Intracellular utilization of superoxide anion by indoleamine 2,3-dioxygenase of rabbit enterocytes.

The participation of superoxide anion (O2-) in the intracellular indoleamine 2,3-dioxygenase activity was studied using the dispersed cell suspension of the rabbit small intestine. The dioxygenase activity was assayed by measuring [14C]formate released from DL-[ring-2-14C]tryptophan. The addition of diethyldiethiocarbamate, a superoxide dismutase inhibitor, markedly accelerated the intracellular dioxygenase activity while the superoxide dismutase activity decreased concomitantly. Furthermore, substrates of xanthine oxidase such as inosine, adenosine, and hypoxanthine also increased the dioxygenase activity in the cells, particularly in the presence of methylene blue. This increase was completely abolished by the addition of allopurinol, a specific inhibitor of xanthine oxidase. These results, taken together, indicate that the intracellular accumulation of O2- results in acceleration of the in situ dioxygenase activity, and that indoleamine 2,3-dioxygenase utilizes O2- in the isolated intestinal cells.     

The roles of superoxide anion and methylene blue in the reductive activation of indoleamine 2,3-dioxygenase by ascorbic acid or by xanthine oxidase-hypoxanthine

To clarify the roles of superoxide anion (O2.-) and methylene blue in the reductive activation of the heme protein indoleamine 2,3-dioxygenase, effects of xanthine oxidase-hypoxanthine used at various oxidase concentration levels as an O2.- source and an electron donor on the catalytic activity of the dioxygenase have been examined in the presence and absence of either methylene blue or superoxide dismutase using L- and D-tryptophan as substrates. In the absence of methylene blue, initial rates of the product N-formylkynurenine formation are enhanced in parallel with the xanthine oxidase level up to approximately 100 and approximately 50% of the apparent maximal activity (approximately 2 s-1) for L- and D-Trp, respectively. Superoxide dismutase effectively inhibits the reactions by 80-98% for both isomers. Additions of methylene blue (25 microM) help to maintain the linearity of the product formation that would be rapidly lost a few minutes after the start of the reaction without the dye, especially for L-Trp. Additions of methylene blue also enhance the activity to the maximal level for D-Trp. In the presence of methylene blue, the inhibitory effects of superoxide dismutase are considerably decreased with the increase in xanthine oxidase concentration, and at near maximal dioxygenase activity levels superoxide dismutase is totally without effect. In separate anaerobic experiments leuco-methylene blue, generated either by photoreduction or by ascorbate reduction, is shown to be able to reduce the ferric dioxygenase up to 25-40%. Substrate Trp and heme ligands (CO, n-butyl isocyanide) help to shift a ferric form----ferrous form equilibrium to the right. Thus, under aerobic conditions leuco-methylene blue might similarly be able to reduce the dioxygenase in the presence of an electron donor with the aid of substrate and O2. These results strongly suggest that indoleamine 2,3-dioxygenase can be activated through different pathways either by O2.- or by an electron donor-methylene blue system. For the latter case, the dye is acting as an electron mediator from the donor to the ferric dioxygenase.     

Properties of 2-nitropropane dioxygenase of Hansenula mrakii. Formation and participation of superoxide.

2-Nitropropane dioxygenase, purified to homogeneity from a yeast, Hansenula mrakii, is significantly inhibited by superoxide dismutase and various scavengers for superoxide anion such as cytochrome c, epinephrine, NADH, thiols, and polyhydric phenols. The reduction of cytochrome c and the oxidation of epinephrine and NADH are concomitant with the inhibition of enzymatic oxygenation. Neither the oxidation nor the reduction occursin the presence of superoxide dismutase or in the absence of 2-nitropropane or oxygen. Superoxide anion added externally induces the oxygenation. These findings indicate the generation of superoxide anion and its participation in the oxygenation of 2-nitropropane. The difference spectrum of the binding of NADH to 2-nitropropane dioxygenase exhibits a negative peak at 353 nm. One mole of NADH is bound to 1 mol of the enzyme and the pro-R hydrogen of the nicotinamide moiety of bound NADH predominantly is transferred to superoxide anion formed enzymatically or given externally. Thus, the diastereotopic hydrogen of NADH is discriminated by the enzyme, although not completely.     

