Synthesis of a superoxide dismutase derivative that circulates bound to albumin and accumulates in tissues whose pH is decreased

Protection of tissues from oxidative stress is one of the major prerequisites for aerobic life. Since intravenously injected Cu2+/Zn2+-type superoxide dismutase (SOD) disappears from the circulation with a short half-life of 5 min, its clinical use as a scavenger for superoxide radical is limited. We synthesized a human erythrocyte type SOD derivative (SM-SOD) by linking 2 mol of hydrophobic organic anion, alpha-4-[( 6-(N-maleimido)hexanoyloxymethyl]cumyl]half-butyl-esterified poly(styrene-co-maleic acid) (SM), to the cysteinyl residues of the dimeric enzyme without decreasing enzymic activity. SM-SOD, but not SOD, bound to an albumin-Sepharose column; the bound SM-SOD was eluted by a buffer solution containing 0.5% sodium dodecyl sulfate or 10 mM warfarin, suggesting that SM-SOD reversibly binds to the warfarin site on albumin. Due to the amphipathic nature of the SMI moiety, SM-SOD bound also to cell membranes particularly when the pH was decreased. In vivo analysis in the rat revealed that intravenously injected SM-SOD circulated bound to albumin with a half-life of 6 h. Postischemic reperfusion arrhythmias were almost completely prevented by a single dose of SM-SOD, but not SOD. Thus, the prolonged half-life of SM-SOD in the circulation and its preferential accumulation in an injured site with decreased pH appeared to be responsible for preventing myocardial injury. These results suggest that superoxide radical and/or its metabolite(s) would play an important role in the pathogenesis of postischemic reperfusion arrhythmias and that SM-SOD may be useful for decreasing tissue injury in ischemic heart disease.     

Inhibition of stress-induced gastric mucosal injury by a long acting superoxide dismutase that circulates bound to albumin

To determine whether oxygen-derived free radicals play an important role in the pathogenesis of stress-induced tissue injury, the effect of a superoxide dismutase derivative, which binds to albumin and circulates with a half-life of 6 h in intact rats, on acute gastric mucosal lesion was observed in rats which were given water-immersion-restraint. This enzyme derivative also circulated bound to albumin with a half-life of 8 h in rats which were challenged with water-immersion-restraint. This treatment significantly perturbed systemic circulation of animals by decreasing the effective volume of circulating blood, increased vascular permeability of the gastric mucosa, and induced acute gastric mucosal lesion. Intravenous administration of this enzyme derivative normalized both systemic circulation and vascular permeability of the gastric mucosa and prevented the occurrence of stress-induced gastric injury. These findings suggest that the superoxide radical and/or its metabolite(s) plays an important role in the pathogenesis of stress-induced acute gastric mucosal lesion.     

Inhibition of carrageenin-induced paw edema by a superoxide dismutase derivative that circulates bound to albumin.

Although the possible involvement of superoxide radical and its metabolite(s) in the pathogenesis of various types of edema have been suggested, direct evidence supporting this concept is lacking. Since intravenously administered Cu2+Zn2(+)-type superoxide dismutase (SOD) rapidly disappeared from the circulation with a half-life of 4 min, the enzyme could not be used to test whether superoxide radicals played a critical role in the modulation of vascular permeability. We previously synthesized a SOD derivative (SM-SOD) by linking poly(styrene co-maleic acid butyl ester) (SM) to the enzyme (Ogino, T., Inoue, M., Ando, Y., Awai, M., Maeda, H. and Morino Y. (1988) Int. J. Pept. Protein Res. 32, 1583-1588); SM-SOD circulates bound to albumin with a half-life of 6 h. To test whether superoxide radicals play an important role in the regulation of vascular permeability, the effect of SM-SOD on experimental paw edema was studied in the rat. Subcutaneous injections of carrageenin to the paw rapidly induced local edema by increasing vascular permeability. Intravenous administration of SM-SOD markedly inhibited the carrageenin-induced increase in vascular permeability and suppressed the development of paw edema. In contrast, the same dose of SOD showed no such inhibitory effect. These results suggest that superoxide radical and/or its metabolite(s) might play a critical role in the pathogenesis of carrageenin-induced vasogenic edema.     

