A study of the supposed hydroxylation of tyrosine catalysed by peroxidase.

The claim that peroxidase (rather than tyrosinase) is the enzyme responsible for the conversion of tyrosine into dopa (3,4-dihydroxyphenylalanine) in melanogenesis was investigated. The spectral changes that occurred during the action of horseradish peroxidase in the presence of H2O2 on dopa, tyrosine and mixtures of dopa with tyrosine or other phenolic compounds were studied. The effect of ascorbic acid or dihydroxyfumaric acid on some of these changes was also investigated. No evidence was found that tyrosine was hydroxylated by peroxidase in the presence of H2O2 and dopa as cofactor, although tyrosine or other phenolic compounds increased the rate of oxidation of dopa to dopachrome (indoline-5,6-quinone-2-carboxylic acid). Peroxidase was, however, effective in oxidizing tyrosine to dopa in the presence of dihydroxyfumaric acid and oxygen.     

Action of biologically-relevant oxidizing species upon uric acid. Identification of uric acid oxidation products

Uric acid is an end-product of purine metabolism in Man, and has been suggested to act as an antioxidant in vivo. Products of attack upon uric acid by various oxidants were measured by high performance liquid chromatography. Hypochlorous acid rapidly oxidized uric acid, forming allantoin, oxonic/oxaluric and parabanic acids, as well as several unidentified products. HOCl could oxidize all these products further. Hydrogen peroxide did not oxidize uric acid at detectable rates, although it rapidly oxidized oxonic acid and slowly oxidized allantoin and parabanic acids. Hydroxyl radicals generated by hypoxanthine/xanthine oxidase or Fe2(+)-EDTA/H2O2 systems also oxidized uric acid to allantoin, oxonic/oxaluric acid and traces of parabanic acid. Addition of ascorbic acid to the Fe2(+)-EDTA/H2O2 system did not increase formation of oxidation products from uric acid, possibly because ascorbic acid can 'repair' the radicals resulting from initial attack of hydroxyl radicals upon uric acid. Mixtures of methaemoglobin or metmyoglobin and H2O2 also oxidized uric acid: allantoin was the major product, but some parabanic and oxonic/oxaluric acids were also produced. Caeruloplasmin did not oxidize uric acid under physiological conditions, although simple copper (Cu2+) ions could, but this was prevented by albumin or histidine. The possibility of using oxidation products of uric acid, such as allantoin, as an index of oxidant generation in vivo in humans is discussed.     

Evidence for a radical mechanism of halogenation of monochlorodimedone catalyzed by chloroperoxidase

A radical species of monochlorodimedone has been characterized by its high reactivity with molecular O2. Horseradish peroxidase greatly accelerated O2 uptake by acidic solutions of this substrate; the enzymatic reaction required exogenous H2O2 only with freshly prepared substrate solutions, and the total substrate oxidized was equal to the sum of H2O2 added and O2 consumed. However, with excess Br- and horseradish peroxidase, or high Br- or Cl- and chloroperoxidase, a 1:1 stoichiometry between H2O2 and substrate was observed. In the absence of halide, the stoichiometry of the chloroperoxidase-catalyzed oxidation of monochlorodimedone changed to two molecules of the organic donor per H2O2. Moreover, in the absence of halide, at substrate:H2O2 ratios greater than 2.0, chloroperoxidase catalyzed significant O2 uptake; this enzyme-dependent autoxidation of monochlorodimedone also occurred in the presence of Cl- or Br-, when H2O2 was limiting. These data, and recent evidence from this laboratory for free hypohalous acid as the first product of chloroperoxidase-catalyzed halide oxidation [B. W. Griffin (1983) Biochem. Biophys. Res. Commun. 116, 873-879], strongly support a mixed enzymatic/nonenzymatic radical chain process as the mechanism for halogenation of monochlorodimedone by chloroperoxidase. Both horseradish peroxidase and chloroperoxidase can catalyze either bromination or oxidation of this substrate, depending on the experimental conditions. Implications of these results for the mechanism of HOCl formation catalyzed by chloroperoxidase are considered.     

