Oxidation of 3-hydroxykynurenine catalyzed by methemoglobin with hydrogen peroxide.

Methemoglobin (metHb) with H2O2 catalyzed the oxidation of 3-hydroxykynurenine (3-HKY) in the reaction mixture of metHb, 3-HKY, and H2O2. The spectrophotometric experiments suggest the following mechanism for the 3-HKY oxidation by metHb with H2O2. MetHb first reacts with H2O2 to form the ferryl complex of Hb. This species then oxidizes 3-HKY, while it returns to metHb. 3-HKY was more reactive with the ferryl complex than glutathione but less reactive than ascorbic acid. Scavengers of the hydroxyl radical, dimethyl sulfoxide and ethanol, scarcely inhibited the 3-HKY oxidation by metHb with H2O2. Desferrioxamine, a metal chelator, hardly suppressed the 3-HKY oxidation. These results indicate that the hydroxyl radical is not involved in the 3-HKY oxidation by metHb with H2O2.     

Detection of radical species in mixtures of some retinoids with hematin by using the ESR spin-trapping technique

Radical species were detected in mixtures of some retinoids with hematin by using the ESR spin-trapping technique. The rates of radical formation were approximately proportional to the oxygen consumption during the incubation of the retinoids with hematin. HPLC analyses of the incubation mixtures of the retinoids with hematin showed that 5,6-epoxides of the retinoids were formed. The amounts of the epoxides formed were proportional to both oxygen consumption and the amounts of radicals formed. These results suggest that the 5,6-epoxidations proceed via radical intermediates.     

Development of free radical products during the greening reaction of caffeic acid esters (or chlorogenic acid) and a primary amino compound.

ESR spectra were measured directly on a marked greening reaction mixture of Et-caffeate and a primary amino compound in alkali solution under aeration. A clear hyperfine structure was commonly detected early in the greening reaction with different amino compounds. Its hyperfine spectrum split into seven peaks was analyzed and found to be due to the oxidized free radical product of the Et-caffeate using an authentic sample system. Another type of hyperfine ESR spectrum was observed later in the reaction, and was altered with different amino compounds. The hyperfine structure for n-butylamine split into 12 lines. The latter type of free radical products were assumed to be a semiquinone type radical compound of the trihydroxy benzacridine derivative, which was identified as the principal structure of the green and yellow pigments formed by this greening reaction system. A formation mechanism of the green pigment and related products involving these free radical products is proposed.     

Isolation and identification of alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone radical adducts formed by the decomposition of the hydroperoxides of linoleic acid, linolenic acid, and arachidonic acid by soybean lipoxygenase.

alpha-(4-Pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) radical adducts, which are formed in the reactions of soybean lipoxygenase with linoleic acid, arachidonic acid, and linolenic acid, were isolated using HPLC-ESR spectroscopy. Both linoleic acid and arachidonic acid gave one radical adduct, whereas in the case of linolenic acid, two radical adducts were isolated. These radical adducts all showed virtually identical uv spectra with lambda max at 292 and 220 nm in hexane. The absence of absorbance with lambda max at 234 nm indicates that a conjugated diene structure is not contained in these radical adducts. The mass spectra of the radical adducts formed from linoleic and arachidonic acids were identical and contained a molecular ion of m/z 264, consistent with the trapping of the pentyl radical by 4-POBN. Indeed, authentic 4-POBN pentyl radical adduct obtained from the reaction between pentylhydrazine and 4-POBN gave the same mass spectrum as the product obtained from the reaction of linoleic acid and arachidonic acid with 4-POBN. The two 4-POBN radical adducts formed in the linolenic acid reaction were shown by mass spectrometry to be isomers of pentenyl radicals. The 4-POBN-pentyl radical adduct was also detected in the reaction mixture of 13-hydroperoxy-linoleic acid, soybean lipoxygenase, and 4-POBN, indicating that the pentyl radical and pentenyl radical are formed by the decomposition of the hydroperoxides.     

Detection of the Ethyl-and Pentyl-Radical Adducts of α-(4-Pyridyl-1-Oxide)-N-Ter J-Butylnitrone in Rat-Liver Microsomes Treated with ADP,NADPH and Ferric Chloride

HPLC-EPR analyses of the reaction mixtures of microsomal suspensions incubated with ADP, ferric chloride, NADPH and α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) were performed. In the elution pattern of the reaction mixture, three peaks (peaks 1, 2 and 3) were detected. The radical adducts (1 and 3) were identified as being the pentyl- and ethyl-radical adducts of 4-POBN by comparing their retention times with those of the authentic radical adducts.     