Inhibition of ischemia and reflow-induced liver injury by an SOD derivative that circulates bound to albumin

Ischemia followed by reflow often results in tissue injury. Although reactive oxygens seem to play an important role in the pathogenesis of postischemic reflow-induced tissue injury, the mechanism and an efficient way to inhibit oxidative injury are not known. We studied the mechanism by which hepatic transport function was inhibited by a transient occlusion followed by reflow of the portal vein and hepatic artery by using a superoxide dismutase (SOD) derivative (SM-SOD) which circulates bound to albumin with a half-life of 6 h. Occlusion of the hepatic vessels for 20 min followed by reflow for 60 min significantly inhibited transhepatic transport of cholephilic ligands, such as bromosulfophthalein (BSP) and taurocholic acid. Intravenous administration of SM-SOD markedly inhibited the reflow-induced decrease in transhepatic transport of these ligands. Thiobarbituric acid - reactive metabolites (TBAR) in the liver and plasma remained unchanged during occlusion and reflow, while TBAR in the bile increased significantly. Intravenous injection of SM-SOD inhibited the reflow-induced increase in biliary TBAR. Xanthine oxidase activity in plasma also increased during occlusion and reflow by an SM-SOD-inhibitable mechanism. Polymorphonuclear leukocyte-dependent chemiluminescence of the peripheral blood remained unchanged during occlusion, but increased markedly with time after reflow. SM-SOD also inhibited the increase in chemiluminescence almost completely. These and other results suggested that the superoxide radical and/or its metabolite(s) might play an important role in the pathogenesis of the reflow-induced liver injury and that SM-SOD might be useful for studying the mechanism for tissue injury caused by oxygen toxicity.     

Mechanism for enterohepatic injury caused by circulatory disturbance of hepatic vessels in the rat

We reported previously that a transient occlusion followed by reperfusion of the portal vein and the hepatic artery of the rat significantly decreased the transhepatic transport of a cholephilic compound, and that this decrease was prevented by pretreating animals with poly(styrene co-maleic acid butyl ester)-conjugated superoxide dismutase (SM-SOD). To elucidate the mechanism for oxidative injury of the liver and the site for the generation of superoxide radicals, the effect of a portosystemic bypass on the liver function was examined in the rat whose hepatic vessels were temporarily occluded. A portosystemic bypass inhibited the reperfusion-induced decrease in hepatic transport of bromosulfophthalein as effectively as did SM-SOD. Kinetic analysis using 125I-labeled albumin revealed that the permeability of the small intestine markedly increased after a transient occlusion. The increase in intestinal permeability was also inhibited either by SM-SOD or by the portosystemic bypass. Xanthine oxidase activity in portal plasma markedly increased during occlusion and reperfusion, while it remained within normal ranges in the bypassed group. Thus, superoxide radical, and/or its metabolite(s), might play a critical role in increasing the intestinal permeability and in the pathogenesis of reperfusion-induced liver injury.     

Superoxide dismutase-inhibitible reduction of cytochrome c by the alloxan radical. Implications for alloxan cytotoxicity.

Cytochrome c was reduced when superoxide was generated from xanthine oxidase in the presence of alloxan, and by the reaction of alloxan and with reduced glutathione. In each case, most of the reduction was inhibited by superoxide dismutase, but considerably more enzyme was required than with superoxide alone. This indicates that the superoxide dismutase-inhibitible cytochrome c reduction was mainly due to a direct reaction with the alloxan radical, and implies that other reactions that are inhibited by superoxide dismutase could be due to either alloxan radicals or superoxide.     

Chemical modification of superoxide dismutase. Extension of plasma half life of the enzyme through its reversible binding to the circulating albumin

Protection of organisms from oxidative stress is one of the major prerequisites for aerobic life. Since intravenously injected Cu++/Zn++-type superoxide dismutase (SOD) rapidly undergoes renal glomerular filtration and appears in urine in its intact form, its clinical use as a scavenger for superoxide radicals has been highly limited. To test whether reversible interaction of SOD with plasma albumin might decrease the rate of disappearance of the enzyme from the circulation, the lysyl residues of the human erythrocyte-type enzyme were covalently linked with poly-(styrene-co-maleic acid) butyl ester (SMA) via amide linkage. Affinity chromatographic analysis by an albumin-Sepharose column revealed that the enzyme samples labeled with SMA (SMA-SOD) tightly bound to the column, while unmodified SOD was eluted in the unbound fractions. SMA-SOD bound to the column could be eluted by the buffer solution containing 0.1% sodium dodecylsulfate. In vivo analysis revealed that intravenously administered SMA-SOD circulated bound to albumin with an extremely long half-life (6 h), while unmodified SOD rapidly underwent renal glomerular filtration with a plasma half-life of 4 min. Thus, SMA-SOD may effectively dismutase superoxide radicals in the circulation.     

