Flavonoids and Some Other Phenolics as Substrates of Peroxidase: Physiological Significance of the Redox Reactions

2 O₂ without accumulating oxidation products of phenolics. Scavenging of H₂O₂ by the systems can proceed in vacuoles and the apoplast, because phenolics, AA and POX are normal components of the compartments. AA seems to control lignification because it reduces radicals of lignin monomers which are formed by POX-dependent reactions. On lignification, oxidation of sinapyl alcohol is enhanced by radicals of coniferyl alcohol and hydroxycinnamic acid esters when apoplastic POX rapidly oxidizes coniferyl alcohol and the esters but slowly oxidizes sinapyl alcohol. POX seems to participate in the browning of tobacco leaves and onion scales on aging. H₂O₂, which is required for the POX-dependent reactions, can be formed by autooxidation of the phenolics that are transformed to brown components. It is discussed that browning involves the formation of antimicrobial substances. Received 5 June 2000/ Accepted in revised form 1 July 2000     

Oxidation of vacuolar and apoplastic phenolic substrates by peroxidase: Physiological significance of the oxidation reactions

Phenolic components and peroxidases are localized in vacuoles. Vacuolar peroxidase can oxidize phenolics when H₂O₂ is formed in vacuoles or tonoplasts, or when H₂O₂ formed outside of vacuoles is diffused into the organelles. In a mixture of phenolics containing a good and a poor substrate for peroxidase, a radical transfer reaction is possible from the radicals of the good substrate to the poor substrate, resulting in the enhancement of oxidation of the poor substrate. Phenoxyl radicals formed by peroxidase-dependent reactions are reduced by ascorbate in vacuoles. So, as long as ascorbate is present in vacuoles, the accumulation of oxidation products of phenolics is not significant. This suggests that ascorbate/phenolics/peroxidase systems in the vacuoles can scavenge H₂O₂. During aging, some phenolics are accumulated in vacuoles and the apoplast, and the accumulated phenolics are oxidized to brown components by peroxidase-dependent reactions. The brown components can produced O₂ ^? and H₂O₂ by autooxidation. The significance and the mechanisms of browning are discussed in tobacco leaves and onion scales.

     

Deglucosidation of quercetin glucosides to the aglycone and formation of antifungal agents by peroxidase-dependent oxidation of quercetin on browning of onion scales

Outer scales of yellow onion bulbs turn brown during maturing. The brown outer scales contain an antifungal component, 3,4-dihydroxybenzoic acid. An aim of the present study is to elucidate the mechanism of formation of the benzoic acid. In a browning scale, the scale was divided into three areas; fleshy, drying and dried brown areas. Levels of quercetin glucosides in dried brown areas were less than 10% of the glucosides in fleshy and drying areas, whereas levels of quercetin were high in dried brown areas. This result suggests that quercetin was formed by deglucosidation of quercetin glucosides on the border between drying and dried brown areas. Peroxidase (POX) activity of dried brown areas was about 10% of those of fleshy and drying areas. Quercetin was oxidized by autooxidation, and cell-free extracts of drying areas and POX isolated from onion scales enhanced the oxidation even in the absence of externally added hydrogen peroxide. The enhancement of quercetin oxidation was suppressed by catalase. No tyrosinase-like activity was detected in the cell-free extracts and the POX preparation. These results suggest that, during the enhanced oxidation of quercetin, hydrogen peroxide is formed. 3,4-Dihydroxybenzoic acid and 2,4,6-trihydroxyphenylglyoxylic acid, which were the oxidation products of quercetin, were found in dried brown area. These results suggest that an antifungal agent 3,4-dihydroxybenzoic acid is formed by POX-dependent oxidation of quercetin on browning of onion scales.     