Monooxygenase activities of dioxygenases. Benzphetamine demethylation and aniline hydroxylation reactions catalyzed by indoleamine 2,3-dioxygenase

Benzphetamine demethylase and aniline hydroxylase activities were determined with various hemoproteins including indoleamine 2,3-dioxygenase in a cytochrome P-450-like reconstituted system containing NADPH, NADPH-cytochrome P-450 reductase, and O2. The highest specific activities, almost comparable to those of liver microsomal cytochrome P-450, were detected with indoleamine 2,3-dioxygenase from the rabbit intestine. The indoleamine 2,3-dioxygenase-catalyzed benzphetamine demethylation reaction was inhibited by catalase but not by superoxide dismutase. Exogenous H2O2 or organic hydroperoxides was able to replace the reducing system and O2. The stoichiometry of H2O2 added to the product formed was essentially unity. These results indicate that the dioxygenase catalyzes the demethylation reaction by the so-called "peroxygenation" mechanism using H2O2 generated in the reconstituted system. On the other hand, the dioxygenase-catalyzed aniline hydroxylation reaction was not only completely inhibited by catalase but also suppressed by superoxide dismutase by about 60%. Although the O2- and H2O2-generating system (e.g. hypoxanthine-xanthine oxidase) was also active as the reducing system, neither exogenous H2O2 nor the generation of O2- in the presence of catalase supported the hydroxylation reaction, indicating that both H2O2 and O2- were essential for the hydroxylation reaction. However, typical scavengers for hydroxyl radical and singlet oxygen were not inhibitory. These results suggest that a unique, as yet unidentified active oxygen species generated by H2O2 and O2- participates in the dioxygenase-mediated aniline hydroxylation reaction.     

Permeation of the erythrocyte stroma by superoxide radical.

Superoxide anion, generated by xanthine oxidase within vesicles formed from washed erythrocyte ghosts, crosses the vesicle membrane to reduce cytochrome c in the medium (Lynch, R. E., and Fridovich, I. (1978) J. Biol. Chem, 253, 1838-1845). To determine whether O2- could travel through the membrane in the "channel" for the exchange of stable anions, the effects of two specific inhibitors of anion exchange, 4-acetamido-4'-isothiocyano-2,2' disulfonic acid stilbene (SITS) and 4,4'-diisothiocyano-2,2' disulfonic acid stilbene (DIDS), on the escape of O2- from vesicles were studied. The reduction of external cytochrome c, caused by O2- produced by the enzymic turnover of internal xanthine oxidase, was 85 to 90% inhibited by SITS and DIDS. If SITS impeded the egress of O2- from vesicles, it should enhance the internal effects of O2- and antagonize the inhibition of these effects by external superoxide dismutase. External superoxide dismutase inhibited the lysis of vesicles containing xanthine oxidase. SITS (60 micron) partially reversed this inhibition. It appears that O2- can cross the membrane of the erythrocyte in the anion channel.     

Evidence for superoxide-dependent reduction of Fe3+ and its role in enzyme-generated hydroxyl radical formation.

This report describes studies yielding additional evidence that superoxide anion (O2) production by some biological oxidoreductase systems is a potential source of hydroxyl radical production. The phenomenon appears to be an intrinsic property of certain enzyme systems which produce superoxide and H2O2, and can result in extensive oxidative degradation of membrane lipids. Earlier studies had suggested that iron (chelated to maintain solubility) augmented production of the hydroxyl radical in such systems according to the following reaction sequence: O2 + Fe3+ leads to O2 + Fe2+ Fe2+ + H2O2 leads to Fe3+ + HO-+OH-. The data reported below provide additional support for the occurrence of these reactions, especially the reduction of Fe3+ by superoxide. Because the conditions for such reactions appear to exist in animal tissues, the results indicate a mechanism for the initiation and promotion of peroxidative attacks on membrane lipids and also suggest that the role of antioxidants in intracellular metabolism may be to inhibit initiation of degradative reactions by the highly reactive radicals formed extraneously during metabolic activity. This report presents the following new information: (1) Fe3+ is reduced to Fe2+ during xanthine oxidase activity and a significant part of the reduction was oxygen dependent. (2) Mn2+ appears to function as an efficient superoxide anion scavenger, and this function can be inhibited by EDTA. (3) The O2-dependent reduction of Fe3+ to Fe2+ by xanthine oxidase activity is inhibited by Mn2+, which, in view of statement 2 above, is a further indication that the reduction of the iron involves superoxide anion. (4) Free radical scavengers prevent or reverse the Fe3+ inhibiton of cytochrome c3+ reduction by xanthine oxidase. (5) The inhibition of xanthine oxidase-catalyzed reduction of cyt c3+ by Fe3+ does not affect uric acid production by the xanthine oxidase system. (6) The reoxidation of reduced cyt c in the xanthine oxidase system is markedly enhanced by Fe3+ and is apparently due to enhanced HO-RADICAL formation since the Fe3+-stimulated reoxidation is inhibited by free radical scavengers, including those with specificity for the hydroxyl radical.     