The oxidation of arachidonic acid by the cyclooxygenase activity of purified prostaglandin H synthase: spin trapping of a carbon-centered free radical intermediate

The ESR spin trapping technique was used to study the first detectable radical intermediate in the oxidation of arachidonic acid by purified prostaglandin H synthase. The holoenzyme and the apoenzyme, reconstituted with either hematin or Mn2+ protoporphyrin IX, were investigated. Depending on the different types of enzyme activity present, arachidonic acid was oxidized to at least two free radicals. One of these radicals is thought to be the first ESR detectable radical intermediate in the conversion of arachidonic acid to prostaglandin G2 and was detected previously in incubations of ram seminal vesicle microsomes, which are rich in prostaglandin H synthase. The ESR findings correlated with oxygen incorporation into arachidonic acid and prostaglandin formation, where the spin trap inhibits oxygen incorporation and prostaglandin formation by apparently competing with oxygen for the carbon-centered radical. Substitution of arachidonic acid by octadeuterated (5, 6, 8, 9, 11, 12, 14, 15)-arachidonic acid confirmed that the radical adduct contained arachidonic acid that is bound to the spin trap at one of these eight positions. An attempt was made to explain the apparent time lag between the metabolic activity observed in the oxygraph measurements and the appearance of the trapped radical signals.     

Tetramer-dimer equilibrium of oxyhemoglobin mutants determined from auto-oxidation rates.

One of the main difficulties with blood substitutes based on hemoglobin (Hb) solutions is the auto-oxidation of the hemes, a problem aggravated by the dimerization of Hb tetramers. We have employed a method to study the oxyHb tetramer-dimer equilibrium based on the rate of auto-oxidation as a function of protein concentration. The 16-fold difference in dimer and tetramer auto-oxidation rates (in 20 mM phosphate buffer at pH 7.0, 37 degrees C) was exploited to determine the fraction dimer. The results show a transition of the auto-oxidation rate from low to high protein concentrations, allowing the determination of the tetramer-dimer dissociation coefficient K4,2 = [Dimer] 2/[Tetramer]. A 14-fold increase in K4,2 was observed for addition of 10 mM of the allosteric effector inositol hexaphosphate (IHP). Recombinant hemoglobins (rHb) were genetically engineered to obtain Hb with a lower oxygen affinity than native Hb (Hb A). The rHb alpha2beta2 [(C7) F41Y/(G4) N102Y] shows a fivefold increase in K4,2 at pH 7.0, 37 degrees C. An atmosphere of pure oxygen is necessary in this case to insure fully oxygenated Hb. When this condition is satisfied, this method provides an efficient technique to characterize both the tetramer-dimer equilibrium and the auto-oxidation rates of various oxyHb. For low oxygen affinity Hb equilibrated under air, the presence of deoxy subunits accelerates the auto-oxidation. Although a full analysis is complicated, the auto-oxidation studies for air equilibrated samples are more relevant to the development of a blood substitute based on Hb solutions. The double mutants, rHb alpha2beta2 [(C7) F41Y/(G4) N102A] and rHb alpha2beta2 [(C7) F41Y/(E10) K66T], show a lower oxygen affinity and a higher rate of oxidation than Hb A. Simulations of the auto-oxidation rate versus Hb concentration indicate that very high protein concentrations are required to observe the tetramer auto-oxidation rate. Because the dimers oxidize much more rapidly, even a small fraction dimer will influence the observed oxidation rate.     

Lipid radicals: properties and detection by spin trapping

Unsaturated lipids are rapidly oxidized to toxic products such as lipid hydroperoxides, especially when transition metals such as iron or copper are present. In a Fenton-type reaction Fe2+ converts lipid hydroperoxides to the very short-lived lipid alkoxyl radicals. The reaction was started upon the addition of Fe2+ to an aqueous linoleic acid hydroperoxide (LOOH) emulsion and the spin trap in the absence of oxygen. Even when high concentrations of spin traps were added to the incubation mixture, only secondary radical adducts were detected, probably due to the rapid re-arrangement of the primary alkoxyl radicals. With the commercially available nitroso spin trap MNP we observed a slightly immobilized ESR spectrum with only one hydrogen splitting, indicating the trapping of a methinyl fragment of a lipid radical. With DMPO or 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) adducts were detected with carbon-centered lipid radical, with acyl radical, and with the hydroxyl radical. We also synthesized lipophilic derivatives of the spin trap DEPMPO in order to detect lipid radical species generated in the lipid phase. With all spin traps studied a lipid-derived carbon-centered radical was obtained in the anaerobic incubation system Fe2+/LOOH indicating the trapping of a lipid radical, possibly generated as a secondary reaction product of the primary lipid alkoxyl radical formed. Under aerobic conditions an SOD-insensitive oxygen-centered radical adduct was formed with DEPMPO and its lipophilic derivatives. The observed ESR parameters were similar to those of alkoxyl radical adducts, which were independently synthesized in model experiments using Fe3+-catalyzed nucleophilic addition of methanol or t-butanol to the respective spin trap.     