Methylene blue competes with paraquat for reduction by flavo-enzymes resulting in decreased superoxide production in the presence of heme proteins

Methylene blue competes 100 to 600 times more effectively than paraquat for reduction by three different flavo-containing enzymes; xanthine oxidase, NADH cytochrome c reductase, and NADPH cytochrome c reductase. Paraquat and methylene blue both interact with deflavo xanthine oxidase, indicating that neither electron acceptor reacted at the FAD site of the enzyme where molecular oxygen is reduced to superoxide. As the paraquat radical also directly reduced acetylated cytochrome c the hemeprotein could not be utilized for measuring superoxide production in the presence of the herbicide. In the presence of cytochrome c the methylene blue caused a sharp decrease in both paraquat-induced superoxide and hydroxyl radical production.     

Permeability of bilayer phospholipid membranes to superoxide oxygen radicals

Lecithin monolayer liposomes (1000 A in diameter) loaded with cytochrome c were placed into the external solution, in which O2 superoxide radicals were regenerated by the xanthine-xanthine oxidase system. The penetration of superoxide radicals across the liposomal membranes was followed by cytochrome c reduction in the interval volume of the liposomes. The effects of lipid membrane modifiers and temperature on this process were investigated. The results obtained were used for calculation of the permeability coefficients of bilayer lipid membranes for O(2) (P'O(2) = (7.6 +/- 0.3) . 10(-8) cm . s-1) or HO . 2(P'HO(2) = 4.9 x 10(-4) cm . s-1). The effect of the transmembrane electric potential (concentration gradient of H+, valinomycin) on the permeability of liposomal membranes for the superoxide radical was studied. The superoxide radical was down to penetrate across the bilayer lipid membranes in an unloaded state. Using an intramolecular cholesterol-amphotericin B-complex, the superoxide radicals were shown to penetrate across the bilayer lipid membranes, predominantly via the anionic channels.     

Inhibition of posthemorrhagic transfusion-induced gastric injury by a long-acting superoxide dismutase derivative

Although oxygen-free radicals have been postulated to play an important role in the pathogenesis of gastric mucosal injury induced by posthemorrhagic blood transfusion, direct evidence supporting this hypothesis is lacking. Superoxide dismutase (SOD) has been shown to inhibit oxygen toxicity in vitro in various types of cell injury. However, in some cases, oxidative tissue injury cannot be decreased efficiency predominantly due to its rapid elimination by renal glomerular filtration. To overcome such frustrating situations, we have synthesized a SOD derivative that circulates bound to albumin with a half-life of 6 hr. When blood was withdrawn from the rat (22 ml/kg) for 30 min followed by transfusion of the extracted blood, marked gastric mucosal lesions occurred within 30 min after transfusion. Intravenously injected SOD derivative markedly decreased gastric mucosal injury. Kinetic analysis using 125I-labeled albumin revealed that the vascular permeability of the stomach increased significantly after transfusion by a SOD derivative inhibitable mechanism. Thus, superoxide radical and/or its metabolite(s) play a critical role in the pathogenesis of posthemorrhagic transfusion-induced gastric injury.     

Cytoprotective function of nitric oxide: inactivation of superoxide radicals produced by human leukocytes.

The oxygen-derived free radical superoxide anion (.O2-) plays an important role in the pathogenesis of various diseases. Recent demonstrations that .O2- inactivates the potent vasodilator endothelium-derived relaxing factor (EDRF) and that EDRF is probably nitric oxide (NO) suggest that EDRF(NO) may act as an endogenous free radical scavenger. This hypothesis was tested in an in vitro system by analyzing the effect of authentic NO (dilutions of a saturated aqueous solution) on .O2- production (detected spectrophotometrically as reduction of cytochrome c) by fMet-Leu-Phe-activated human leukocytes (PMN). NO depressed the rate of reduction of cytochrome c by .O2- released from PMN's or generated from the oxidation of hypoxanthine by xanthine oxidase. This effect was concentration-dependent and occurred at dilutions of the saturated NO solution (1:250 to 1:10) which inhibited platelet aggregation. NO had no direct effect on cytochrome c or on xanthine oxidase. These observations indicate that NO(EDRF) can be regarded as a scavenger of superoxide anion and they suggest that EDRF(NO) may provide a chemical barrier to cytotoxic free radicals (.O2-).     