Mechanisms of 1,3-butadiene oxidations to butadiene monoxide and crotonaldehyde by mouse liver microsomes and chloroperoxidase

NADPH-dependent oxidation of 1,3-butadiene by mouse liver microsomes or H2O2-dependent oxidation by chloroperoxidase produced both butadiene monoxide and crotonaldehyde; methyl vinyl ketone and 2,3- and 2,5- dihydrofuran were not detected. The crotonaldehyde to butadiene monoxide ratio remained constant over time in both the microsomal and the chloroperoxidase reactions; however, much more crotonaldehyde was produced by chloroperoxidase than microsomes; crotonaldehyde was not detected when reference samples of butadiene monoxide were used in control incubations containing NADPH and microsomes or H2O2 and chloroperoxidase. Moreover, incubations of 1,3-butadiene with horseradish peroxidase and H2O2, or microsomes and H2O2 or arachidonic acid did not result in the oxidation of 1,3-butadiene. In microsomes, metabolite formation was dependent on incubation time, NADPH, and protein concentrations and did not change when the 1,3-butadiene pressure was varied between 24 and 52 cm Hg. Inclusion of the cytochrome P450 inhibitor 1-benzylimidazole inhibited 1,3-butadiene metabolism, but inclusion of KCN, catalase, or superoxide dismutase had no effect. These results support the role of cytochrome P450 in 1,3-butadiene oxidation by mouse liver microsomes. The formation of crotonaldehyde but not methyl vinyl ketone by cytochrome P450 or chloroperoxidase indicates regioselectivity in the oxygen transfer from the hemoproteins to 1,3-butadiene. The intermediates formed may undergo either ring closure to form butadiene monoxide or a hydrogen shift to form 3-butenal which tautomerizes to produce crotonaldehyde. Evidence for this tautomerization was obtained by the finding that 3-buten-1-ol, an alternative precursor of 3-butenal, was oxidized to crotonaldehyde under incubation conditions similar to that used for 1,3-butadiene.     

Expression of the Caldariomyces fumago chloroperoxidase in Aspergillus niger and characterization of the recombinant enzyme

The Caldariomyces fumago chloroperoxidase was successfully expressed in Aspergillus niger. The recombinant enzyme was produced in the culture medium as an active protein and could be purified by a three-step purification procedure. The catalytic behavior of recombinant chloroperoxidase (rCPO) was studied and compared with that of native CPO. The specific chlorination activity (47 units/nmol) of rCPO and its pH optimum (pH 2.75) were very similar to those of native CPO. rCPO catalyzes the oxidation of various substrates in comparable yields and selectivities to native CPO. Indole was oxidized to 2-oxindole with 99% selectivity and thioanisole to the corresponding R-sulfoxide (enantiomeric excess >98%). Incorporation of (18)O from labeled H(2)18O(2) into the oxidized products was 100% in both cases.     

The reaction of hyaluronic acid and its monomers, glucuronic acid and N-acetylglucosamine, with reactive oxygen species

Synovial fluid is a approximately 0.15% (w/v) aqueous solution of hyaluronic acid (HA), a polysaccharide consisting of alternating units of GlcA and GlcNAc. In synovial fluid of patients suffering from rheumatoid arthritis, HA is thought to be degraded either by radicals generated by Fenton chemistry (Fe2+/H2O2) or by NaOCl generated by myeloperoxidase. We investigated the course of model reactions of these two reactants in physiological buffer with HA, and with the corresponding monomers GlcA and GlcNAc. meso-Tartaric acid, arabinuronic acid, arabinaric acid and glucaric acid were identified by GC-MS as oxidation products of glucuronic acid. When GlcNAc was oxidised, erythronic acid, arabinonic acid, 2-acetamido-2-deoxy-gluconic acid, glyceric acid, erythrose and arabinose were formed. NaOCl oxidation of HA yielded meso-tartaric acid; in addition, arabinaric acid and glucaric acid were obtained by oxidation with Fe2+/H2O2. These results indicate that oxidative degradation of HA proceeds primarily at glucuronic acid residues. meso-Tartaric acid may be a useful biomarker of hyaluronate oxidation since it is produced by both NaOCl and Fenton chemistry.     

