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.     

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-.     

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.     

The influence of porphyrins on iron-catalysed generation of hydroxyl radicals.

Uroporphyrin I, haematoporphyrin and haematoporphyrin derivative had no effect on O2-. generation during oxidation of hypoxanthine by xanthine oxidase and on the formation of hydroxyl radicals (OH.) in the hypoxanthine/xanthine oxidase/Fe3+-EDTA/deoxyribose system. On the other hand, these porphyrins strongly inhibited O2-. formation in a horseradish peroxidase/H2O2/NADPH mixture, whereas they augmented OH. generation in this system after addition of Fe3+-EDTA. Experimental evidence suggests that these observations should be ascribed to the formation of a porphyrin anion radical in the horseradish peroxidase/NADPH system. The formation of this anion radical was confirmed by e.s.r. spectroscopy. This radical is apparently unable to reduce cytochrome c, but it can replace O2-. in the OH.-generating Haber-Weiss reaction.     

NADPH oxidation catalyzed by the peroxidase/H2O2 system. Iodide-mediated oxidation of NADPH to iodinated NADP

Oxidation of NADPH catalyzed by the peroxidase/H2O2 system is known to require the presence of mediating molecules. Using either lactoperoxidase or horseradish peroxidase, we demonstrated that in the peroxidase/H2O2 system, NADPH oxidation was mediated by iodide. The oxidation product was the iodinated NADP. This product was shown to possess spectral characteristics different from those of NADP+ and NADPH, since for iodinated NADP, increased absorbance was observed in the 280-nm region and was directly proportional to the rate of iodination. It is suggested that oxidation and iodination of NADPH proceed via a single reaction between the intermediary iodide oxidation species and NADPH. Experiments with different molecules of NADPH analogues indicated that iodination occurred in the nicotinamide part of the NADPH molecule.     

A mechanistic study of the formation of hydroxyl radicals induced by horseradish peroxidase with NADH

During the oxidation of NADH by horseradish peroxidase (HRP-Fe(3+)), superoxide (O(-)(2)) is produced, and HRP-Fe(3+) is converted to compound III. Superoxide dismutase inhibited both the generation of O(-)(2) and the formation of compound III. In contrast, catalase inhibited only the generation of O(-)(2). Under anaerobic conditions, the formation of compound III did not occur in the presence of NADH, thus indicating that compound III is produced via formation of a ternary complex consisting of HRP-Fe(3+), NADH and oxygen. The generation of hydroxyl radicals was dependent upon O(-)(2) and H(2)O(2) produced by HRP-Fe(3+)-NADH. The reaction of compound III with H(2)O(2) caused the formation of compound II without generation of hydroxyl radicals. Only HRP-Fe(3+)-NADH (but not K(+)O(-)(2) and xanthine oxidase-hypoxanthine) was able to induce the conversion of metmyoglobin to oxymyoglobin, thus suggesting the participation of a ternary complex made up of HRP-Fe(2+…)O(2)(…)NAD(.) (but not free O(-)(2) or H(2)O(2)) in the conversion of metmyoglobin to oxymyoglobin. It appears that a cyclic pathway is formed between HRP-Fe(3+), compound III and compound II in the presence of NADH under aerobic conditions, and a ternary complex plays the central roles in the generation of O(-)(2) and hydroxyl radicals.     

Plasma Membrane Redox Enzyme Is Involved in the Synthesis of O2- and H2O2 by Phytophthora Elicitor-Stimulated Rose Cells

An elicitor prepared from the autoclaved cell walls of Phytophthora sp. induced O2- generation and H2O2 accumulation by cultured cells of Rosa damascena Mill. cv Gloire de Guilan. N,N-Diethyldithiocarbamate, a superoxide dismutase inhibitior, blocked H2O2 accumulation and caused a dramatic accumulation of O2- by elicitor-treated rose cells. In the absence of N,N-diethyldithiocarbamate no detectable O2- was accumulated. Diphenyleneiodonium, quinacrine, pyridine, and imidazole, inhibitors of the mammalian neutrophil NADPH oxidase responsible for the generation of O2- during phagocytosis, inhibited O2- generation by elicitor-treated rose cells. Diphenyleneiodonium also inhibited NADH-dependent O2- production by plasma membranes isolated from rose cells. None of the four compounds inhibited the peroxidase activity in the cell-suspension medium. These results demonstrate that elicitor-stimulated accumulation of H2O2 comes only from superoxide dismutase-catalyzed dismutation of O2-. The data are inconsistent with the hypothesis that the synthesis of O2- is catalyzed by extracellular peroxidase and suggest that the enzyme responsible for the synthesis of O2- by elicitor-treated rose cells might be similar to the mammalian neutrophil NADPH oxidase.     