Properties of Chlorogenic Acid Quinone: Relationship between Browning and the Formation of Hydrogen Peroxide from a Quinone Solution

Chlorogenic acid is the major polyphenol in foods derived from plants and is a good substrate for polyphenol oxidase. Chlorogenic acid quinone (CQA-Q), which is an oxidative product of chlorogenic acid by polyphenol oxidase, is an important intermediate compound in enzymatic browning. CQA-Q was prepared, and its properties and the relationship with browning were examined. The quinone solution was yellow or orange, and its molecular absorption coefficient was estimated to be 1.7×10³ for 325 nm and 9.7×10² for 400 nm in an acidic aqueous solution. Chlorogenic acid and H₂O₂ were spontaneously generated in the CQA-Q solution as the yellowish color of the solution gradually faded. A pale colored polymer was the major product in the reaction solution. Amino acids such as lysine and arginine added to CQA-Q solution did not repress the fading of the yellowish color of the solution. We concluded from these results that CQA-Q itself and a mixture of CQA-Q and amino acids did not form intensive brown pigments in the acidic aqueous solution. H₂O₂ spontaneously formed in the CQA-Q solution, and other polyphenols might have played an important role in the formation of the brown color by enzymatic browning.     

Age-Dependent Changes in Levels of Ascorbic Acid and Chlorogenic Acid, and Activities of Peroxidase and Superoxide Dismutase in the Apoplast of Tobacco leaves: Mechanism of the Oxidation of Chlorogenic Acid in the Apoplast

In order to understand browning in tobacco plants during aging,age-dependent changes in the levels of ascorbic acid (AA) andchlorogenic acid (CGA) and its isomers were investigated inthe apoplast and the symplast of the leaves. Also activitiesof peroxidase (POX) and superoxide dismutase (SOD) were determined.AA decreased during aging until it was no longer detectablein the apoplast, while symplastic AA remained although the leveldecreased on aging. In contrast, levels of CGA and its isomersand activity of POX in the apoplast increased on aging, whilethose in the symplast remained nearly constant in mature andold leaves. The activity of SOD in the apoplast increased duringaging, while that in the symplast decreased. Oxidation of CGAby the apoplastic solution was observed in the absence of externallyadded H₂O₂ and the oxidation was inhibited by SOD and catalase.Brown components, which contained caffeic acid moieties, accumulatedin the apoplast on aging and the components produced O–₂and H₂O₂ by autooxidation. From these results, we conclude (i)that brown components are formed in the apoplast by the CGA/POXsystem, (ii) that the H₂O₂ required for the reaction can beprovided by the CGA/POX system itself and by autooxidation ofthe brown components, and (iii) that apoplastic SOD functionsto generate H₂O₂ from apoplastically formed O–₂. (Received February 8, 1999; Accepted May 7, 1999)     

Characterization of oxygen free radicals generated during vanadate-stimulated NADH oxidation

The oxidation of NADH and accompanying reduction of oxygen to H₂O₂ stimulated by polyvanadate was markedly inhibited by SOD and cytochrome c. The presence of decavanadate, the polymeric form, is necessary for obtaining the microsomal enzyme-catalyzed activity. The accompanying activity of reduction of cytochrome c was found to be SOD-insensitive and therefore does not represent superoxide formation. The reduction of cytochrome c by vanadyl sulfate was also SOD-insensitive. In the presence of H₂O₂ all the forms of vanadate were able to oxidize reduced cytochrome c, which was sensitive to mannitol, tris and also catalase, indicating H₂0₂-dependent generation of hydroxyl radicals. Using ESR and spin trapping technique only hydroxyl radicals, but not superoxide anion radicals, were detected during polyvanadate-dependent NADH oxidation.     

Thiol Oxidation Induced by Oxidative Action of Adriamycin

To clarify the mechanism of the cardiotoxic action of adriamycin (ADM), the participation of free radicals from ADM in cardiotoxicity was investigated through the protective action of glutathione (GSH) or by using electron spin resonance (ESR). Oxidation of ADM by horseradish peroxidase and H₂O₂ (HRP-H₂O₂) was blocked by GSH concentration dependently. Inactivation of creatine kinase (CK) induced during interaction of ADM with HRP-H₂O₂ was also protected by GSH. Other anthracycline antitumor drugs that have a p-hydroquinone structure in the B ring also inactivated CK, and GSH inhibited the inactivation of CK. These results suggest that ADM was activated through oxidation of the p-hydroquinone in the B ring by HRP-H₂O₂. Although ESR signals of the oxidative ADM B ring semiquinone were not detected, glutathionyl radicals were formed during the interaction of ADM with HRP-H₂O₂ in the presence of GSH. ADM may be oxidized to the ADM B ring semiquinone and then reacts with the SH group. However, ESR signals of ADM C ring semiquinone, which was reductively formed by xanthine oxidase (XO) and hypoxanthine (HX) under anaerobic conditions, were not diminished by GSH, but they completely disappeared with ferric ion. These results indicate that oxidative ADM B ring semiquinones oxidized the SH group in CK, but reductive ADM C ring semiquinone radicals may participate in the oxidation of lipids or DNA and not of the SH group.     