Contribution of cytochromes and proteins to the effect of ascorbic acid on artificial and microsomal hydroxylation systems containing oxygen and hydrogen peroxide.

Hydroxylation systems containing cytochromes, proteins and ascorbic acid were studied at physiological pH (7.4) under O2 or N2 with added H2O2. Proteins inhibited aromatic hydroxylation of p-nitrophenol or oxidative demethylation of ethylmorphine in ascorbic acid-containing systems incubated under O2, but strongly activated the systems containing H2O2. Cytochrome c and partially purified cytochrome P-450 from rat liver microsomal preparations activated the system in either O2 or H2O2. The systems needed ascorbic acid (or other enol structures) for activation. Cytochrome iron participated probably in the activation of O2, whereas cytochrome protein participated in a free radical activation of H2O2 (or of O2).     

Altered production of the active oxygen species is involved in enhanced cytotoxic action of acylated derivatives of ascorbate to tumor cells

Our previous study shows that 6-O-acyl derivatives of L-ascorbic acid inhibits more markedly cell growth of mouse Ehrlich carcinoma than ascorbic acid. The present study shows that 6-O-palmitoyl ascorbic acid but not ascorbic acid prolongs the lifespan of mice into which tumors such as Meth A fibrosarcoma, MM46 mammary carcinoma, Ehrlich carcinoma and sarcoma 180 are implanted. The potentiated cytotoxicity of 6-O-palmitoyl ascorbic acid is not due to an increase in duration time of the cytotoxic action, because 6-O-palmitoyl ascorbic acid is gradually inactivated during contact with tumor cells and exhibits a similar action time curve to that of ascorbic acid as shown by clonal growth assay. Cytotoxicity of 6-O-palmitoyl ascorbic acid is markedly diminished by combined addition of catalase and superoxide dismutase (SOD), as shown by dye exclusion assay, whereas the cytotoxicity was slightly reduced by either enzyme alone but not by the specifically inactivated or heat-denatured enzymes. In contrast, cytotoxicity of ascorbic acid is abolished by catalyse but not SOD. Autooxidation of 6-O-palmitoyl ascorbic acid was not inhibited by catalase plus SOD. The results indicate that cytotoxicity of 6-O-palmitoyl ascorbic acid is attributed at least partly to both hydrogen peroxide (H2O2) and superoxide (O2-.) generated at the early stage. Cytotoxicity of 6-O-palmitoyl ascorbic acid is also appreciably attenuated by singlet oxygen (1O2) scavengers such as hydroquinone, 1,4-diazobicyclo-2,2,2-octane or sodium azide, but not by hydroxyl radical scavengers including butylated hydroxytoluene, D-mannitol, benzoic acid and ethanol. Thus, in contrast to cytotoxicity of ascorbic acid mediated entirely by H2O2 initially generated, acylated ascorbic acid produces a diversity of active oxygen species including H2O2, O2-. and other species secondarily generated via disproportion, which may be additively involved in the enhanced cytotoxic action.     

Ascorbic acid and dihydroxyfumaric acid dependent hydroxylase activity of immobilized haemoglobin

Aniline hydroxylase activity of ascorbic acid and dihydroxyfumaric acid-haemoglobin systems has been studied. Hydroxylase activity of haemoglobin immobilized by crosslinking with glutaraldehyde as insoluble particles is reported. Activity yields after immobilization and kinetic constants were estimated. A peroxidative mechanism is postulated in which ascorbic acid and dihydroxyfumaric acid are electron donors as well as competitive substrates.     

Peroxide oxidation of indole to oxindole by chloroperoxidase catalysis.

In the presence of chloroperoxidase, indole was oxidized by H2O2 to give oxindole as the major product. Under most conditions oxindole was the only product formed, and under optimal conditions the conversion was quantitative. This reaction displayed maximal activity at pH 4.6, although appreciable activity was observed throughout the entire pH range investigated, namely pH 2.5-6.0. Enzyme saturation by indole could not be demonstrated, up to the limit of indole solubility in the buffer. The oxidation kinetics were first-order with respect to indole up to 8 mM, which was the highest concentration of indole that could be investigated. On the other hand, 2-methylindole was not affected by H2O2 and chloroperoxidase, but was a strong inhibitor of indole oxidation. The isomer 1-methylindole was a poor substrate for chloroperoxidase oxidation, and a weak inhibitor of indole oxidation. These results suggest the possibility that chloroperoxidase oxidation of the carbon atom adjacent to the nitrogen atom in part results from hydrogen-bonding of the substrate N-H group to the enzyme active site.     

