Hydroxylation of p-coumaric acid by horseradish peroxidase. The role of superoxide and hydroxyl radicals.

1. In the presence of dihydroxyfumarate, horseradish peroxidase catalyses the conversion of p-coumaric acid into caffeic acid at pH 6. This hydroxylation is completely inhibited by superoxide dismutase. 2. Dihydroxyfumarate cannot be replaced by ascorbate H2O2, NADH, cysteine or sulphite. Peroxidase can be replaced by high (10 mM) concentrations of FeSO4, but this reaction is almost unaffected by superoxide dismutase. 3. Hydroxylation by the peroxidase/dihydroxyfumarate system is completely inhibited by low concentrations of Mn2+ or Cu2+. It is proposed that this is due to the ability of these metal ions to react with the superoxide radical O2--. 4. Hydroxylation is partially inhibited by mannitol, Tris or ethanol and completely inhibited by formate. This seems to be due to the ability of these reagents to react with the hydroxyl radical -OH. 5. It is concluded that O2-- is generated during the oxidation of dihydroxyfumarate by peroxidase and reacts with H2O2 to produce hydroxyl radicals, which then convert p-coumaric acid into caffeic acid.     

Mechanism of hydrogen peroxide formation catalyzed by NADPH oxidase in thyroid plasma membrane

The thyroid plasma membrane contains a Ca2(+)-regulated NADPH-dependent H2O2 generating system which provides H2O2 for the thyroid peroxidase-catalyzed biosynthesis of thyroid hormones. The plasma membrane fraction contains a Ca2(+)-independent cytochrome c reductase activity which is not inhibited by superoxide dismutase. But it is not known whether H2O2 is produced directly from molecular oxygen (O2) or formed via dismutation of super-oxide anion (O2-). Indirect evidence from electron scavenger studies indicate that the H2O2 generating system does not liberate O2-, but studies using the modified peroxidase, diacetyldeuteroheme horseradish peroxidase, to detect O2- indicate that H2O2 is provided via the dismutation of O2-. The present results provide indirect evidence that the cytochrome c reductase activity is not a component of the NADPH-dependent H2O2 generator, since it was removed by washing the plasma membranes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid without affecting H2O2 generation. Spectral studies with diacetyldeuteroheme-substituted horseradish peroxidase showed that the thyroid NADPH-dependent H2O2 generator does not catalyze superoxide anion formation. The O2- adduct compound (compound III) was formed but was completely inhibited by catalase, indicating that the initial product was H2O2. The rate of NADPH oxidation also increased in the presence of diacetylheme peroxidase. This increase was blocked by catalase and was greatly enhanced by superoxide dismutase. The O2- adduct compound (compound III) was produced in the presence of NADPH when glucose-glucose oxidase (which does not produce O2-) was used as the H2O2 generator. NADPH oxidation occurred simultaneously and was enhanced by superoxide dismutase. We conclude that O2- formation occurs in the presence of an H2O2 generator, diacetylheme peroxidase and NADPH, but that it is not the primary product of the H2O2 generator. We suggest that O2- formation results from oxidation of NADPH, catalyzed by the diacetylheme peroxidase compound I, producing NADP degree, which in turn reacts with O2 to give O2-.     

NADH oxidation by manganese peroxidase with or without alpha-hydroxy acid

NADH oxidation by manganese peroxidase (MnP) was done in a reaction mixture including either alpha-hydroxy acid or acetate. The oxidation in the former reaction mixture was inhibited by a catalase and was accelerated by exogenous H2O2, while the oxidation in the latter reaction mixture was inhibited by a superoxide dismutase and was not accelerated by the exogenous H2O2. These results indicated that there are significant differences between the two reaction systems, particularly, in the active oxygen species involved in the reactions. Additionally, the experiment of MnP reduction with Mn(II) suggests that MnP has a separate catalytic activity other than an oxidation of Mn(II) to Mn(III) in the reaction mixture including acetate.     

Deficient metabolic utilization of hydrogen peroxide in Trypanosoma cruzi.