Endosomal H2O2 production leads to localized cysteine sulfenic acid formation on proteins during lysophosphatidic acid-mediated cell signaling

Lysophosphatidic acid (LPA) is a growth factor for many cells including prostate and ovarian cancer-derived cell lines. LPA stimulates H₂O₂ production which is required for growth. However, there are significant gaps in our understanding of the spatial and temporal regulation of H₂O₂-dependent signaling and the way in which signals are transmitted following receptor activation. Herein, we describe the use of two reagents, DCP-Bio1 and DCP-Rho1, to evaluate the localization of active protein oxidation after LPA stimulation by detection of nascent protein sulfenic acids. We found that LPA stimulation causes internalization of LPA receptors into early endosomes that contain NADPH oxidase components and are sites of H₂O₂ generation. DCP-Rho1 allowed visualization of sulfenic acid formation, indicative of active protein oxidation, which was stimulated by LPA and decreased by an LPA receptor antagonist. Protein oxidation sites colocalized with LPAR1 and the endosomal marker EEA1. Concurrent with the generation of these redox signaling-active endosomes (redoxosomes) is the H₂O₂- and NADPH oxidase-dependent oxidation of Akt2 and PTP1B detected using DCP-Bio1. These new approaches therefore enable detection of active, H₂O₂-dependent protein oxidation linked to cell signaling processes. DCP-Rho1 may be a particularly useful protein oxidation imaging agent enabling spatial resolution due to the transient nature of the sulfenic acid intermediate it detects.     

Chemical Degradation of Melanins: Application to Identification of Dopamine-melanin

Melanocytes produce two chemically distinct types of melanin pigments, eumelanins and pheomelanins. These pigments can be quantitatively analyzed by acidic KMnO₄ oxidation or reductive hydrolysis with hydriodic acid (HI) to form pyrrole-2,3,5-tricarboxylic acid (PTCA) or aminohydroxyphenylalanine (AHP), respectively. Dark brown melanin-like pigments are also widespread in nature, for example, in the substantia nigra of humans and primates (neuromelanin), in butterfly wings and in the fungus Cryptococcus neoformans. To characterize such diverse types of melanins, we have improved the alkaline H₂O₂ oxidation method of Napolitano et al. (Tetrahedron, 51: 5913–5920, 1995) and re-examined the HI hydrolysis method of Wakamatsu et al. (Neurosci. Lett., 131: 57–60, 1991). The results obtained with H₂O₂ oxidation show that 1) pyrrole-2,3-dicarboxylic acid (PDCA), a specific marker of 5,6-dihydroxyindole units in melanins, is produced in yields ten times higher than by acidic KMnO₄ oxidation, and 2) PTCA is artificially produced from pheomelanins. The results with HI hydrolysis show that dopamine-melanin produces a 1:1 mixture of 3-amino and 4-amino isomers of aminohydroxyphenylethylamine, while the isomer ratio is about 0.2 in melanins prepared from dopamine and cysteine. These results indicate that alkaline H₂O₂ oxidation is useful in characterizing synthetic and natural eumelanins and that reductive hydrolysis with HI can be applied to analyzing oxidation products of dopamine such as neuromelanin.     

Free Radical Formation from the Antineoplastic Agent VP 16-213

Free radical formation from VP 16-213 was studied by ESR spectroscopy. Incubation of VP 16-213 with the one-electron oxidators persulphate-ferrous, myeloperoxidase (MPO)/hydrogen peroxide and horseradish peroxidase (HRP)/hydrogen peroxide readily led to the formation of a free radical. The ESR spectra obtained in the last two cases, were in perfect accord with that of a product obtained by electrochemical oxidation of VP 16-213 at +550 mV. The half-life of the free radical in 1 mM Tris (pH 7.4), 0.1 MNaClat 20°C, was 257 ± 4 s. The signal recorded on incubation with HRP/H₂O₂ or MPO/H₂O₂ did not disappear on addition of 0.3 - 1.2 mg/ml microsomal protein. From incubations with rat liver microsomes in the presence of NADPH, no ESR signals were obtained.     

