generation occurs indirectly. Initial hydroxylation of phenolic estrogen (at either the 2- or 4-position) gives a catechol estrogen in situ; subsequent two-electron oxidation of the catechol to the quinone, followed by reverse disproportionation, leads to the formation of radicals. A competing mechanism for radical production involves autoxidation of the catechol. Results obtained from the estrogen systems have been compared with those from the model compound 5,6,7,8-tetrahydro-2-naphthol.     

Capacity of ascorbyl palmitate to produce the ascorbyl radical in vitro: an electron spin resonance investigation

This study aims to compare the electron spin resonance (ESR) spectra emitted by human blood loaded with either ascorbyl-6-palmitate (AP), a lipid-soluble derivative of ascorbic acid (AA), or with AA. Whole blood of a healthy male individual was equilibrated with equimolar concentrations of AP and AA of 200, 400, and 800 micromol/l. The intensity of the ESR signal, expressed as the peak-to-peak amplitude, reflects the amount of unpaired spins that are created due to the reducing action of AA and is proportional, in relative terms, to the amount of the ascorbyl radical formed. We found that the blood with AP emitted an ESR signal whose singlet shape, width, and location precisely correlate with the known characteristics of the ascorbyl radical in vitro. The signal magnitude increased linearly with increasing concentrations of AP and was similar to that of AA. We conclude that AP is biologically active, as it generates the ascorbyl radical, an action that also underlies the scavenging process by ascorbic acid. To this end, ascorbyl-6-palmitate might have potential advantages, due to its ability to penetrate biomembranes and to act at the lipid-related molecular target sites.     

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.     

An electron spin resonance study of the novel radical cation produced during the horseradish peroxidase-catalyzed oxidation of tetramethylhydrazine

Thrombin stimulation of [³²P]-prelabeled platelets induces a rapid decrease of the radioactivity from phosphatidylinositol-4,5-bisphosphate. No significant change is observed in phosphatidylinositol-4-monophosphate. The initial, thrombin-induced decrease of phosphatidylinositol-4,5-bisphosphate is not inhibited by cytochalasin D or by compounds that interfere with the mobilization of Ca²⁺ such as 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, the calmodulin-antagonist, trifluoperazine, prostacyclin and cyclic AMP. Our information indicates that the rapid loss of phosphatidylinositol-4,5-bisphosphate is linked to receptor activation and insensitive to Ca²⁺-mobilization.     

An electron spin resonance study on the free radicals produced from aclacinomycin a and its derivatives: analysis of hyperfine structure of the spectra by means of molecular orbital method

Quinone-containing carcinostatics, aclacinomycin A and its derivatives were investigated on the convertibility to free radical under a mild reducing condition. The hyperfine structures of electron spin resonance (ESR) spectra were satisfactorily reproduced by computer simulations, using the hyperfine coupling constants calculated by the Intermediate Neglect of Differential Overlap Molecular Orbital (INDO MO) method. This verifies the reliability of molecular orbital calculations and opens a way to analyze theoretically the correlation between chemical structures and carcinostatic activities. By analyzing hyperfine structures of ESR spectra, the free radical produced from aclacinomycin was identified as a neutral form of semiquinone radical of intact aclacinomycin. Taking into account the previous finding that 7-deoxyaklavinone (C1) is formed reductively by cytochrome P-450 reductase (EC 1.6.2.4; Komiyama et al., 1979), it is postulated that two types of semi-quinone radicals exist in vivo.     

An electron spin resonance study of free radical intermediates in the oxidation of indole acetic acid by horseradish peroxidase

The oxidation of indole-3-acetic acid by horseradish peroxidase was studied using the spin traps t-nitrosobutane and 5,5-dimethyl-1-pyrroline N-oxide to trap free radical intermediates. The major free radical metabolite of indole acetic acid was unambiguously determined by the use of indole-3-[2,2-2H2]acetic acid to be the skatole carbon-centered free radical. In the presence of oxygen, superoxide was also trapped.     