The glutathione peroxidase-glutathione reductase system, an alternative pathway for metabolic utilization of H2O2 [Chance, Sies & Boveris (1979) Physiol. Rev. 59, 527-605], was investigated in Trypanosoma cruzi, an organism lacking catalase and deficient in peroxidase [Boveris & Stoppani (1977) Experientia 33, 1306-1308]. The presence of glutathione (4.9 +/- 0.7 nmol of reduced glutathione/10(8) cells) and NADPH-dependent glutathione reductase (5.3 +/- 0.4 munit/10(8) cells) was demonstrated in the cytosolic fraction of the parasite, but with H2O2 as substrate glutathione peroxidase activity could not be demonstrated in the same extracts. With t-butyl hydroperoxide or cumene hydroperoxide as substrate, a very low NADPH-dependent glutathione peroxidase activity was detected (equivalent to 0.3-0.5 munit of peroxidase/10(8) cells, or about 10% of glutathione reductase activity). Blank reactions of the glutathione peroxidase assay (non-enzymic oxidation of glutathione by hydroperoxides and enzymic oxidation of NADPH) hampered accurate measurement of peroxidase activity. The presence of superoxide dismutase and ascorbate peroxidase activity in, as well as the absence of catalase from, epimastigote extracts was confirmed. Ascorbate peroxidase activity was cyanide-sensitive and heat-labile, but no activity could be demonstrated with diaminobenzidine, pyrogallol or guaiacol as electron donor. The summarized results support the view that T. cruzi epimastigotes lack an adequate enzyme defence against H2O2 and H2O2-related free radicals.     

Oxidation of 2-nitropropane by horseradish peroxidase. Involvement of hydrogen peroxide and of superoxide in the reaction mechanism

Incubation of aqueous solutions of 2-nitropropane in air causes a slow oxidation reaction that generates H(2)O(2). Purified horseradish peroxidase catalyses the oxidation of such preincubated 2-nitropropane solutions according to the equation: [Formula: see text] The pH optimum is 4.5 and K(m) for 2-nitropropane is 16mm. Other nitroalkanes or nitro-aromatics tested are not oxidized at significant rates by peroxidase. H(2)O(2) or 2,4-dichlorophenol increases the rate of 2-nitropropane oxidation by peroxidase. Catalase inhibits the reaction completely. Superoxide dismutase or mannitol, a scavenger of the hydroxyl radical, OH(.), each inhibits partially. Aniline and guaiacol are also powerful inhibitors of 2-nitropropane oxidation. It is suggested that peroxidase uses the traces of H(2)O(2) generated during preincubation of 2-nitropropane to catalyse oxidation of this substrate into a radical species that can reduce O(2) to the superoxide ion, O(2) (-.).O(2) (-.), or OH(.) derived from it, then appears to react with more nitropropane, generating further radicals and H(2)O(2) to continue the oxidation. Inhibition by aniline and guaiacol seems to be due to a competition for H(2)O(2).     

Superoxide-dependent oxidation of extracellular reducing agents by isolated neutrophils

Incubation of stimulated neutrophils with sulfhydryl (RSH) compounds or ascorbic acid (ascorbate) results in rapid superoxide (O2-)-dependent oxidation of these reducing agents. Oxidation of RSH compounds to disulfides (RSSR) is faster than the rate of O2- production by the neutrophil NADPH-oxidase, whereas about one ascorbate is oxidized per O2-. Ascorbate is oxidized to dehydroascorbate, which is also oxidized but at a slower rate. Oxidation is accompanied by a large increase in oxygen (O2) uptake that is blocked by superoxide dismutase. Lactoferrin does not inhibit, indicating that ferric (Fe3+) ions are not required, and Fe3+-lactoferrin does not catalyze RSH or ascorbate oxidation. Two mechanisms contribute to oxidation: 1) O2- oxidizes ascorbate or reduced glutathione and is reduced to hydrogen peroxide (H2O2), which also oxidizes the reductants. O2- reacts directly with ascorbate, but reduced glutathione oxidation is mediated by the reaction of O2- with manganese (Mn2+). The H2O2-dependent portion of oxidation is mediated by myeloperoxidase-catalyzed oxidation of chloride to hypochlorous acid (HOCl) and oxidation of the reductants by HOCl. 2) O2- initiates Mn2+-dependent auto-oxidation reactions in which RSH compounds are oxidized and O2 is reduced. Part of this oxidation is due to the RSH-oxidase activity of myeloperoxidase. This activity is blocked by superoxide dismutase but does not require O2- production by the NADPH-oxidase, indicating that myeloperoxidase produces O2- when incubated with RSH compounds. It is proposed that an important role for O2- in the cytotoxic activities of phagocytic leukocytes is to participate in oxidation of reducing agents in phagolysosomes and the extracellular medium. Elimination of these protective agents allows H2O2 and products of peroxidase/H2O2/halide systems to exert cytotoxic effects.     