Characterization of the structure and reactions of free radicals from serotonin and related indoles

The ESR spectra of the free radicals formed by the autoxidation of serotonin, 5-hydroxyindole, and 5-hydroxytryptophan in 1 N NaOH are presented. The analysis of the hyperfine splitting constants in H2O and D2O characterize these free radicals as semiquinone-imines, the one-electron oxidation product of the corresponding indole. At alkaline pH, autoxidation of these compounds ultimately leads to solid precipitate and unresolved ESR spectra characteristic of polymeric material. The reduction of cytochrome c at pH 7.4 by a wide variety of indoles correlates with the amplitude of the ESR signal in 1 N NaOH, as do other processes thought to be related to 5-hydroxyindole free radical formation. Relative to the rate of cytochrome c reduction, neither serotonin nor the serotonin free radical appears to react with oxygen to form superoxide. In the presence of NAD(P)H, the serotonin radical most probably oxidizes NAD(P)H to form the NAD(P). radical. The NAD(P). radical then reacts with oxygen to form superoxide, which ultimately reduces cytochrome c.     

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.     

Generation and recycling of radicals from phenolic antioxidants

Hindered phenols are widely used food preservatives. Their pharmacological properties are usually attributed to high antioxidant activity due to efficient scavenging of free radicals. Butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) also cause tissue damage. Their toxic effects could be due to the production of phenoxyl radicals. If phenoxyl radicals can be recycled by reductants or electron transport, their potentially harmful side reactions would be minimized. A simple and convenient method to follow phenoxyl radical reactions in liposomes and rat liver microsomes based on an enzymatic (lipoxygenase + linolenic acid) oxidation system was used to generate phenoxyl radicals from BHT and its homologues with substitutents in m- and p-positions. Different BHT-homologues display characteristic ESR signals of their radical species. In a few instances the absence of phenoxyl radical ESR signals was found to be due to inhibition of lipoxygenase by BHT-homologues. In liposome or microsome suspensions addition of ascorbyl palmitate resulted in disappearance of the ESR signal of phenoxyl radicals with concomittant appearance of the ascorbyl radical signal. After exhaustion of ascorbate, the phenoxyl radical signal reappears. Comparison of the rates of ascorbyl radical decay in the presence or absence of BHT-homologues showed that temporary elimination of the phenoxyl radical ESR signal was due to their reduction by ascorbate. Similarly, NADPH or NADH caused temporary elimination of ESR signals as a result of reduction of phenoxyl radicals in microsomes. Since ascorbate and NADPH might generate superoxide in the incubation system used, SOD was tested. SOD shortened the period, during which the phenoxyl radicals ESR signal could not be observed. Both ascorbyl palmitate and NADPH exerted sparing effects on the loss of BHT-homologues during oxidation. These effects were partly diminished by SOD. These data indicate that reduction of phenoxyl radicals was partly superoxide-dependent. It is concluded that redox recycling of phenoxyl radicals can occur by intracellular reductants like ascorbate and microsomal electron transport.     

Binding of arylazidocytochrome c derivatives to beef heart cytochrome c oxidase: cross-linking in the high- and low-affinity binding sites

Two arylazidocytochrome c derivatives, one modified at lysine-13 and the second modified at lysine-22, were reacted with beef heart cytochrome c oxidase. The lysine-13 modified arylazidocytochrome c was found to cross-link both to the enzyme and with lipid bound to the cytochrome c oxidase complex. The lysine-22 derivative reacted only with lipids. Cross-linking to protein was through subunit II of the cytochrome c oxidase complex, as first reported by Bisson et al. [Bisson, R., Azzi, A., Gutweniger, H., Colonna, R., Monteccuco, C., & Zanotti, A. (1978) J. Biol. Chem. 253, 1874]. Binding studies show that the cytochrome c derivative covalently bound to subunit II was in the high-affinity binding site for the substrate. Evidence is also presented to suggest that cytochrome c bound to the lipid was in the low-affinity binding site [as defined by Ferguson-Miller et al. [Ferguson-Miller, S., Brautigan, D. L., & Margoliash, E. (1976) J. Biol. Chem. 251, 1104]]. Covalent binding of the cytochrome c derivative into the high-affinity binding site was found to inhibit electron transfer even when native cytochrome c was added as a substrate. Inhibition was almost complete when 1 mol of the Lys-13 modified arylazidocytochrome c was covalently bound to the enzyme per cytochrome c oxidase dimer (i.e., congruent to 280 000 daltons). Covalent binding of either derivative with lipid (low-affinity site) had very little effect on the overall electron transfer activity of cytochrome c oxidase. These results are discussed in terms of current theories of cytochrome c-cytochrome c oxidase interactions.     

Measurement of free radical oxygen generation by cytochrome c reduction requirement for cytochrome c oxidase blockade

Data is presented showing that one commercial preparation of cytochrome c, used to trap and measure free radical superoxide anion, can be contaminated with cytochrome c oxidase activity. This activity can vary from lot to lot, can introduce variability into the measurement of superoxide anion and can result in falsely low estimations of free radical formation. This cytochrome c oxidase activity can be inhibited by low (0.2 mM) concentrations of KCN. Blockade of the cytochrome c oxidase activity allows reproducible measurement of superoxide anion formation at low levels by red cells.     