Oxidation of hydroquinones by the versatile ligninolytic peroxidase from Pleurotus eryngii. H2O2 generation and the influence of Mn2+.

Formation of H2O2 during the oxidation of three lignin-derived hydroquinones by the ligninolytic versatile peroxidase (VP), produced by the white-rot fungus Pleurotus eryngii, was investigated. VP can oxidize a wide variety of phenols, including hydroquinones, either directly in a manner similar to horseradish peroxidase (HRP), or indirectly through Mn3+ formed from Mn2+ oxidation, in a manner similar to manganese peroxidase (MnP). From several possible buffers (all pH 5), tartrate buffer was selected to study the oxidation of hydroquinones as it did not support the Mn2+-mediated activity of VP in the absence of exogenous H2O2 (unlike glyoxylate and oxalate buffers). In the absence of Mn2+, efficient hydroquinone oxidation by VP was dependent on exogenous H2O2. Under these conditions, semiquinone radicals produced by VP autoxidized to a certain extent producing superoxide anion radical (O2*-) that spontaneously dismutated to H2O2 and O2. The use of this peroxide by VP produced quinone in an amount greater than equimolar to the initial H2O2 (a quinone/H2O2 molar ratio of 1 was only observed under anaerobic conditions). In the presence of Mn2+, exogenous H2O2 was not required for complete oxidation of hydroquinone by VP. Reaction blanks lacking VP revealed H2O2 production due to a slow conversion of hydroquinone into semiquinone radicals (probably via autooxidation catalysed by trace amounts of free metal ions), followed by O2*- production through semiquinone autooxidation and O2*- reduction by Mn2+. This peroxide was used by VP to oxidize hydroquinone that was mainly carried out through Mn2+ oxidation. By comparing the activity of VP to that of MnP and HRP, it was found that the ability of VP and MnP to oxidize Mn2+ greatly increased hydroquinone oxidation efficiency.     

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.     

Generation of hydrogen peroxide during the oxidation of L-phenylalanine by Proteus mirabilis isolated membranes

In the process of L-phenylalanine oxidation by Proteus mirabilis cytoplasmic membrane, hydrogen peroxide was produced at a rate corresponding to 1-3 per cent of the total electron flow (30-110 nmoles min-1mg-1). Peroxide was estimated using a fluorimetric assay with horseradish peroxidase, or by anodic oxidation on a platinum electrode. When using the former method, superoxide dismutase decreased the apparent yield of peroxide, a fact suggesting that H2O2 was in part the dismutation product of superoxide radicals. However the superoxide dismutase effect could be an artefact due to the generation of some superoxide during the peroxidatic reaction in the assay. Adrenaline was the reagent used for the detection of superoxide. There was no significant emergence of superoxide as the result of phenylalanine oxidation by the membrane (specific activity lower than 1-2 nmoles min-1mg-1). Thus it seemed that superoxide was not an intermediate for the bulk of H2O2 formed in this system. According to the results, peroxide was probably formed at a stage of electron transport earlier than the cytochrome level. The membrane phenylalanine dehydrogenase could be a site where peroxide was evolved in these experiments.     

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).     

Hydroxyl-radical production in physiological reactions. A novel function of peroxidase.