Radiation resistance and the CuZn superoxide dismutase, Mn superoxide dismutase, catalase, and glutathione peroxidase activities of seven human cell lines

CuZn superoxide dismutase, Mn superoxide dismutase, catalase, and glutathione peroxidase form the primary enzymic defense against toxic oxygen reduction metabolites in cells. To test the importance of these protective enzymes in the cellular radiation response, the enzymic activities of seven different human cell lines were determined in parallel with their clonogenic survival characteristics. A positive correlation between the content of glutathione peroxidase in cell lines and their extrapolation numbers (n) and quasithreshold doses (Dq) was detected. Between the cellular contents of the other enzymes and D0, n, and Dq no positive correlations could be established. An interesting finding was a very high Mn superoxide dismutase content in a malignant mesothelioma cell line P7, which had an extremely high D0, 5.0 Gy.     

Detection of hydrogen peroxide with Amplex Red: interference by NADH and reduced glutathione auto-oxidation

We report here that reduced pyridine nucleotides and reduced glutathione result in an oxidation of Amplex Red by dioxygen that is dependent on the presence of horseradish peroxidase (HRP). Concentrations of NADH and glutathione typically found in biological systems result in the oxidation of Amplex Red at a rate comparable to that produced, for example, by respiring mitochondria. The effects of NADH and glutathione in this assay system are likely to be the result of H(2)O(2) generation via a superoxide intermediate because both catalase and superoxide dismutase prevent the oxidation of Amplex Red. These results suggest caution in the assay of H(2)O(2) production in biological systems using the Amplex Red/HRP because the assay will also report the mobilization of NADH or glutathione. However, the interruption of this process by the addition of superoxide dismutase offers a simple and reliable method for establishing the source of the oxidant signal.     

Role of superoxide in endothelial-cell modification of low-density lipoproteins

Cultured endothelial cells and arterial smooth muscle cells have been shown to modify LDL in a way that leads to rapid uptake by macrophages. Previous studies have demonstrated that this modification involves free radical peroxidation of LDL, and that the role of the cells was to accelerate oxidation under conditions where it otherwise would occur slowly. The objective of the present study was to determine whether the modification was mediated by oxygen-derived free radicals, and whether the ability of a given cell type of line to modify LDL was related to its secretion rate of O2- or H2O2. The results showed that modification required the presence of oxygen, and could be specifically inhibited by superoxide dismutase but not by catalase or by mannitol, a hydroxyl radical scavenger. Rabbit aortic endothelial cells, rabbit arterial smooth muscle cells, monkey arterial smooth muscle cells and human skin fibroblasts were all found to modify LDL, and all of these cell types generated more O2- (superoxide dismutase-inhibitable cytochrome c reduction) than a line of bovine aortic endothelial cells that did not modify LDL. The content of superoxide dismutase and catalase was higher in bovine aortic endothelial cells than in the cell lines that modified LDL, but glutathione peroxidase levels were not different. It was concluded that cells that were capable of modifying LDL produced superoxide or a substance that could be converted to superoxide in the medium, and that superoxide was an important, though possibly indirect, mediator of the modification of LDL by cells.     

Hydroxyl radicals is not a significant intermediate in the vanadate-stimulated oxidation of NAD(P)H by O2

The vanadate-stimulated oxidation of NADH by an enzymatic flux of O2- is inhibited by superoxide dismutase, but not by catalase. Keller et al. (1989, Free Radical Biol. Med. 6, 15-22) observed inhibition by catalase presumably because they used a commercial preparation contaminated with superoxide dismutase. Their proposal, that H2O2 and hydroxyl radical play significant roles in vanadate-stimulation of NAD(P)H oxidation, may be discounted on the basis of these and of previously reported results.     