An electron spin resonance (ESR) study on the mechanism of ascorbyl radical production by metal-binding proteins

The mechanism of ascorbate oxidation by metal-binding proteins (ceruloplasmin, albumin and transferrin) was investigated in vitro and in isolated plasma by the measurement of the ascorbyl free radicals (AFR) by electron spin resonance (ESR). In plasma of 13 healthy volunteers, a spontaneous and variable pro-duction of AFR was detected, which was increased by a 10 M ascorbate overloading; however, this increase was not correlated to the intensity of the spontaneous AFR signal. The addition of Cu and ceruloplasmin to plasma increased the ESR signal, while the addition of transferrin decreased the signal intensity in a dose-dependent manner. In vitro, we demonstrated that ascorbate was oxidized by human serum albumin and by ceruloplasmin, and that this oxidase-like activity was lost by trypsin or heat treatment of these proteins. These two proteins positively interacted in the oxidation of ascorbate, since addition of crude albumin to a solution of ascorbate and ceruloplasmin increased the intensity of ESR signal in a dose-dependent manner. The treatment of albumin by a metal chelator (DDTC) abolished these positive inter-actions. The respective roles of copper and iron in ascorbate oxidation were studied and showed a dose-dependent effect of these ions on ascorbate oxidation. The role of iron was confirmed by the inhibiting effect of metal-free transferrin on iron-dependent ascorbate oxidation. Concerted actions between iron carrying albumin and copper carrying ceruloplasmin appear responsible for the production of AFR in vitro and in vivo. © Rapid Science 1998     

EPR spin trapping of a radical intermediate in the urate oxidase reaction

Urate oxidase, or uricase (EC 1.7.3.3), is a peroxisomal enzyme that catalyses the oxidation of uric acid to allantoin. The chemical mechanism of the urate oxidase reaction has not been clearly established, but the involvement of radical intermediates was hypothesised. In this study EPR spectroscopy by spin trapping of radical intermediates has been used in order to demonstrate the eventual presence of radical transient urate species. The oxidation reaction of uric acid by several uricases (Porcine Liver, Bacillus Fastidiosus, Candida Utilitis) was performed in the presence of 5-diethoxyphosphoryl-5-methyl-pyrroline-N-oxide (DEPMPO) as spin trap. DEPMPO was added to reaction mixture and a radical adduct was observed in all cases. Therefore, for the first time, the presence of a radical intermediate in the uricase reaction was experimentally proved.     

Identification of phosphorylation-induced changes in vimentin intermediate filaments by site-directed spin labeling and electron paramagnetic resonance

Phosphorylation drives the disassembly of the vimentin intermediate filament (IF) cytoskeleton at mitosis. Chromatographic analysis has suggested that phosphorylation produces a soluble vimentin tetramer, but little has been determined about the structural changes that are caused by phosphorylation or the structure of the resulting tetramer. In this study, site-directed spin labeling and electron paramagnetic resonance (SDSL-EPR) were used to examine the structural changes resulting from protein kinase A phosphorylation of vimentin IFs in vitro. EPR spectra suggest that the tetrameric species resulting from phosphorylation is the A11 configuration. EPR spectra also establish that the greatest degree of structural change was found in the linker 2 and the C-terminal half of the rod domain, despite the fact that most phosphorylation occurs in the N-terminal head domain. The phosphorylation-induced changes notably affected the proposed "trigger sequences" located in the linker 2 region, which have been hypothesized to mediate the induction of coiled-coil formation. These data are the first to document specific changes in IF structure resulting from a physiologic regulatory mechanism and provide further evidence, also generated by SDSL-EPR, that the linker regions play a key role in IF structure and regulation of assembly/disassembly.     

Electron spin resonance investigation of the interaction of the anion and glucose transport inhibitor, p-azidobenzylphlorizin, with the human red cell membrane

The membrane perturbations caused by the interaction of p-azidobenzylphlorizin (p-AzBPhz), a potential photoaffinity labeling agent of the anion and D-glucose transporters in the human erythrocyte, have been studied using electron spin resonance (ESR) spectrometry. Two lipid-specific spin labels have been employed; one of these agents, a hexadecyl-quarternary amine with the nitroxide reporter group covalently attached to the cationic nitrogen, (CAT-16), has been used to monitor changes in the physical state of the membrane's extracellular phospholipid/water interface. The other spin label, 5-doxylstearic acid (5-NS), is designed to examine the order and motion of the lipid bilayer near the cell surface. In separate experiments, intact human red cells labeled with these lipid-specific spin labels were exposed to small amounts of the phlorizin azide. A dose-dependent alteration in CAT-16 motion was observed, but the p-AzBPhz interaction with the membrane had no effect on the spectrum of 5-NS. The half-maximal effect of the phlorizin derivative on the CAT-16 spectrum occurred when about 2 million molecules were bound to each cell. This is also the combined amount of band 3 and band 4.5 present in the red cell membrane and represents the concentration necessary to inhibit both anion and glucose transport. Our results suggest that the first p-AzBPhz molecules binding to the red cell membrane interact with the anion and sugar transporters, and not with the bulk lipid bilayer.     