Peroxidases catalyze the dehydrogenation by hydrogen peroxide (H2O2) of various phenolic and endiolic substrates in a peroxidatic reaction cycle. In addition, these enzymes exhibit an oxidase activity mediating the reduction of O2 to superoxide (O2.-) and H2O2 by substrates such as NADH or dihydroxyfumarate. Here we show that horseradish peroxidase can also catalyze a third type of reaction that results in the production of hydroxyl radicals (.OH) from H2O2 in the presence of O2.-. We provide evidence that to mediate this reaction, the ferric form of horseradish peroxidase must be converted by O2.- into the perferryl form (Compound III), in which the haem iron can assume the ferrous state. It is concluded that the ferric/perferryl peroxidase couple constitutes an effective biochemical catalyst for the production of .OH from O2.- and H2O2 (iron-catalyzed Haber-Weiss reaction). This reaction can be measured either by the hydroxylation of benzoate or the degradation of deoxyribose. O2.- and H2O2 can be produced by the oxidase reaction of horseradish peroxidase in the presence of NADH. The .OH-producing activity of horseradish peroxidase can be inhibited by inactivators of haem iron or by various O2.- and .OH scavengers. On an equimolar Fe basis, horseradish peroxidase is 1-2 orders of magnitude more active than Fe-EDTA, an inorganic catalyst of the Haber-Weiss reaction. Particularly high .OH-producing activity was found in the alkaline horseradish peroxidase isoforms and in a ligninase-type fungal peroxidase, whereas lactoperoxidase and soybean peroxidase were less active, and myeloperoxidase was inactive. Operating in the .OH-producing mode, peroxidases may be responsible for numerous destructive and toxic effects of activated oxygen reported previously.     

Superoxide anion is the initial product in the hydrogen peroxide formation catalyzed by NADPH oxidase in porcine thyroid plasma membrane

The plasma membrane fraction from porcine thyroid is known to exhibit an NADPH-dependent production of hydrogen peroxide (H2O2), which is utilized for the oxidative biosynthesis of thyroid hormones catalyzed by thyroid peroxidase. The H2O2 formation is cyanide-insensitive, ATP-activatable, and Ca2+-dependent (Nakamura, Y., Ogihara, S., and Ohtaki, S. (1987) J. Biochem. (Tokyo) 102, 1121-1132). It remains unknown, however, whether H2O2 is produced directly from molecular oxygen (O2) or formed via dismutation of superoxide anion (O2-). We therefore attempted to analyze the mechanism of H2O2 formation by utilizing a new method for the simultaneous measurement of O2- and H2O2, in which diacetyldeuteroheme-substituted horseradish peroxidase was employed as the trapping agent for both oxygen metabolites. When NADPH was incubated with the membrane fraction in the presence of the heme-substituted peroxidase, a massive O2 consumption was observed together with the formation of compound III, and O2- adduct of the peroxidase. The amounts of compound III formed and O2 consumed were stoichiometric with each other, while formation of compound II, an indicative of H2O2, was not observed during the reaction. On the other hand, when an excess amount of superoxide dismutase was included in the reaction mixture, compound II was produced with complete suppression of the compound III formation. NADH minimally supported both O2 consumption and formation of compound III or II. These results indicate that the NADPH oxidase in the plasma membrane of thyroid produces O2- as the primary metabolite of O2 and hence that H2O2 required for the thyroid hormone synthesis provided through the dismutation of O2-.     

Evidence for free radical formation during the oxidation of 2'-7'-dichlorofluorescin to the fluorescent dye 2'-7'-dichlorofluorescein by horseradish peroxidase: possible implications for oxidative stress measurements.

The oxidation of 2'-7'-dichlorofluorescin (DCFH) to the fluorescent 2'-7'-dichlorofluorescein (DCF) by horseradish peroxidase (HRP) was investigated by fluorescence, absorption, and electron spin resonance spectroscopy (ESR). As has been previously reported, HRP/H2O2 oxidized DCFH to the highly fluorescent DCF. However, DCF fluorescence was still observed when H2O2 was omitted, although its intensity was reduced by 50%. Surprisingly, the fluorescence increase, in the absence of exogenous H2O2, was still strongly inhibited by catalase, demonstrating that H2O2 was present and necessary for DCF formation. H2O2 was apparently formed during either chemical or enzymatic deacetylation of 2'-7'-dichlorofluorescin diacetate (DCFH-DA), probably by auto-oxidation. Spectrophotometric measurements clearly showed that DCFH could be oxidized either by HRP-compound I or HRP-compound II with the obligate generation of the DCF semiquinone free radical (DCF*-). Oxidation of DCF*- to DCF by oxygen would yield superoxide (O2*-). ESR spectroscopy in conjunction with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) revealed the presence of both superoxide and hydroxyl radicals in the DCFH/H2O2/HRP system. Both radicals were also detected in the absence of added H2O2, although the intensities of the resultant adducts were decreased. This work demonstrates that DCF fluorescence cannot be used reliably to measure O2*- in cells because O2*- itself is formed during the conversion of DCFH to DCF by peroxidases. The disproportionation of superoxide forms H2O2 which, in the presence of peroxidase activity, will oxidize more DCFH to DCF with self-amplification of the fluorescence. Because the deacetylation of DCFH-DA, even by esterases, can produce H2O2, the use of this probe to measure H2O2 production in cells is problematic.     