Oxygen toxicity in Streptococcus sanguis. The relative importance of superoxide and hydroxyl radicals

Streptococcus sanguis, whose growth appears to be independent of the availability of iron, makes no hemes, contains neither catalase nor peroxidase, and can accumulate millimolar concentration levels of H2O2 during aerobic growth. It possesses a single manganese-containing superoxide dismutase whose concentration can be varied over a 50-100-fold range by manipulating the availability of oxygen during growth. Cell extracts contain a soluble NADH-plumbagin diaphorase which mediates O2- production in vitro and presumably also in vivo. Plumbagin increased oxygen consumption by S. sanguis and imposed an oxygen-dependent toxicity. Cells grown aerobically and containing elevated levels of superoxide dismutase were resistant to this toxicity. Dimethyl sulfoxide, which was shown to permeate S. sanguis freely, was used as an indicating scavenger of OH. An in vitro enzymic source of O2- plus H2O2 generated formaldehyde from dimethyl sulfoxide, an indication of OH. production. Either superoxide dismutase or catalase inhibited this OH. production and iron salts augmented it. Intact, aerobic cells of S. sanguis also gave evidence of OH. production, in the presence of plumbagin, but all of it appeared to be generated outside the cells. In addition, 0.5 M dimethyl sulfoxide did not diminish the oxygen-dependent toxicity of plumbagin. We conclude that, in S. sanguis, O2- can exert a toxic effect independent of the production of OH..     

Catalase, superoxide dismutase, and the production of O2-sensitive mutants of Bacillus coagulans

A number of facultatively anaerobic members of the genus Bacillus were screened for their catalase, diaminobenzidine peroxidase, and superoxide dismutase activities. A strain of Bacillus coagulans (7050) lacking peroxidatic activity and containing single catalatic and superoxide dismutase activities was selected. Responses of the superoxide dismutase activity and catalase level to the partial pressure of oxygen, and Fe and Mn levels, as well as to aerobic and fermentative metabolism, were determined. There appeared to be a relationship between high endogenous catalase levels and the high H2O2 evolution and KCN insensitivity of B. coagulans respiration. Bacillus coagulans 7050 was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine and screened for the expression of oxygen intolerance. All of the 38 stable oxygen sensitive mutants obtained had very low or completely absent catalatic activity and catalase protein. No mutant lacked superoxide dismutase, although five showed significantly lowered levels of the enzyme. Exogenous bovine liver catalase restored aerotolerance and reduced cell pleomorphism in the mutants.     

Coisolation of glutathione peroxidase, catalase and superoxide dismutase from human erythrocytes

Glutathione peroxidase (GSH-Px; glutathione: hydrogen peroxide oxidoreductase; EC 1.11.1.9), catalase (H2O2: H2O2 oxidoreductase; EC 1.11.1.6) and superoxide dismutase (superoxide: superoxide oxidoreductase; EC 1.15.1.1) were coisolated from human erythrocyte lysate by chromatography on DEAE-cellulose. Glutathione peroxidase was separated from superoxide dismutase and catalase by thiol-disulfide exchange chromatography and then purified to approximately 90% homogeneity by gel permeation chromatography and dye-ligand affinity chromatography. Catalase and superoxide dismutase were separated from each other and purified further by gel permeation chromatography. Catalase was then purified to approximately 90% homogeneity by ammonium sulfate precipitation and superoxide dismutase was purified to apparent homogeneity by hydrophobic interaction chromatography. The results for glutathione peroxidase represent an improvement of approximately 10-fold in yield and 3-fold in specific activity compared with the established method for the purification of this enzyme. The yields for superoxide dismutase and catalase were high (45 mg and 232 mg, respectively, from 820 ml of washed packed cells), and the specific activities of both enzymes were comparable to values found in the literature.     