Methemoglobin Formation from Butylated Hydroxyanisole and Oxyhemoglobin. Comparison with Butylated Hydroxytoluene and P-Hydroxyanisole

The widely used food additives butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) react with oxyhemoglobin, thereby forming methemoglobin. The reaction rates were measured using visible spectroscopy, and second order rate constants were established for BHA and compared with p-hydroxyanisole. Using ESR we investigated the involvement of free radical reaction intermediates. The expected one-electron oxidation product of BHA and BHT, the phenoxyl radical, could only be detected with pure 3-t-butyl-4-hydroxyanisole and oxyhemoglobin. With the commercial mixture of 2- and 3-t-butyI-4-hydroxyanisole a very strong ESR signal of a secondary free radical species was observed, similar to the one observed earlier with p-hydroxyanisole and dependent on the presence of free thiol groups, so that we assumed the intermediate existence of a perferryl species, the MetHb-H₂O₂ adduct. In a second series of experiments we investigated the reactivity of this postulated intermediate with BHA and BHT, starting with a pure MetHb/H₂O₂-phenol mixture in a stopped-flow apparatus linked to the ESR spectrometer, detecting the expected phenoxyl radicals from BHA and p-hydroxyanisole. Due to the low solubility and decreased reactivity of BHT only traces of the phenoxyl type radical were found together with a high concentration of unreacted perferryl species. The reactivity of BHA, BHT and p-hydroxyanisole with free thiol groups is demonstrated by an increased reaction rate in the presence of the thiol group blocking substance NEM.     

Peroxidative metabolism of apigenin and naringenin versus luteolin and quercetin: glutathione oxidation and conjugation

GSH was readily depleted by a flavonoid, H(2)O(2), and peroxidase mixture but the products formed were dependent on the redox potential of the flavonoid. Catalytic amounts of apigenin and naringenin but not kaempferol (flavonoids that contain a phenol B ring) when oxidized by H(2)O(2) and peroxidase co-oxidized GSH to GSSG via a thiyl radical which could be trapped by 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) to form a DMPO-glutathionyl radical adduct detected by ESR spectroscopy. On the other hand, quercetin and luteolin (flavonoids that contain a catechol B ring) or kaempferol depleted GSH stoichiometrically without forming a thiyl radical or GSSG. Quercetin, luteolin, and kaempferol formed mono-GSH and bis-GSH conjugates, whereas apigenin and naringenin did not form GSH conjugates. MS/MS electrospray spectroscopy showed that mono-GSH conjugates for quercetin and luteolin had peaks at m/z 608 [M + H](+) and m/z 592 [M + H](+) in the positive-ion mode, respectively. (1)H NMR spectroscopy showed that the GSH was bound to the quercetin A ring. Spectral studies indicated that at a physiological pH the luteolin-SG conjugate was formed from a product with a UV maximum absorbance at 260 nm that was reducible by potassium borohydride. The quercetin-SG conjugate or kaempferol-SG conjugate on the other hand was formed from a product with a UV maximum absorbance at 335 nm that was not reducible by potassium borohydride. These results suggest that GSH was oxidized by apigenin/naringenin phenoxyl radicals, whereas GSH conjugate formation involved the o-quinone metabolite of luteolin or the quinoid (quinone methide) product of quercetin/kaempferol.     