Thianthrene 5-oxide as a probe of the electrophilicity of hemoprotein oxidizing species.

Thianthrene 5-oxide (T-5-O), which is oxidized to the 5,10- and 5,5-dioxides, respectively, by electrophilic and nucleophilic agents, has been used to determine the electronic properties of hemoprotein oxidizing species. Cytochrome P450 oxidizes T-5-O to the 5,10- rather than the 5,5-dioxide but oxidizes the 5,5-dioxide rapidly and the 5,10-dioxide slowly to the 5,5,10-trioxide. Chloroperoxidase oxidizes T-5-O to the 5,10-dioxide but very poorly oxidizes it further to the 5,5,10-trioxide. It does, however, readily oxidize the 5,5-dioxide to the trioxide. The oxidizing species of cytochrome P450 and chloroperoxidase are thus comparably electrophilic, but the former is more powerful. T-5-O is not detectably oxidized by horseradish peroxidase/H2O2 but is oxidized exclusively to the 5,5-dioxide when the peroxide is replaced by dihydroxyfumaric acid (DHFA). The oxygen incorporated into the 5,5-dioxide in this reaction derives from molecular oxygen. This is consistent with the involvement of a DHFA-derived co-oxidizing species. Oxidation of T-5-O by human hemoglobin and H2O2 yields the 5,5- and 5,10-dioxides and the 5,5,10-trioxide. The oxygen in these products derives primarily (greater than 80%) from H2O2. Hemoglobin and H2O2 thus form both a P450-like electrophilic oxidant (5,10-dioxide) and a peroxide-derived nucleophilic oxidant (5,5-dioxide). A large difference in the cis:trans ratios of the 5,10-dioxides produced from T-5-O by cytochrome P450 (1.3:1) and chloroperoxidase (2.5:1) vs hemoglobin (0.1:1) suggests that the hemoglobin active site severely constrains the geometry of the electrophilic oxidation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Peroxygenase metabolism of N-acetylbenzidine by prostaglandin H synthase. Formation of an N-hydroxylamine.

Synthesis of prostaglandin H2 by prostaglandin H synthase (PHS) results in a two-electron oxidation of the enzyme. An active reduced enzyme is regenerated by reducing cofactors, which become oxidized. This report examines the mechanism by which PHS from ram seminal vesicle microsomes catalyzes the oxidation of the reducing cofactor N-acetylbenzidine (ABZ). During the conversion of 0.06 mM ABZ to its final end product, 4'-nitro-4-acetylaminobiphenyl, a new metabolite was observed when 1 mM ascorbic acid was present. Similar results were observed whether 0.2 mM arachidonic acid or 0.5 mM H2O2 was used as the substrate. This metabolite co-eluted with synthetic N'-hydroxy-N-acetylbenzidine (N'HA), but not with N-hydroxy-N-acetylbenzidine. The new metabolite was identified as N'HA by electrospray ionization/MS/MS. N'HA represented as much as 10% of the total radioactivity recovered by high pressure liquid chromatography. When N'HA was substituted for ABZ, PHS metabolized N'HA to 4'-nitro-4-acetylaminobiphenyl. Inhibitor studies demonstrated that metabolism was due to PHS, not cytochrome P-450. The lack of effect of 5,5-dimethyl-1-pyrroline N-oxide, mannitol, and superoxide dismutase suggests the lack of involvement of one-electron transfer reactions and suggests that hydroxyl radicals and superoxide are not sources of oxygen or oxidants. Oxygen uptake studies did not demonstrate a requirement for molecular oxygen. When [18O]H2O2 was used as the substrate, 18O enrichment was observed for 4'-nitro-4-acetylaminobiphenyl, but not for N'HA. A 97% enrichment was observed for one atom of 18O, and a 17 +/- 7% enrichment was observed for two 18O atoms. The rapid exchange of 18O-N'HA with water was suggested to explain the lack of enrichment of N'HA and the low enrichment of two 18O atoms into 4'-nitro-4-acetylaminobiphenyl. Results demonstrate a peroxygenase oxidation of ABZ and N'HA by PHS and suggest a stepwise oxidation of ABZ to N'-hydroxy, 4'-nitroso, and 4'-nitro products.     