NADPH oxidation catalyzed by the peroxidase/H2O2 system. Guaiacol-mediated and scopoletin-mediated oxidation of NADPH to NADPH+

We have examined the respective roles played by guaiacol and scopoletin in NADPH oxidation catalyzed by the peroxidase/H2O2 system. It was shown that NADPH was not oxidized by either the horseradish or lactoperoxidase/H2O2 systems alone; oxidation occurred immediately after the addition of guaiacol or scopoletin. In both cases, the oxidation product was enzymatically active NADP+. Differences were observed in the NADPH oxidation mechanism depending on whether guaiacol or scopoletin was the mediator molecule. In guaiacol-mediated NADPH oxidation, the stoichiometry between H2O2 and oxidized NADPH was about 1; superoxide dismutase did not affect the oxidation rate. In scopoletin-mediated oxidation, the stoichiometry was much higher (1:14 in the present experiments); superoxide dismutase considerably increased the oxidation rate. It is concluded that catalysis of NADPH oxidation by the horse radish peroxidase/H2O2 system requires the presence of a mediator molecule. The NADPH oxidation mechanism depends on the intermediary oxidation state of this molecule.     

Antioxidative enzyme activity and metabolism of peroxide compounds in the crystalline lens during cataractogenesis

Lens antioxidative enzyme activity (catalase, superoxide dismutase, glutathione peroxidase) in cataract as well as the possibility of cataract induction by the lipid peroxidation products and their influence on the content of reduced thiols (oxy-red balance) were studied. It was shown that the rate of the H2O2 decomposition by the human cataract lenses is lowered in comparison with the normal lenses. This is not due to the lowered catalase or glutathione-peroxidase 1 activity, but depends on the deficiency of reduced glutathione in the lens. Activity of superoxide dismutase and glutathione peroxidase metabolizing organic hydroperoxides is significantly lowered in the cataract lenses. Lipid peroxidation products injected into the rabbit vitreous induce posterior subcapsular cataract, which is accompanied by depletion of reduced glutathione level in the lens. The conclusion is made that two interrelated processes: accumulation of H2O2 and of lipid peroxides induce aggregation of the soluble proteins and the fragmentation of the membrane structures in cataract lenses.     

A method for measuring H2O2 based on the potentiation of peroxidative NADPH oxidation by superoxide dismutase and scopoletin.

NADPH oxidation catalyzed by horseradish peroxidase is considerably increased by scopoletin and superoxide dismutase. These effects were used to develop a method for measuring H2O2 in a horseradish peroxidase, superoxide dismutase, and scopoletin system by measuring the NADPH oxidation rate. The optimal concentration of each reactant was determined. H2O2 could be detected and measured when it was present free in the medium or when it was produced by an H2O2-generating system, such as glucose-glucose oxidase or NADPH oxidase from thyroid plasma membranes. H2O2 was measured either by taking aliquots of the incubation medium or by placing NADPH directly in the medium and following the kinetics of NADPH oxidation. This latter approach required smaller amounts of biological material. In contrast to other methods, the H2O2 which is measured is regenerated. This method is 10 times more sensitive than the standard scopoletin method for H2O2 measurement and will detect a H2O2 production rate as low as 0.2 nmol per hour. The method is particularly suitable for biological systems in which small quantities of biological material are available.     

Role of Antioxidant Systems in Wheat Genotypes Tolerance to Water Stress

The role of plant antioxidant systems in stress tolerance was studied in leaves of three contrasting wheat genotypes. Drought imposed at two different stages after anthesis resulted in an increase in H2O2 accumulation and lipid peroxidation and decrease in ascorbic acid content. Antioxidant enzymes like superoxide dismutase, ascorbate peroxidase and catalase significantly increased under water stress. Drought tolerant genotype C 306 which had highest ascorbate peroxidase and catalase activity and ascorbic acid content also showed lowest H2O2 accumulation and lipid peroxidation (malondialdehyde content) under water stress in comparison to susceptible genotype HD 2329 which showed lowest antioxidant enzyme activity and ascorbic acid content and highest H2O2 content and lipid peroxidation. HD 2285 which is tolerant to high temperature during grain filling period showed intermediate behaviour. Superoxide dismutase activity, however, did not show significant differences among the genotypes under irrigated as well as water stress condition. It seems that H2O2 scavenging systems as represented by ascorbate peroxidase and catalase are more important in imparting tolerance against drought induced oxidative stress than superoxide dismutase alone. This revised version was published online in July 2006 with corrections to the Cover Date.     