Partial purification and kinetics of indoleacetic Acid oxidase from tobacco roots

Extracts from roots of Nicotiana tabacum L var. Bottom Special contain oxidative enzymes capable of rapid degradation of indoleacetic acid (IAA) in the presence of Mn²⁺ and 2, 4-dichlorophenol. Purification of IAA oxidase was attempted by means of ammonium sulfate fractionation and elution through a column of SE-Sephadex. Two distinct fractions, both causing rapid oxidation of IAA in the absence of H₂O₂, were obtained. One fraction exhibited high peroxidase activity when guaiacol was used as the electron donor; the other did not oxidase guaiacol. Both enzyme fractions caused similar changes in the UV spectrum of IAA; absorption at 280 mμ was reduced, while major absorption peaks appeared at 254 and 247 mμ. The kinetics of IAA oxidation by both fractions were followed by measuring the increase in absorption at 247 mμ. The peroxidase-containing fraction showed no lag or a slight lag which could be eliminated by addition of H₂O₂ (3 μmoles/ml). The peroxidase-free fraction showed a longer lag, but addition of similar amounts of H₂O₂ inhibited the rate of IAA oxidation and did not remove the lag. With purified preparations, IAA oxidation was stimulated only at low concentrations of H₂O₂ (0.03 μmole/ml). A comparison of K_m values for IAA oxidation by the peroxidase-containing and peroxidase-free fractions suggests that tobacco roots contain an IAA oxidase which may have higher affinity for IAA and may be more specific than the general peroxidase system previously described from other plant sources. A similar oxidase is present in commercial preparations of horseradish peroxidase. It is suggested that oxidation of IAA by horseradish peroxidase may be due to a more specific component.     

Gallic acid oxidation by turnip peroxidase

Both ellagic and gallic acids non competitively inhibited guaiacol oxidation by turnip peroxidase. The K_i values were 3 and 26 μm for ellagic and gallic acid respectively. Enzymatic oxidation of gallic acid by the isolated major turnip peroxidase was characterized with respect to spectral behaviour, affinity constant and pH effect. The K_m for H₂O₂ and gallic acid are 2.5 and 8.0 mM for turnip peroxidase. The pH optimum for gallic acid oxidation is about 6.5 and the rate constant k₄ decreased with the increase of pH in presence of both guaiacol and Gallic acid. When the gallic acid oxidation products were subjected to chromatographic analysis, it was found to be converted mainly to ellagic and an unknown quinone.     

Study on the reaction of 2,3-dihydroxybenzoic acid with molybdenum in aqueous solution. I. Synthesis and characterization of the oligomeric complexes formed

Dimeric or oligomeric oxo-complexes of Mo(VI) with 2,3-dihydroxybenzoic acid were prepared in aqueous solutions in the presence or not of K₂S₂O₅ (acting as a reducing agent) in various conditions. The complexes were found to contain the cis-(Mo₂O₅)²⁺ core and the ligands in the catecholate, semiquinonate or mixed valence oxidation form, depending on the reaction conditions and especially on the presence or not of the reductant. The isolated complexes in the presence or absence of reductant and the oxidation products in solution in the presence of air were studied via elemental, thermogravimetric and electrochemical analysis, Infrared, Raman, NMR and ESR spectroscopies and Electrospray Mass Spectra. The general molecular formula for the complexes is {[(PPh₄)₂(Mo₂O₅L₂X₂] · xH₂O)}_n, where the coordinated ligand’s L oxidation form varies and X involves coordinated water or hydroxyl group depending on the ligand oxidation state.     