Deactivation mechanisms of chloroperoxidase during biotransformations

Inactivation mechanisms of chloroperoxidase (CPO) from Caldariomyces fumago have been investigated with the aim of improving the practical utility of CPO for hydrocarbon oxidation. Deactivation studies in the presence of oxidants (i.e., hydrogen peroxide and t-butyl hydroperoxide) indicated that CPO lost oxidation activity toward hydrocarbon substrates during dismutation of hydrogen peroxide. The loss of enzyme activity was accompanied by the apparent destruction of the heme rather than aggregation or denaturation of the apo-protein. The decrease of enzyme activity was significantly retarded by adding the radical scavenger t-butyl alcohol at pH 4.1, or by optimizing the reaction pH. CPO retained greatest oxidation activity at pH 5-6, which may produce a more favorable ionization state of the key amino acid (Glu-183) and thus reduce radical formation. As a result of higher activity at pH 5-6, the total turnover numbers (TTN, defined as the amount of product produced over the catalytic lifetime of the enzyme) for the oxidation of toluene and o-, m-, p-xylenes in substrate/aqueous emulsion systems ranged from ca. 10% to 110% higher at pH 5.5 (20,000 to 45,000 mol product/mol enzyme) compared to pH 4.1. Furthermore, TTNs of CPO increased with increasing turnover frequencies, indicating that higher activity toward reducing substrates reduces radical formation and stabilizes CPO toward inactivation by H(2)O(2). These findings demonstrate the important relationship between CPO stability and activity, and illustrate that large improvements in CPO activity and stability can be achieved through solvent engineering.     

Kinetics of the oxidation of ascorbic acid, ferrocyanide and p-phenolsulfonic acid by chloroperoxidase compounds I and II

For the first time elementary reactions involving chloroperoxidase compounds I and II have been investigated. A multi-mixing stopped-flow apparatus was used to study the kinetics of the reactions of compounds I and II with ascorbic acid, ferrocyanide and p-phenolsulfonic acid. The second-order rate constants of the reactions of both compounds with all three substrates were determined between pH 3 and pH 7. In all cases the rate constants decrease with increasing pH. The reactions of p-phenolsulfonic acid are influenced by a catalytically important group on both compounds I and II with a pKa of 3.7 +/- 0.2. With ascorbic acid and ferrocyanide as substrates, a decrease in rate was observed upon ionization of the substrate. Comparisons with horseradish peroxidase show that chloroperoxidase is a much less efficient peroxidatic enzyme. The kinetic data were used to calculate the percentage composition of the mixture of chloroperoxidase species which contribute to the spectra measured during the turnover with ascorbate as substrate.     

Purification and characterization of nitrite-oxidizing enzyme from heterotrophic Bacillus badius 1-73, with special concern to catalase

Nitrite-oxidizing enzyme I (NiOx I) was purified from a heterotrophic bacterium, Bacillus badius I-73. The enzyme was a homotetramer of a heme-containing peptide, and was similar to catalases from various sources in its N-terminal amino acid sequence. The purified enzyme also catalyzed H2O2 degradation. The nitrite oxidation reaction required ascorbic acid and oxygen. Successive H2O2 feeding could be substituted for ascorbic acid. These indicated that NiOx I is a catalase and nitrite was oxidized by a peroxidase-like reaction.     