Hydrogen peroxide formation and iron ion oxidoreduction linked to NADH oxidation in radish plasmalemma vesicles

Previously, we showed the presence in radish (Raphanus sativus L.) plasmalemma vesicles of an NAD(P)H oxidase, active at pH 4.5-5.0, which elicits the formation of anion superoxide (Vianello and Macrí (1989) Biochim. Biophys. Acta 980, 202-208). In this work, we studied the role of hydrogen peroxide and iron ions upon this oxidase activity. NADH oxidation was stimulated by ferrous ions and, to a lesser extent, by ferric ions. Salicylate and benzoate, two known hydroxyl radical scavengers, inhibited both basal and iron-stimulated NADH oxidase activity. The iron chelators EDTA (ethylenediaminetetraacetic acid) and DFA (deferoxamine melysate) at high concentrations (2 mM) inhibited the NADH oxidation, whereas they were ineffective at lower concentrations (80 microM); the subsequent addition of ferrous ions caused a rapid and limited increase of oxygen consumption which later ceased. Hydrogen peroxide was not detected during NADH oxidation but, in the presence of salicylate, its formation was found in significant amounts. NADH oxidase activity was also associated to a Fe2+ oxidation which was only partially inhibited by salicylate. Ferrous ion oxidation was partially inhibited by catalase and prevented by superoxide dismutase, while ferric ion reduction was abolished by catalase and unaffected by superoxide dismutase. These results show that during NADH oxidation iron ions undergo oxidoreduction and that hydrogen peroxide is produced and rapidly consumed. As previously suggested, this oxidation appears linked to the univalent oxidoreduction of iron ions by a reduced flavoprotein of radish plasmalemma which is then converted to a radical form. The latter, reacting with oxygen generates the superoxide anion which dismutases to H2O2. Hydrogen peroxide, through a Fenton's reaction, may react with Fe2+ to produce hydroxyl radicals, or with Fe3+ to generate the superoxide anion.     

The mechanism of potentiation of horseradish peroxidase-catalysed oxidation of NADPH by porphyrins.

Several porphyrins, including HpD (haematoporphyrin derivative), potentiate the oxidation of NADPH by horseradish peroxidase/H2O2. To elucidate the mechanism of potentiation, the following observations are relevant. During peroxidase-catalysed NADPH oxidation, O2-.(superoxide radical) is generated, as judged from superoxide dismutase-inhibitable cytochrome c reduction. This generation of O2-. is suppressed by HpD. Peroxidase-catalysed NADPH oxidation is stimulated by superoxide dismutase and by anaerobic conditions. Under anaerobic conditions HpD has no influence on peroxide-catalysed NADPH oxidation. Previous studies have shown that horseradish peroxidase is inhibited by O2-.. Thus the experimental results indicate that the potentiating effect of HpD can be explained by its ability to inhibit O2-. generation in the horseradish peroxidase/H2O2/NADPH system.     

Oxidation of arachidonic acid in micelles by superoxide and hydrogen peroxide

Arachidonic acid was co-oxidized by xanthine oxidase. Both superoxide radical and hydrogen peroxide were required for oxidation, as shown by essentially complete inhibition caused by superoxide dismutase or by catalase. Pure arachidonate, free of lipid hydroperoxides, was susceptible to this co-oxidation, and the presence of lipid hydroperoxides did not accelerate the process. The role of trace metals was indicated by the stimulatory effect of EDTA-Fe and by the inhibitory effect of diethylenetriamine pentaacetate. Initiation of arachidonate co-oxidation was due to a potent oxidant generated by the interaction of H2O2 and O2- in the presence of Fe, rather than to either O2- or H2O2 per se. Hence, mannitol, a scavenger of OH ., but not of O2- or H2O2, also inhibited oxidation. Arachidonic acid autoxidation, a much slower process than xanthine oxidase co-oxidation, was barely detectable on the time scale of these observations. Unlike the co-oxidation, autoxidation was autocatalytic and therefore accelerated by hydroperoxide products. Marked quantitative differences in the distribution of isomeric hydroperoxide products of enzymic co-oxidation, as compared to the autoxidation, were noted and their significance was discussed.