高铁离子自由基与亚油酸反应的初步研究

利用停留仪快速反应动力学方法和自旋捕集ＥＳＲ技术监测高铁离子自由基和自旋捕捉剂ＰＯＢＮ的反应,发现高铁离子自由基本身不被自旋捕捉剂ＰＯＢＮ捕捉,但是ＰＯＢＮ可以捕捉到停流仪第三相中的ＯＨ,可能来自剩余的Ｆｅｎｔｏｎ试剂或高铁离子自由基的衰变。以含两个双键的不饱和脂肪酸－亚油酸（ＬＨ）作为模型化合物,测定高铁离子自由基与亚油酸分子的反应速率。ＥＳＲ结果表明,高铁离子自由基可能在一定程度上启动了亚油酸体系的脂质过氧化。     

One-electron oxidation of Trolox C (a vitamin E analogue) by peroxidases

The oxidation mechanism of Trolox C (a vitamin E analogue) by peroxidases was examined by stopped flow and ESR techniques. The results revealed that during the oxidation of Trolox C, peroxidase Compound II was the catalytic intermediate. The rate constants for the reaction of Compound II with Trolox C, which should be the rate-determining step, were estimated to be 2.1 X 10(4) and 7.2 X 10(3) M-1.s-1 for horseradish peroxidase and lactoperoxidase, respectively, at pH 6.0. The formation of the Trolox C radical was followed by ESR. The time course of the signal was similar to that of the optical absorbance changes at 440 nm, assigned as the peak of the Trolox C radical. The signal exhibited a hyperfine structure characteristic of phenoxyl radicals. From an estimation of the radical concentration in the steady state and the velocity of the radical formation, the dismutation constant was calculated to be 5 X 10(5) M-1.s-1. The concentration of the signal in the steady state was reduced by the addition of GSH. The spectrum changed from that of the Trolox C radical to that of the ascorbate radical when the reaction was carried out in the presence of ascorbate.     

Reaction of hematin with allylic fatty acid hydroperoxides: identification of products and implications for pathways of hydroperoxide-dependent epoxidation of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene

Reaction of 10-hydroperoxyoctadec-8-enoic acid (10-OOH-18:1) (50 microM) with hematin (0.5 microM) in sodium phosphate buffer containing Tween 20 (200 microM) generates 10-oxooctadec-8-enoic acid, 10-oxodec-8-enoic acid (10-oxo-10:1), and 10-hydroxyoctadec-8-enoic acid in relative yields of 79, 4, and 17%, respectively. The product profile and relative distribution are unaffected by 1 mM butylated hydroxyanisole. Approximately 5% of the hydroperoxide isomerizes from the 10- to the 8-position. 10-Oxo-10:1 most likely arises via beta-scission of an intermediate alkoxyl radical to the aldehyde and the n-octyl radical. To test this, 10-hydroperoxyoctadeca-8,12-dienoic acid was reacted with hematin under identical conditions. 10-Oxooctadeca-8,12-dienoic acid, 10-oxodec-8-enoic acid, and 10-hydroxyoctadeca-8,12-dienoic acid are formed in relative yields of 50, 45, and 5%, respectively. The product ratios are constant with time and hydroperoxide to catalyst ratio and unaffected by inclusion of phenolic antioxidants. The higher yield of 10-oxo-10:1 from 10-OOH-18:2 compared to 10-OOH-18:1 is due to the higher rate of beta-scission of the intermediate alkoxyl radical from the former to the resonance-stabilized octenyl radical. Two products of reaction of the 2-octenyl radical with O2, octenal and octenol, were detected in 10% yield relative to 10-oxo-10:1. Inclusion of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) led to epoxidation by both 10-OOH-18:1 and 10-OOH-18:2. Studies with isotopically labeled hydroperoxide or O2 indicated approximately 65% of the epoxide oxygen was derived from O2 and 35% from hydroperoxide oxygen, consistent with the involvement of peroxyl free radicals as the oxidizing agents. The available evidence indicates that hematin reduces the fatty acid hydroperoxides homolytically to alkoxyl radicals that are oxidized to ketones, reduced to alcohols, or undergo beta-scission to aldehydes. Carbon radicals generated during these reactions couple to O2, generating peroxyl free radicals that epoxidize BP-7,8-diol. The smaller percentage of epoxidation that results from hydroperoxide oxygen may arise from oxidation of the hydroperoxide group to peroxyl radicals or from heterolytic cleavage of the hydroperoxide to alcohol and an iron-oxo complex.     

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.     