The accumulation of superoxide radical during the aerobic action of xanthine oxidase. A requiem for H2O4.

The action of xanthine oxidase upon acetaldehyde or xanthine at pH 10.2 has been shown to be accompanied by substantial accumulation of O2- during the first few minutes of the reaction. H2O2 decreases this accumulation of O2- presumably because of the Haber-Weiss reaction (H2O2+O2- leads to OH- +OH+O2) and very small amounts of superoxide dismutase eliminate it. This accumulation of O2- was demonstrated in terms of a burst of reduction of cytochrome c, seen when the latter compound was added after aerobic preincubation of xanthine oxidase with its substrate. The kinetic peculiarities of the luminescence seen in the presence of luminol, which previously led to the proposal of H2O4-, can now be satisfactorily explained entirely on the basis of known radical intermediates.     

From Haemocuprein to Copper-Zinc Superoxide Dismutase: A History on the Fiftieth Anniversary of the Discovery of Haemocuprein and the Twentieth Anniversary of the Discovery of Superoxide Dismutase

Haemocuprein was discovered fifty years ago by T. Mann and D. Keilin as a copper protein of red blood cells, later named erythrocuprein. Superoxide dismutase was discovered twenty years ago by J.M. McCord and I. Fridovich as an enzymatic activity in preparations of carbonic anhydrase or myoglobin that inhibited the aerobic reduction of cytochrome c by xanthine oxidase. Astonishingly the superoxide dismutase proved to be haemocuprein. Around this time zinc was found in haemocuprein, in equimolar amount to the copper. Haemocuprein thus became copper-zinc superoxide dismutase after thirty years as an obscure cupropro-tein of red blood cells. This historical article is a tribute to the achievement of J.M. McCord and I. Fridovich. Their discovery of superoxide dismutase revolutionized the study of oxygen free-radicals in biochemistry.     

Chemiluminescence by Listeria monocytogenes.

Listeria monocytogenes cells suspended in brain heart infusion broth or in carbonated saline solution emitted light (chemiluminescence) that could be detected by a liquid scintillation spectrometer. This chemiluminescence was inhibited by superoxide dismutase and catalase but not by the hydroxyl radical scavengers mannitol and benzoate; it was also dependent upon and proportional to the carbonate ion concentration in the medium. Organisms suspended in carbonated saline solution which had ceased to chemiluminesce immediately began to chemiluminesce again when acetaldehyde was added but not when glucose, sucrose, or xanthine was added. Acetaldehyde-induced chemiluminescence was inhibited by suproxide dismutase and catalase but not by allopurinol. Our data indicate that the superoxide anion, hydrogen peroxide, and the carbonate ion are involved in chemiluminescence by L. monocytogenes. Chemiluminescence is apparently initiated by the extracellular generation of superoxide anon by this organism. The mechanism for the production of the superoxide anion is not known, but xanthine oxidase does not appear to be involved.     

Thioureas react with superoxide radicals to yield a sulfhydryl compound. Explanation for protective effect against paraquat

Thiourea and superoxide dismutase were effective antidotes to paraquat toxicity in an HL60 cell culture system, whereas other hydroxyl scavengers were ineffective. The efficacy of thioureas was not due to blockage of intracellular paraquat uptake, inhibition of NADPH-P-450 reductase, or reaction with the paraquat radical. Thiourea also competitively inhibited the reduction of cytochrome c by the xanthine/xanthine oxidase superoxide-generating system, and the release of iron from ferritin by superoxide radicals. The reaction of superoxide with thiourea produced a sulfhydryl compound distinct from products formed by hydrogen peroxide or hydroxyl radicals. Spectrophotometric and chromatographic studies indicated the carbon-sulfide double bond was converted to a sulfhydryl group which reacted with Ellman's reagent. Additional confirmatory evidence for the sulfhydryl compound was obtained with carbon-13 NMR and mass spectroscopies. Thus, thioureas are direct scavengers of superoxide radicals as well as hydroxyl radicals and hydrogen peroxide. The rate constant for the reduction of thiourea by superoxide was estimated at 1.1 x 10(3) M-1 s-1. The implication of this finding on free radical studies, the mechanism of paraquat toxicity, and the metabolism of thioureas is discussed.     