Studies on the mechanism of NADPH oxidation by the granule fraction isolated from human resting polymorphonuclear blood cells.

Various factor affecting NADPH-oxidation by resting human leucocyte granules (LG) at acid pH, have been investigated. It was found that: 1) oxidation of NADPH by LG was increasingly inhibited by increased cyanide concentrations in the medium and was abolished by 4 mM cyanide. 2) with or without cyanide in the incubation medium, LG omitted, Mn++ in the presence of NADPH induced superoxide anion (O- WITH 2) production, as evidenced by oxygen consumption and H2O2 production, which were abolished (in the absence of cyanide) by cytochrome C (a potent O- with 2 scavenger). 3) Both NADPH oxidation in the presence of 2 mM cyanide (cyanide-resistant) and in its absence (cyanide-sensitive) by LG occurred only in the presence of Mn++, and both were inhibited by superoxide dismutase. 4) Cyanide-resistant NADPH oxidation by LG generated H2O2, was inhibited by H2O2 and was not modified by "active" catalase. The ratio of cyanide-resistant NADPH oxidation/O2 uptake was 1 up to 1.25 mM NADPH, and increased above this concentration. 5) Cyanide-sensitive NADPH oxidation was inhibited by catalase and increased upon addition of H2O2. The ratio of cyanide-sensitive NADPH oxidation/O2 uptake was 2. It was concluded that after initiation by O - with 2, produced independently of LG, two sequential types of LG dependent NADPH oxidations occur. First, an O - with 2-dependent protein mediated NADPH oxidation (cyanide-resistant) which generates H2O2 and O - with 2 occurs. Second, NADPH peroxidation (cyanide-sensitive) which utilizes H2O2 takes place.     

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.     

An electron spin resonance study of free radicals from catechol estrogens

Electron spin resonance spectroscopy has been used to demonstrate production of semiquinone free radicals from the oxidation of the catechol estrogens 2- and 4-hydroxyestradiol and 2,6- and 4,6-dihydroxyestradiol. Radicals were generated by horseradish peroxidase/H2O2 or tyrosinase/O2, or by autoxidation, and were detected as their complexes with spin-stabilizing metal ions (Zn2+ and/or Mg2+). Radical production occurs via one- or two-electron oxidation of catechol estrogens, depending on the type of activating system. Autoxidation of catechol estrogens produces superoxide and H2O2 at physiological pH values. The present results also indicate a difference in the reactivity of quinones derived from 2- and 4-hydroxyestradiol. The toxicological significance of these reactions is discussed.     

The mechanism of action of lymphokines. IX. The enzymatic basis of hydrogen peroxide production by lymphokine-activated macrophages

The purpose of this study was to elucidate the biochemical basis of the enhanced hydrogen peroxide (H2O2) production by guinea pig peritoneal macrophages (MP) cultured in lymphokine (LK)-containing medium. The markedly augmented H2O2 generation by these cells, demonstrable by the horseradish peroxidase (HRP)-catalyzed oxidation of phenol red, is distinguished by its lack of dependence on a second stimulus. We demonstrate that H2O2 production is truly spontaneous and is not caused by a stimulant present among the H2O2 assay reagents. The principal candidate for such a role was HRP type II (a mixture of five isoenzymes) that was reported to be capable of eliciting an oxidative burst in MP. Four distinct HRP isoenzymes that were found incapable of provoking an oxidative response were nevertheless adequate for demonstrating H2O2 production by LK-activated MP. Blocking the MP receptor for mannose by the addition of mannan to the assay system resulted in enhanced detection of H2O2 by low concentrations of HRP type II and by three out of four HRP isoenzymes. Treatment of MP with LK-containing medium for 72 hr did not result in a significant change in the activity of cellular superoxide dismutase (SOD) compared with MP cultured for the same length of time in control medium. By using the specific inhibitor of copper, zinc-containing SOD, sodium diethyldithiocarbamate (DDC), and the universal SOD inhibitor, sodium nitroprusside, we found that the predominant enzyme in guinea pig peritoneal MP is probably manganese-containing SOD. Incubation of LK-activated MP with nitroprusside resulted in almost total inhibition of H2O2 production and a simultaneous switch to superoxide (O2-) liberation. Similar exposure to DDC had no effect. These data indicate that H2O2 produced by LK-activated MP is derived exclusively by enzymatic dismutation of O2- mediated by a manganese-containing SOD. The increase in spontaneous H2O2 production induced by LK is therefore secondary to augmented O2- production that occurs at a cellular location where O2- is accessible to SOD. The enzymatic basis of the enhanced oxygen radical production was investigated by determining the kinetic parameters of the O2- -forming NADPH oxidase of resting LK-treated MP in a cellfree system in which O-2 production was induced by sodium dodecyl sulfate. The Km for NADPH and the Vmax of the enzyme of LK-treated MP were not different from those of the enzyme of MP incubated in control medium. We conclude that LK treatment of MP does not modulate the NADPH oxidase itself but, most likely, a process related to activation of the enzyme.     