The homolytic and heterolytic fatty acid hydroperoxide lyase-like activities of hematin

Pentenols and pentene dimers are biosynthetized in plants by homolytic fatty acid hydroperoxide lyase (HPL) or HPL-like enzymes. It has been found that these compounds can modify the flavor of olive oil. Reactions between hematin and 13-hydroperoxyoctadecatrienoic acid resulted in the formation of the same compounds via a free radical reaction in which an alkoxyl radical derived from linolenic acid hydroperoxide undergoes a beta-scission. (Z)-3-Hexenal has also been detected as a minor product of the reaction. It is bioconversed from the same substrate in plants by heterolytic HPL. Thanks to the redox cycle of its central iron, hematin has both homolytic and heterolytic HPL-like activities.     

Effect of Hb-encapsulation with vesicles on H2O2 reaction and lipid peroxidation

We encapsulated a purified and concentrated hemoglobin (Hb) solution with a phospholipid bilayer membrane to form Hb vesicles (particle diameter, ca. 250 nm) for the development of artificial oxygen carriers. Reaction of Hb inside the vesicle with hydrogen peroxide (H(2)O(2)) is one of the important safety issues to be clarified and compared with a free Hb solution. During the reaction of the Hb solution with H(2)O(2), metHb (Fe(III)) and ferrylHb (Fe(IV)=O) are produced, and H(2)O(2) is decomposed by the catalase-like reaction of Hb. The aggregation of discolored Hb products due to heme degradation is accompanied by the release of iron (ferric ion). On the other hand, the concentrated Hb within the Hb vesicle reacts with H(2)O(2) that permeated through the bilayer membrane, and the same products as the Hb solution are formed inside the vesicle. However, there is no turbidity change, no particle diameter change of the Hb vesicles, and no peroxidation of lipids comprising the vesicles after the reaction with H(2)O(2). Furthermore, no free iron is detected outside the vesicle, though ferric ion is released from the denatured Hb inside the vesicle, indicating the barrier effect of the bilayer membrane against the permeation of ferric ion. When vesicles composed of egg york lecithin (EYL) as unsaturated lipids are added to the mixture of Hb and H(2)O(2), the lipid peroxidation is caused by ferrylHb and hydroxyl radical generated from reaction of the ferric iron with H(2)O(2), whereas no lipid peroxidation is observed in the case of the Hb vesicle dispersion because the saturated lipid membrane of the Hb vesicle should prevent the interaction of the ferrylHb or ferric iron with the EYL.     

Conversion of linoleic acid hydroperoxide to hydroxy, keto, epoxyhydroxy, and trihydroxy fatty acids by hematin

We have carried out a study of the reaction of 13-hydroperoxy-9-cis,11-trans-octadecadienoic acid (linoleic acid hydroperoxide) with hematin. The major products are erythro-11-hydroxy-12,13-epoxy-9-octadecenoic acid, threo-11-hydroxy-12,13-epoxy-9-octadecenoic acid, 9,12,13-trihydroxy-10-octadecenoic acid, 13-keto-9,11-octadecadienoic acid, and 13-hydroxy-9,11-octadecadienoic acid. Several minor products have also been identified, including 9-hydroxy-12,13-epoxyoctadecenoic acid, 11-hydroxy-9,10-epoxy-12-octadecenoic acid, 9-hydroxy-10,12-octadecadienoic acid, and 9-keto-10,12-octadecadienoic acid. Oxygen labeling studies indicate that the observed products arise by at least two pathways. In the major pathway, hematin reduces 13-hydroperoxy-9,11-octadecadienoic acid by one electron to an alkoxyl radical that cyclizes to an adjacent double bond to form an epoxy allylic radical. The allylic radical either couples to the hydroxyl radical coordinated to hematin or diffuses from the solvent cage and couples to O2, forming a peroxyl radical. In the minor pathway, the hydroperoxide is oxidized by one electron to a 13-peroxyl radical that undergoes beta-scission to a pentadienyl radical and O2. Exchange of hydroperoxide-derived O2 for dissolved O2 occurs at this stage followed by coupling of O2 to either terminus of the pentadienyl radical. Both pathways of hydroperoxide metabolism generate significant quantities of peroxyl radicals that epoxidize the isolated double bonds of dihydroaromatic molecules. The products of hydroperoxide reaction with hematin and the oxygen labeling patterns are very similar to the products of unsaturated fatty acid hydroperoxide metabolism by platelets, aorta, and lung. Our results not only provide a mechanism for the formation of a series of mammalian metabolites of linoleic and arachidonic acids but also offer an estimate of the yield of peroxyl radicals generated during the process.     