Nitric oxide irreversibly inhibits cytochrome oxidase at low oxygen concentrations: evidence for inverse oxygen concentration-dependent peroxynitrite formation

The present study shows that nitric oxide (NO) irreversibly inhibits purified cytochrome oxidase in a reverse oxygen concentration-dependent manner. The inhibition is dramatically protected by a peroxynitrite scavenger, suggesting that peroxynitrite is formed from the reaction of NO with cytochrome oxidase at low oxygen concentration, and that peroxynitrite is involved in irreversible cytochrome oxidase inactivation. Production of nitroxyl anion or superoxide was tested as potential mechanisms underlying the conversion of NO to peroxynitrite. A nitroxyl anion scavenger potently protected the irreversible inhibition, whereas a superoxide dismutase did not provide protective effect, suggesting that the peroxynitrite was formed from nitroxyl anion rather than the reaction of NO with superoxide.     

Cytochrome c reduction by semiquinone radicals can be indirectly inhibited by superoxide dismutase

The inhibition by superoxide dismutase of cytochrome c reduction by a range of semiquinone radicals has been studied. The semiquinones were produced from the parent quinones by reduction with xanthine and xanthine oxidase. Most of the quinones studied were favored over O₂ as the enzyme substrate, and in air as well as N₂, semiquinone radicals rather than superoxide were produced and they caused the cytochrome c reduction. With all but one of the quinones (benzoquinone), cytochrome c reduction in air was inhibited by superoxide dismutase, but the amount of enzyme required for inhibition was up to 100 times greater than that required to inhibit reduction by superoxide. It was highest for the quinones with the highest redox potential. These results demonstrate how superoxide dismutase can inhibit cytochrome c reduction by species other than superoxide. They can be explained by the dismutase displacing the equilibrium: semiquinone + O₂ ? quinone + O₂^? to the right, thereby allowing the forward reaction to out-compete other reactions of the semiquinone. The implication from these findings that superoxide dismutase-inhibitable reduction of cytochrome c may not be a specific test for superoxide production is discussed.     

How relevant is the reoxidation of ferrocytochrome c by hydrogen peroxide when determining superoxide anion production?

In a recent publication [(1987) FEBS Lett. 210, 195-198] the authors claim the use of cytochrome c to detect superoxide anion underestimates the real rate of superoxide anion formation on the basis that: (i) the rate of uric acid formation by xanthine oxidase is about 4-fold faster than the rate of cytochrome c reduction and (ii) hydrogen peroxide formed upon dismutation of the superoxide anion generated by xanthine oxidase is capable of reoxidizing ferrocytochrome c. That paper may have been misleading for readers not very familiar with the field of oxygen radicals, since both assumptions are, in fact, incorrect. In this report we demonstrate that the build up in concentration of H2O2 during most reactions in which superoxide anion is being produced is not enough to affect the rate of cytochrome c reduction. Our results suggest that the authors may have been misled by an artifact due to exposure of the samples containing H2O2 to UV light, which generates hydroxyl radicals by photolysis.     

Superoxide generation by NADPH-cytochrome P-450 reductase: the effect of iron chelators and the role of superoxide in microsomal lipid peroxidation

Superoxide generation, assessed as the rate of acetylated cytochrome c reduction inhibited by superoxide dismutase, by purified NADPH cytochrome P-450 reductase or intact rat liver microsomes was found to account for only a small fraction of their respective NADPH oxidase activities. DTPA-Fe3+ and EDTA-FE3+ greatly stimulated NADPH oxidation, acetylated cytochrome c reduction, and O(2) production by the reductase and intact microsomes. In contrast, all ferric chelates tested caused modest inhibition of acetylated cytochrome c reduction and O(2) generation by xanthine oxidase. Although both EDTA-Fe3+ and DTPA-Fe3+ were directly reduced by the reductase under anaerobic conditions, ADP-Fe3+ was not reduced by the reductase under aerobic or anaerobic conditions. Desferrioxamine-Fe3+ was unique among the chelates tested in that it was a relatively inert iron chelate in these assays, having only minor effects on NADPH oxidation and/or O(2) generation by the purified reductase, intact microsomes, or xanthine oxidase. Desferrioxamine inhibited microsomal lipid peroxidation promoted by ADP-Fe3+ in a concentration-dependent fashion, with complete inhibition occurring at a concentration equal to that of exogenously added ferric iron. The participation of O(2) generated by the reductase in NADPH-dependent lipid peroxidation was also investigated and compared with results obtained with a xanthine oxidase-dependent lipid peroxidation system. NADPH-dependent peroxidation of either phospholipid liposomes or rat liver microsomes in the presence of ADP-Fe3+ was demonstrated to be independent of O(2) generation by the reductase.     