Homogentisic acid autoxidation and oxygen radical generation: implications for the etiology of alkaptonuric arthritis

The metabolic disorder, alkaptonuria, is distinguished by elevated serum levels of 2,5-dihydroxyphenylacetic acid (homogentisic acid), pigmentation of cartilage and connective tissue and, ultimately, the development of inflammatory arthritis. Oxygen radical generation during homogentisic acid autoxidation was characterized in vitro to assess the likelihood that oxygen radicals act as molecular agents of alkaptonuric arthritis in vivo. For homogentisic acid autoxidized at physiological pH and above, yielding superoxide (O2-)2 and hydrogen peroxide (H2O2), the homogentisic acid autoxidation rate was oxygen dependent, proportional to homogentisic acid concentration, temperature dependent and pH dependent. Formation of the oxidized product, benzoquinoneacetic acid was inhibited by the reducing agents, NADH, reduced glutathione, and ascorbic acid and accelerated by SOD and manganese-pyrophosphate. Manganese stimulated autoxidation was suppressed by diethylenetriaminepentaacetic acid (DTPA). Homogentisic acid autoxidation stimulated a rapid cooxidation of ascorbic acid at pH 7.45. Hydrogen peroxide was among the products of cooxidation. The combination of homogentisic acid and Fe3+-EDTA stimulated hydroxyl radical (OH.) formation estimated by salicylate hydroxylation. Ferric iron was required for the reaction and Fe3+-EDTA was a better catalyst than either free Fe3+ or Fe3+-DTPA. SOD accelerated OH. production by homogentisic acid as did H2O2, and catalase reversed much of the stimulation by SOD. Catalase alone, and the hydroxyl radical scavengers, thiourea and sodium formate, suppressed salicylate hydroxylation. Homogentisic acid and Fe3+-EDTA also stimulated the degradation of hyaluronic acid, the chief viscous element of synovial fluid. Hyaluronic acid depolymerization was time dependent and proportional to the homogentisic acid concentration up to 100 microM. The level of degradation observed was comparable to that obtained with ascorbic acid at equivalent concentrations. The hydroxyl radical was an active intermediate in depolymerization. Thus, catalase and the hydroxyl radical scavengers, thiourea and dimethyl sulfoxide, almost completely suppressed the depolymerization reaction. The ability of homogentisic acid to generate O2-, H2O2 and OH. through autoxidation and the degradation of hyaluronic acid by homogentisic acid-mediated by OH. production suggests that oxygen radicals play a significant role in the etiology of alkaptonuric arthritis.     

Electron transfer in chromaffin-vesicle ghosts containing peroxidase.

In chromaffin vesicles, the enzyme dopamine beta-monooxygenase converts dopamine to norepinephrine. It is believed that reducing equivalents for this reaction are supplied by intravesicular ascorbic acid and that the ascorbate is regenerated by importing electrons from the cytosol with cytochrome b-561 functioning as the transmembrane electron carrier. If this is true, then the ascorbate-regenerating system should be capable of providing reducing equivalents to any ascorbate-requiring enzyme, not just dopamine beta-monooxygenase. This may be tested using chromaffin-vesicle ghosts in which an exogenous enzyme, horseradish peroxidase, has been trapped. If ascorbate and peroxidase are trapped together within chromaffin-vesicle ghosts, cytochrome b-561 in the vesicle membrane is found in the reduced form. Subsequent addition of H2O2 causes the cytochrome to become partially oxidized. H2O2 does not cause this oxidation if either peroxidase or ascorbate are absent. This argues that the cytochrome is oxidized by semidehydroascorbate, the oxidation product of ascorbate, rather than by H2O2 or peroxidase directly. The semidehydroascorbate must be internal because the ascorbate from which it is formed is sequestered and inaccessible to external ascorbate oxidase. This shows that cytochrome b-561 can transfer electrons to semidehydroascorbate within the vesicles and that the semidehydroascorbate may be generated by any enzyme, not just dopamine beta-monooxygenase.     