Characterization of radicals arising from oxidation of commercially-important essential oils

Inappropriate use of essential oils may entail risks to human health due to mutational events, carcinogenic effects, genetic damages and sensitizing effect caused by generation of reactive oxygen species. In order to detect radicals that are expected to form during their oxidation, we measured the electron spin resonance (ESR) spectra of a standard reaction mixture (I) containing 25?μM flavin mononucleotide, 0.018% several essential oils (or 0.015% geraniol), 1.9 M acetonitrile, 20?mM phosphate buffer (pH 7.4), 0.1 M α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) and 1.0?mM FeSO₄(NH₄)₂SO₄ irradiated with 436?nm visible light (7.8 J/cm²). The ESR peak heights of the standard reaction mixture (I) of the essential oils increased in the following order: tea tree?>?palmarosa?>geranium?>?clary sage?>?petitgrain?>?lavender?>?bergamot?>?frankincense?>?ravintsara?>?ylang ylang?>?lemongrass?>?niaouli?>?eucalyptus globulus?>?peppermint. The ESR peak height of the standard reaction mixture (I) of geraniol, a main component of palmarosa, was comparable to the one of palmarosa (97?±?19% of palmarosa). Furthermore, high performance liquid chromatography (HPLC)-ESR analyses of the standard reaction mixture (I) of palmarosa and geraniol gave the same peaks. The results suggest that the radicals formed in the standard reaction mixture (I) of palmarosa are derived from geraniol. HPLC-ESR-mass spectrometry analyses detected m/z 294 ions, 4-POBN/5-hydroxy-3-methyl-3-pentenyl radical adducts and m/z 320 ions, 4-POBN/C₇O₂H₉ radical adducts in the standard reaction (I) of geraniol. The 5-hydroxy-3-methyl-3-pentenyl and C₇O₂H₉ radicals may be implicated in the sensitizing effect of palmarosa.     

Hydroxyl radical scavenging action of capsaicin

We recently reported that capsaicin (CAP) is capable of scavenging peroxyl radicals derived from 2,2'-azobis(2,4-dimethylvaleronitrile) as measured by electron spin resonance (ESR) spectroscopy. The present study describes the hydroxyl radical (HO*) scavenging ability of CAP as measured by DNA strand scission assay and by an ESR spin trapping technique with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The Fenton reaction [Fe(II)+ H(2)O(2) --> Fe(III) + HO* + HO(-)] was used as a source of HO*. The incubation of DNA with a mixture of FeSO(4) and H(2)O(2) caused DNA strand scission. The addition of CAP to the incubation mixture decreased the strand scission in a concentration-dependent manner. To understand the antioxidative mechanism of CAP, we used an ESR spin trapping technique. Kinetic competition studies using different concentrations of DMPO indicated that the decrease of the oxidative DNA damage was mainly due to the scavenging of HO* by CAP, not to the inhibition of the HO* generation system itself. We estimated the second order rate constants in the reaction of CAP and common HO* scavengers with HO* by kinetic competition studies. By comparison with the common HO* scavengers, CAP was found to scavenge HO* more effectively than mannitol, deoxyribose and ethanol, and to be equivalent to DMSO and benzoic acid, demonstrating that CAP is a potent HO* scavenger. The results suggest that CAP may act as an effective HO* scavenger as well as a peroxyl radical scavenger in biological systems.     

Light-induced changes of absorbance and electron spin resonance in small photosystem II particles.

Photosystem II reaction center components have been studied in small system II particles prepared with digitonin. Upon illumination the reduction of the primary acceptor was indicated by absorbance changes due to the reduction of a plastoquinone to the semiquinone anion and by a small blue shifts of absorption bands near 545 nm (C550) and 685 nm. The semiquinone to chlorophyll ratio was between 1/20 and 1/70 in various preparations. The terminal electron donor in this reaction did not cause large absorbance changes but its oxidized form was revealed by a hitherto unknown electron spin resonance (ESR) signal, which had some properties of the well-known signal II but a linewidth and g-value much nearer to those of signal I. Upon darkening absorbance and ESR changes decayed together in a cyclic or back reaction which was stimulated by 3-(3,4 dichlorophenyl)-1,1-dimethylurea. The donor could be oxidized by ferricyanide in the dark. Illumination in the presence of ferricyanide induced absorbance and ESR changes, rapidly reversed upon darkening, which may be ascribed to the oxidation of a chlorophyll a dimer, possibly the primary electron donor of photosystem II. In addition an ESR signal with 15 to 20 gauss linewidth and a slower dark decay was observed, which may have been caused by a secondary donor.