The respiratory burst oxidase of neutrophils. Separation of an FAD enzyme and its characterization

Pig blood neutrophils were briefly activated by various fatty acids and then fractionated into membrane vesicles with different NADPH oxidase activities. Treatment of these membranes with a detergent, octyl glucoside, resulted in a high yield of solubilized oxidase, which was subjected to isoelectric focusing on gels (pI 4.0-8.0). 1) A distinct band staining with NADPH-nitroblue tetrazolium focused at pI 5.0. The enzyme (pI 5.0) showed high specificity for NADPH and similar characteristics to the oxidase involved in the respiratory burst. 2) The enzyme was extracted from gel slices and analyzed. When measured promptly after its extraction, its NADPH oxidase activity was high, but there was apparent superoxide dismutase-insensitive cytochrome c reduction, probably due to direct electron transfer to the heme protein. However, it could produce superoxide anion (O2-) under some micelle conditions. 3) Therefore, the formation of the enzyme-substrate complex of yeast cytochrome c peroxidase was employed for the detection of H2O2. A fresh extract of stimulated cells catalyzed equimolar NADPH oxidation and H2O2 production of 306 and 300 nmol min-1 (mg protein)-1, respectively. The Km value of the enzyme for NADPH was 30 +/- 13 (S.D.) microM. The recovery of the extract (pI 5.0) was 19% of the total activity. 4) The enzyme extract contained 1.1-1.9 nmol of FAD/mg of protein, giving a turnover number of 300-600 min-1 in terms of O2- generation/FAD. No heme protein was found in the enzyme. The enzyme was mainly of 67-kDa molecular mass.     

Phenoxyl free radical formation during the oxidation of the fluorescent dye 2',7'-dichlorofluorescein by horseradish peroxidase. Possible consequences for oxidative stress measurements.

The oxidation of the fluorescent dye 2',7'-dichlorofluorescein (DCF) by horseradish peroxidase was investigated by optical absorption, electron spin resonance (ESR), and oxygen consumption measurements. Spectrophotometric measurements showed that DCF could be oxidized either by horseradish peroxidase-compound I or -compound II with the obligate generation of the DCF phenoxyl radical (DCF(.)). This one-electron oxidation was confirmed by ESR spin-trapping experiments. DCF(.) oxidizes GSH, generating the glutathione thiyl radical (GS(.)), which was detected by the ESR spin-trapping technique. In this case, oxygen was consumed by a sequence of reactions initiated by the GS(.) radical. Similarly, DCF(.) oxidized NADH, generating the NAD(.) radical that reduced oxygen to superoxide (O-(2)), which was also detected by the ESR spin-trapping technique. Superoxide dismutated to generate H(2)O(2), which reacted with horseradish peroxidase, setting up an enzymatic chain reaction leading to H(2)O(2) production and oxygen consumption. In contrast, when ascorbic acid reduced the DCF phenoxyl radical back to its parent molecule, it formed the unreactive ascorbate anion radical. Clearly, DCF catalytically stimulates the formation of reactive oxygen species in a manner that is dependent on and affected by various biochemical reducing agents. This study, together with our earlier studies, demonstrates that DCFH cannot be used conclusively to measure superoxide or hydrogen peroxide formation in cells undergoing oxidative stress.