On the Interaction of Anisyl-3,4-Semiquinone with Oxygen

Pulse radiolysis studies of anisyl-3,4-semiquinone, formed in the metabolic activation of 4-hydroxyanisole, a possible melanocytotoxic drug under current assessment as a treatment for malignant melanoma, have shown this semiquinone to be unreactive towards oxygen (k ≤ 10⁵ M^-1 s^-1), although the reverse reaction of O₂^?_? with anisyl-3,4-quinone is very rapid (k = 8.7 × 10⁸M^-1s^-1). Since 1,4 benzoquinone is also unreactive towards anisyl-3,4-serniquinone (k ≤ 10⁵M^-1s^-1), the one-electron reduction potential, E1/7 (anisyl-3,4-quinone/anisyl-3,4-semiquinone), is likely to be considerably more positive than 0.1V. This suggests that the cytotoxicity mechanism does not involve the generation of O₂^?_? and possible subsequent production of H₂O₂ and/or OH·, leading to lipid peroxidation, as previously proposed, but rather involves as yet unknown reactions of anisyl-3,4-quinone. This quinone is unstable in water and its absorption spectrum was measured immediately (< 0.1s) following disproportionation of anisyl-3,4-semiquinone, before significant decay of the quinone had occurred.     

Effects of chemical disruption on the biological activities of sea urchin egg jelly

In an attempt to relate the biological activities of sea urchin egg jelly to the structural characteristics of the acid glycoprotein molecule, the jelly was oxidized with H₂O₂ and sodium periodate, and digested with trypsin and pronase. The non-dialyzable products of H₂O₂ and periodate oxidation, and a fucose-rich fraction isolated from enzyme-digested jelly by column chromatography, were tested for their capacity to induce sperm agglutination and acrosome reaction in Hemicentrotus pulcherrimus. It was found that a degree of enzyme digestion sufficient to remove about 80% of the amino acids reduced, but failed to eliminate, the capacity of the jelly to elicit agglutination and acrosomal reaction. Mild oxidation with H₂O₂ suppressed sperm agglutination, but more drastic treatment was required to destroy the capacity of the jelly to induce the acrosome reaction. The loss of both these biological activities after periodate oxidation was found to parallel the release of sialic acid.     

Cellular protection of morin against the oxidative stress induced by hydrogen peroxide

Flavonoids are a class of secondary metabolites abundantly found in fruits and vegetables. In addition, flavonoids have been reported as potent antioxidants with beneficial effects against oxidative stress-related diseases such as cancer, aging, and diabetes. The present study was carried out to investigate the cytoprotective effects of morin (2′,3,4′,5,7-pentahydroxyflavone), a member of the flavonoid group, against hydrogen peroxide (H₂O₂)-induced DNA and lipid damage. Morin was found to prevent the cellular DNA damage induced by H₂O₂ treatment, which is shown by the inhibition of 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation (a modified form of DNA base), inhibition of comet tail (a form of DNA strand breakage), and decrease of nuclear phospho histone H2A.X expression (a marker for DNA strand breakage). In addition, morin inhibited membrane lipid peroxidation, which is detected by inhibition of thiobarbituric acid reactive substance (TBARS) formation. Morin was found to scavenge the intracellular reactive oxygen species (ROS) generated by H₂O₂ treatment in cells, which is detected by a spectrofluorometer, flow cytometry, and confocal microscopy after staining of 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA). Morin also induces an increase in the activity of catalase and protein expression. The results of this study suggest that morin protects cells from H₂O₂-induced damage by inhibiting ROS generation and by inducing catalase activation.     

Hydroxyl Radical Generation Depending on O2 or H2O by a Photocatalyzed Reaction in an Aqueous Suspension of Titanium Dioxide

Photocatalytic production of the electron (e^-) and positive hole (h⁺) in an aqueous suspension of TiO₂ (anatase form) under illumination by near-UV light (295-390 nm) generated the superoxide (O₂ ^-) and hydroxyl radical (?OH), which both proceeded linearly with reaction time, while H₂O₂ accumulated non-linearly. Under anaerobic conditions (introduced Ar gas), the yields of three active species of oxygen were decreased to 10-20% of those detected in the air-saturated reaction. The electron spin resonance (ESR) signal characteristics of ?OH were obtained when a spin trap of 5,5-dimthyl-1-pyrroline-N-oxide (DMPO) was included in the illuminating mixture. The intensity of the ESR signal was increased by Cu/Zn superoxide dismutase, and decreased under anaerobic conditions, amounting to only 20% of the intensity detected in the aerobic reaction. The addition of H₂O₂ to the reaction mixture resulted in about an 8-fold increase of ?OH production in the anaerobic reaction, but only about 1.5-fold in the aerobic reaction, indicating that e^- generated by the photocatalytic reaction reduced H₂O₂ to produce ?OH plus OH^-. On the other hand, D₂O lowered the yield of ?OH generation to 18% under air and 40% under Ar conditions, indicating the oxidation of H₂O by h⁺. The addition of Fe(III)-EDTA as an electron acceptor effectively increased ?OH generation, 2.3-fold in the aerobic reaction and 8.4-fold in the anaerobic reaction, the yield in the latter exceeding that in the air-saturated reaction.     