Reaction Properties of the Ascorbic Acid Oxidase from Myrothecium verrucaria

Ascorbic acid oxidase activity in Myrothecium verrucaria extracts resulted in O(2) uptake exceeding 0.5 mole per mole of ascorbic acid and in CO(2) evolution. Measurement of oxidized ascorbic acid at completion of the reaction demonstrated that an average of 10% of the oxidized product disappeared. A comparison of the gas exchange data with the amount of ascorbic acid not accounted for indicated that the reaction could not be explained by independent oxidase and oxygenase systems. Chromatographic examination of the reaction mixtures identified l-threonic acid. Experiments with ascorbic acid-1-(14)C showed that C-1 was partially decarboxylated during the oxidation. Test of the fungal extracts for enzymes that might explain the deviation from expected stoichiometry showed that phenolase, glutathione reductase, cytochrome oxidase, peroxidase and oxalic decarboxylase were not involved. Addition of azide in concentrations sufficient to block catalase increased excess O(2) consumption about 65%. No enzymes were found that could directly attack oxidized ascorbic acid. H(2)O(2) accumulated during oxidation in azide-blocked systems.The O(2) excess could be explained by assuming the enzyme had peroxidative capacity on a reductant other than ascorbic acid. An intermediate of ascorbic acid oxidation appeared to function as the substrate yielding CO(2) and l-threonic acid on degradation. The increase in excess O(2) utilized in azide-blocked systems and the H(2)O(2) accumulation also were explained by the proposed scheme.Another interpretation would involve production of free radicals during ascorbic acid oxidation. Evidence for this was the ability of extracts to oxidize DPNH in the presence of ascorbic acid. Oxygen radicals formed in such reactions were considered possible agents of degradation of ascorbic acid.     

Oxidase-peroxidase enzymes of Datura innoxia. Oxidation of formylphenylacetic acid ethyl ester.

An enzyme system from Datura innoxia roots oxidizing formylphenylacetic acid ethyl ester was purified 38-fold by conventional methods such as (NH4)2SO4 fractionation, negative adsorption on alumina Cy gel and chromatography on DEAE-cellulose. The purified enzyme was shown to catalyse the stoicheiometric oxidation of formylphenylacetic acid ethyl ester to benzoylformic acid ethyl ester and formic acid, utilizing molecular O2. Substrate analogues such as phenylacetaldehyde and phenylpyruvate were oxidized at a very low rate, and formylphenylacetonitrile was an inhilating agents, cyanide, thiol compounds and ascorbic acid. This enzyme was identical with an oxidase-peroxidase isoenzyme. Another oxidase-peroxidase isoenzyme which separated on DEAE-chromatography also showed formylphenylacetic acid ethyl ester oxidase activity, albeit to a lesser extent. The properties of the two isoenzymes of the oxidase were compared and shown to differ in their oxidation and peroxidation properties. The oxidation of formylphenylacetic acid ethyl ester was also catalysed by horseradish peroxidase. The Datura isoenzymes exhibited typical haemoprotein spectra. The oxidation of formylphenylacetic acid ethyl ester was different from other peroxidase-catalysed reactions in not being activated by either Mn2+ or monophenols. The oxidation was inhibited by several mono- and poly-phenols and by catalase. A reaction mechanism for the oxidation is proposed.     

Preparation of dehydro-l-(+)-ascorbic acid dimer by oxidation of ascorbic acid with arsenic acid/iodine and formation of complexes between arsenious acid and ascorbic acid

Ascorbic acid in the presence of a catalytic amount of iodine reduces arsenic acid in methanol giving the arsenious acid bound to the 2-methyl hemi-ketal of dehydroascorbic acid, 5, in 1:1 and in a more stable 2:1 5/As(III) molar ratio. Removal of the As(III) and treating the 2-methyl hemi-ketal of dehydroascorbic acid with refluxing acetonitrile affords the pure, crystalline dehydroascorbic acid dimer in good yields. Ascorbic acid also binds to As(III) of H(3)AsO(3) in a 1:1 and 2:1 ascorbic acid/As(III) molar ratio. The 1:1 complex is not stable and by expulsion of H(3)AsO(3) is transformed to the more stable 2:1 complex. The data do not permit distinguishing the 2:1 complexes between [AsL(2)(H(2)O)](-)H(+) or AsL(LH)(H(2)O) where L is the bis deprotonated and LH is the mono deprotonated 2-methyl hemi-ketal of dehydroascorbic acid or ascorbic acid. The 2:1 ascorbic acid/As(III) complex is oxidized by dioxygen, in a solvent-dependent manner, to dehydroascorbic acid implying dioxygen activation by the bound As(III). With thiophenol the same complex gives quantitatively triphenyl trithioarsenite, As(SPh)(3).