The mechanism of liver microsomal lipid peroxidation.

In the presence of Fe-3+ and complexing anions, the peroxidation of unsaturated liver microsomal lipid in both intact microsomes and in a model system containing extracted microsomal lipid can be promoted by either NADPH and NADPH : cytochrome c reductase or by xanthine and xanthine oxidase. Erythrocuprein effectively inhibits the activity promoted by xanthine and xanthine oxidase but produces much less inhibition of NADPH-dependent peroxidation. The singlet-oxygen trapping agent, 1, 3-diphenylisobenzofuran, had no effect on NADPH-dependent peroxidation but strongly inhibited the peroxidation promoted by xanthine and xanthine oxidase. NADPH-dependent lipid peroxidation was also shown to be unaffected by hydroxyl radical scavengers.. The addition of catalase had no effect on NADPH-dependent lipid peroxidation, but it significantly increased the rate of malondialdehyde formation in the reaction promoted by xanthine and xanthine oxidase. The results demonstrate that NADPH-dependent lipid peroxidation is promoted by a reaction mechanism which does not involve either superoxide, singlet oxygen, HOOH, or the hydroxyl radical. It is concluded that NADPH-dependent lipid peroxidation is initiated by the reduction of Fe-3+ followed by the decomposition of hydroperoxides to generate alkoxyl radicals. The initiation reaction may involve some form of the perferryl ion or other metal ion species generated during oxidation of Fe-2+ by oxygen.     

The reaction of xanthine oxidase with aldehydic products of lipid peroxidation.

A quantitative structure-activity relationship for the reaction of xanthine oxidase with a homologous series of alpha, beta-unsaturated aldehydes, which are known to be products of lipid peroxidation, was investigated. Aldehydes in the series 2-butenal through 2-nonenal and 4-hydroxy-2-nonenal, displayed differential reactivity toward xanthine oxidase as measured by production of the superoxide radical anion. Kinetic parameters for the rate of superoxide production and substrate affinity were determined via the superoxide dismutase-sensitive reduction of cytochrome c. Trends in kinetic parameters as a function of carbon number for the series of trans-2-enals was consistent with a dependence on substrate hydrophobicity. Log kw', a hydrophobicity constant widely employed as a model for the octanol/water partition coefficient, was determined by reversed phase liquid chromatography for the alpha, beta-unsaturated aldehydes in this study. Linear relationships for the correlation of substrate binding (pKm) and efficiency of superoxide production (log kcat/Km) with substrate hydrophobicity (log kw') were found. The mode of inhibition of xanthine oxidation by 2-butenal is shown to be noncompetitive, suggesting distinct binding sites for purine and aldehydic substrates. It is suggested that the reaction of xanthine oxidase with unsaturated aldehydes could be an important route of amplification of oxidative damage in cells.     

The mechanism of the activity-dependent luminescence of xanthine oxidase.

The weak luminescence that accompanies the aerobic xanthine oxidase reaction is inhibited by superoxide dismutase, by catalase, and by scavengers of hydroxyl radicals. It is also entirely dependent upon the presence of carbonate. It thus appears that the O₂ and H₂O₂ produced during the aerobic action of xanthine oxidase interact to generate OH which, in turn, reacts with carbonate to yield the carbonate radical (CO₃^?). The species that is directly responsible for light emission appears to be produced by a dimerization of carbonate radicals, since the light intensity was a function of the square of the carbonate concentration. The data provide no reason to suppose that the light-emitting species is singlet oxygen.     

Decrease in heart mitochondrial creatine kinase activity due to oxygen free radicals.