The Horseradish Peroxidase Catalysed Oxidation of Deoxyribose Sugars

The ability of horseradish peroxidase (E.C. 1.11.1.7. Donor: H₂O₂ oxidoreductase) to catalytically oxidize 2-deoxyribose sugars to a free radical species was investigated. The ESR spin-trapping technique was used to denionstrate that free radical species were formed. Results with the spin trap 3.5-dibronio-4-nitrosoben-zene sulphonic acid showed that horseradish peroxidase can catalyse the oxidation of 2-deoxyribose to produce an ESR spectrum characteristic of a nitroxide radical spectrum. This spectrum was shown to be a composite of spin adducts resulting from two carbon-centered species, one spin adduct being characterized by the hyperfine coupling constants a^N = 13.6Ganda^H_β = 11.0G, and the other by a^N = 13.4G and a^H = 5.8 G. When 2-deoxyribose-5-phosphate was used as the substrate, the spectrum produced was found to be primarily one species characterized by the hyperfine coupling constants a^N = 13.4G and a^H= 5.2. All the radical species produced were carbon-centered spin adducts with a β hydrogen, suggesting that oxidation occurred at the C(2) or C(5) moiety of the sugar. Interestingly, it was found that under the same experimental conditions, horseradish peroxidase apparently did not catalyze the oxidation of either 3-deoxyribose or D-ribose to a free radical since no spin adducts were found in these cases.

It can be readily seen that 2-deoxyribose and 2-deoxyribose-5-phosphate can be oxidized by HRP/H₂O₂ to form a free radical species that can be detected with the ESR spin-trapping technique. There are two probable sites for the formation of a CH type radical on the 2-deoxyribose sugar, these being the C(2) and the C(5) carbons. The fact that there is a species produced from 2-deoxy-ribose, but not 2-deoxy-ribose-5-phosphate, suggests that there is an involvement of the C(5) carbon in the species with the 1 1.0G β hydrogen. In the spectra formed from 2-deoxy-ribose, there is a big difference in the hyperfine splitting of the β hydrogens, suggesting that the radicals are formed at different carbon centers, while the addition of a phosphate group to the C(5) carbon seems to inhibit radical formation at one site. In related work, the chemiluminescence of monosaccharides in the presence of horseradish peroxidase was proposed to be the consequence of carbon-centered free radical formation (10).     

Superoxide dismutase and hydrogen peroxide formation in Campylobacter sputorum subspecies bubulus

Cell-free extracts of Campylobacter sputorum subspecies bubulus contained superoxide dismutase. The enzyme was located in the cytoplasmic fraction and insensitive to cyanide. After centrifuging a cell-free extract at 144000 x g for 1.5 h the total activity in the supernatant fraction was threefold higher than in the crude cell-free extract. The pellet fraction thus obtained was shown to have a lowering effect on superoxide dismutase activities from different sources in the assay method used here. C. sputorum responded to a raised oxygen tension in the culture by an increase in the superoxide dismutase activity. The ability to produce superoxide anion radicals (O₂ ^-·) during oxidation of formate and lactate was demonstrated. Furthermore C. sputorum was found to produce H₂O₂ while oxidizing formate. In experiments in which the reduction of cytochrome c by formate was followed, step-wise kinetics were observed. One of the steady states then obtained was attributed to the oxidizing action of H₂O₂, because it was abolished by the addition of catalase and lengthened by H₂O₂ added in addition to H₂O₂ formed as a product of formate oxidation. An overall reaction for formate oxidation by C. sputorum is discussed.Abbreviations O₂ ^-· superoxide anion radical - NBT p-nitro blue tetrazolium chloride - ABTS 2,2-azino-di-[3-ethylbenzthiazoline sulfonate (6)] - TL-medium tryptose-lactate medium