Hydration, structure, and molecular interactions in the headgroup region of dioleoylphosphatidylcholine bilayers: an electron spin resonance study

The relationship between bilayer hydration and the dynamic structure of headgroups and interbilayer water in multilamellar vesicles is investigated by electron spin resonance methods. Temperature variations of the order parameter of a headgroup spin label DPP-Tempo in DOPC in excess water and partially dehydrated (10 wt % water) show a cusp-like pattern around the main phase transition, Tc. This pattern is similar to those of temperature variations of the quadrupolar splitting of interbilayer D2O in PC and PE bilayers previously measured by 2H NMR, indicating that the ordering of the headgroup and the interbilayer water are correlated. The cusp-like pattern of these and other physical properties around Tc are suggestive of quasicritical fluctuations. Also, an increase (a decrease) in ordering of DPP-Tempo is correlated with water moving out of (into) interbilayer region into (from) the bulk water phase near the freezing point, Tf. Addition of cholesterol lowers Tf, which remains the point of increasing headgroup ordering. Using the small water-soluble spin probe 4-PT, it is shown that the ordering of interbilayer water increases with bilayer dehydration. It is suggested that increased ordering in the interbilayer region, implying a lowering of entropy, will itself lead to further dehydration of the interbilayer region until its lowered pressure resists further flow, i.e., an osmotic phenomenon.     

Electron spin resonance investigation of the cyanyl and azidyl radical formation by cytochrome c oxidase.

Cyanide (CN(-)) is a frequently used inhibitor of mitochondrial respiration due to its binding to the ferric heme a(3) of cytochrome c oxidase (CcO). As-isolated CcO oxidized cyanide to the cyanyl radical ((.)CN) that was detected, using the ESR spin-trapping technique, as the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)/(.)CN radical adduct. The enzymatic conversion of cyanide to the cyanyl radical by CcO was time-dependent but not affected by azide (N(3)(-)). The small but variable amounts of compound P present in the as-isolated CcO accounted for this one-electron oxidation of cyanide to the cyanyl radical. In contrast, as-isolated CcO exhibited little ability to catalyze the oxidation of azide, presumably because of azide's lower affinity for the CcO. However, the DMPO/(.)N(3) radical adduct was readily detected when H(2)O(2) was included in the system. The results presented here indicate the need to re-evaluate oxidative stress in mitochondria "chemical hypoxia" induced by cyanide or azide to account for the presence of highly reactive free radicals.     

The influence of cholesterol on molecular motion in egg lecithin bilayers--a variable-frequency electron spin resonance study of a cholestane spin probe

Electron spin resonance spectra at 9.5, 24. and 35 GHz were obtained for a cholestane spin probe in oriented multibilayers of egg lecithin of varying cholesterol content. In agreement with earlier studies, cholesterol induced a higher degree of spectral anisotropy in the multibilayers—the variation of the hyperfine separations with cholesterol content was in agreement with the model of Lapper et al. (Can. J. Biochem.50, 969 (1972)) where the amplitude of anisotropic probe motion decreased with increasing cholesterol content. Analysis of the electron spin resonance line shapes was done using the relatively simple modified Bloch equation approach, and correlation times for anisotropic probe motion were extracted from the spectra at three frequencies. The data demonstrate that increasing cholesterol content results in a decreased rate of anisotropic motion of the probe, providing further insight into the molecular mechanism of the condensing effect of cholesterol.     

An electron spin resonance study of the formation of a molecular oxygen adduct of the manganese(II) chelate of tetrasodium 3,10,17,24-tetrasulphonato-phthalocyanine

Treatment of the manganese(II) chelate of tetra- sodium 3,10,17,24-tetrasulphonatophthalocyanine (tspc) with alkaline aqueous solution in air followed by the addition of ethanol leads to formation and precipitation of an ESR non-detectable form of the chelate. When the material is dissolved in slightly alkaline aqueous solution and allowed to stand under nitrogen or air for two hours, a product is formed with the ESR spectral characteristics of a manganese(II) molecular oxygen complex. Treatment of this material with ferrocyanide leads to an ESR non- detectable form of manganese tspc formed by a one electron transfer process while titration with sodium ascorbate leads to the formation of low-spin manganese(0) tspc by a two electron transfer.     

An electron spin resonance study of synaptosome opiate receptors. The preparation and use of a spin labeled morphine.

Morphine spin labeled on the phenolic hydroxy group has been prepared using commercially available reagents and characterized by thin layer chromatography, mass spectroscopy, and electron spin resonance spectroscopy. It has been shown that morphine modified in this way retains some opiate activity, does not pass through the blood-brain barrier, and specifically binds to isolate rat brain synaptosomes. Spin labeled morphine has been shown to be an effective biophysical probe complementing radioactive tracer techniques in the study of the narcotic receptor site.