This study was undertaken to examine the effects of oxygen free radicals on mitochondrial creatine kinase activity in rat heart. Xanthine plus xanthine oxidase (superoxide anion radical generating system) reduced mitochondrial creatine kinase activity both in a dose- and a time-dependent manner. Superoxide dismutase showed a protective effect on depression in creatine kinase activity due to xanthine plus xanthine oxidase. Hydrogen peroxide inhibited creatine kinase activity in a dose-dependent manner, this inhibition was protected by the addition of catalase. In order to understand the detailed mechanisms by which oxygen free radicals inhibit mitochondrial creatine kinase activity, the effects of oxygen free radicals on mitochondrial sulfhydryl groups were examined. Mitochondrial sulfhydryl groups contents were decreased by xanthine plus xanthine oxidase or hydrogen peroxide; this depression in sulfhydryl groups contents was prevented by the addition of superoxide dismutase or catalase. N-Ethylmaleimide (sulfhydryl group reagent) expressed inhibitory effects on the creatine kinase activity both in a dose- and a time-dependent manner; dithiothreitol or cysteine (sulfhydryl group reductant) showed protective effects on the creatine kinase activity depression induced by N-ethylmaleimide. Dithiothreitol or cysteine also blocked the depression of mitochondrial creatine kinase activity caused by xanthine plus xanthine oxidase or hydrogen peroxide. These results lead us to conclude that oxygen free radicals may inhibit mitochondrial creatine kinase activity by modifying sulfhydryl groups in the enzyme protein.     

The tiron free radical as a sensitive indicator of chloroplastic photoautoxidation.

Wheat chloroplasts photochemically reduced molecular oxygen, as a Hill oxidant in the Mehler reaction, to superoxide anion which then oxidized added 1,2-dihydroxybenzene-3,5-disulfonate to its semiquinone, a comparatively stable free radical at pH 7. The last mentioned reaction was rapid in aqueous solution, but the rate of formation of 1,2-dihydroxybenzene-3,5-disulfonate semiquinone by the chloroplast system was calculated as T1 of 0.6 s. The Mehler reaction, or more specifically the univalent reduction of oxygen by Photosystem I, was rate-limiting so that the 1,2-dihydroxybenzene-3,5-disulfonate seniquinone was a useful spin probe for superoxide anion production at room temperature. The ESR signal of 1,2-dihydroxybenzene-3,5-disulfonate semiquinone was proportional to its steady state concentration and decayed in the dark with a T1/2 of 5-6 s. This oxygen-dependent signal was enhanced by mediation of chloroplastic oxygen reduction through methyl viologen. The superoxide anion scavengers ascorbate and L-epinephrine competitively obscured 1,2-dihydroxybenzene-3,5-disulfonate semiquinone formation, butadded superoxide dismutase was not as effective in this role. Partial inhibition by superoxide dismutase was achieved only by preincubation of Photosystem I enriched particles with ten times the endogenous concentration of superoxide dismutase. This and the persistence of a small amount of a 1,2-dihydroxybenzene-3,5-disulfonate (Tiron) oxidizing species in the dark supports the concept of Tiron accessibility but not the superoxide dismutase accessibility of superoxide anion bound in its formative enzyme complex. Benzoquinone and naphthoquinone disulfonate also reacted with superoxide anion, and supported both the Hill reaction and the Mehler reaction as final oxidants of both water and superoxide anion.     

Inhibition of xanthine oxidase by pterins

The effect of a panel of pterins on xanthine oxidase was investigated by measuring formation of urate from xanthine as well as formazan production from nitroblue tetrazolium. The pterin derivatives, depending on their chemical structure, decreased urate as well as formazan generation: 200 μM neopterin and biopterin suppressed urate formation (90% from baseline) and formazan production (80% from baseline) as well. Their reduced forms, 7,8-dihydroneopterin and 5,6,7,8-tetrahydrobiopterin, showed a lesser but still strongly diminishing influence (40% from baseline). Another oxidized pterin namely leukopterin showed only a weak inhibitory effect. Xanthopterin, a known substrate of xanthine oxidase, had a strong effect on urate formation (80% inhibition), but a lesser effect on formazan production (30% reduction). When iron-(III)-EDTA complex was added to the reaction mixture all the effects were more pronounced. Superoxide dismutase, which removes superoxide anion by dismutation intooxygen, decreased formazan production in addition to pterin derivatives and had a small but enhancing effect on urate formation. Also the reductant N-acetylcysteine had an additive effect to pterins to diminish formazan production in a dose-dependent way. The results of our study suggest that depending on their chemical structure pterins reduce superoxide anion generation by xanthine oxidase.