Reductase and oxidase activity of rat liver cytochrome P450 with 2,3,5,6-tetramethylbenzoquinone as substrate.

The main objective of the present study was to investigate the proposed role of cytochrome P450 in the reductive metabolism of quinones as well as in the formation of reduced oxygen species in liver microsomes from phenobarbital (PB-microsomes) and beta-naphthoflavone (beta NF-microsomes) pretreated rats. In the present study, 2,3,5,6-tetramethylbenzoquinone (TMQ) was chosen as a model quinone. Anaerobic one-electron reduction of TMQ by PB-microsomes showed relatively strong electron spin resonance (ESR) signals of the oxygen-centered semiquinone free radical (TMSQ), whereas these signals were hardly detectable with beta NF-microsomes. Under aerobic conditions TMSQ formation was diminished and concomitant reduction of molecular oxygen occurred in PB-microsomes. Interestingly, TMQ-induced superoxide anion radicals, measured by ESR (using the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide), and hydrogen peroxide generation was found to occur with beta NF-microsomes as well. Furthermore, SK&F 525-A (a type I ligand inhibitor of cytochrome P450) inhibited TMQ-induced hydrogen peroxide formation in both PB- and beta NF-microsomes. However, metyrapone and imidazole (type II ligand inhibitors of cytochrome P450) inhibited molecular oxygen reduction in beta NF-microsomes and not in PB-microsomes. The present study indicates that cytochrome P450-mediated one-electron reduction of TMQ to TMSQ and subsequent redox cycling of TMSQ with molecular oxygen constitutes the major source for superoxide anion radical and hydrogen peroxide generation in PB-microsomes (i.e. from the reductase activity of cytochrome P450). However, most of the superoxide anion radical formed upon aerobic incubation of TMQ with beta NF-microsomes originates directly from the dioxyanion-ferri-cytochrome P450 complex (i.e. from the oxidase activity of cytochrome P450). In conclusion, both the one-electron reduction of TMQ and molecular oxygen were found to be cytochrome P450 dependent. Apparently, both the reductase and oxidase activities of cytochrome P450 may be involved in the reductive cytotoxicity of chemotherapeutic agents containing the quinoid moiety.     

Relation of the structure and function of ferricytochrome c bound to the phosphoprotein phosvitin

The relationship between the structure and function of ferricytochrome c bound to the phosphoprotein phosvitin was investigated. The rates of reduction of phosvitin-bound ferricytochrome c by cytochrome b2, ascorbate and the superoxide radical generated by xanthine oxidase wer repressed where the binding ratio was less than half the maximum, but at higher ratios they were restored gradually with increase in the ratio. The affinity of cytochrome b2 for cytochrome c was not affected by binding of cytochrome c to phosvitin. The redox potential of the bond form was lower than that of the free form and only decreased with decrease in the ratio. The conformatin around the heme moiety and the electronic structure of the heme group of bound ferricytochrome c were similar to those of free ferricytochrome c, but the conformational stability in the vicinity of the prosthetic group was related to the binding ratio as ratios above half the maximum and was well correlated with the reduction rate. Since the binding of cytochrome c to phosvitin is much stronger at binding ratios below half the maximum, these results suggest that this binding strength exclusively affects the conformational flexibility of the heme crevice in the cytochrome molecule, thus altering the reduction rate.     

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.     

Reductive activation of potential antitumor bis(aziridinyl)benzoquinones by xanthine oxidase: competition between oxygen reduction and quinone reduction

The reduction of a series of 2,5-bis(1-aziridinyl)-1,4-benzoquinone (BABQ) derivatives with various 3,6 substituents by the enzyme xanthine oxidase has been studied. The reduction rate has been assayed by measuring the rate of reduction of cytochrome c, which is very efficiently reduced by reduced BABQ species. Under nitrogen, the reduction rate correlated with the quinone reduction potential and steric parameters. Comparing reduction rates under nitrogen and air demonstrates that at BABQ concentrations greater than 25 microM the competition for electrons from xanthine oxidase between oxygen and the BABQ derivative is dominated by the latter. This is also confirmed by the effect of superoxide dismutase (SOD): in the presence of a BABQ derivative, cytochrome c reduction can be totally inhibited by SOD, although the required amount of SOD depends on the redox potential of the quinones. This indicates that SOD causes the equilibrium between semiquinone and superoxide to shift, resulting in a decrease of the semiquinone concentration. It is concluded that reduction by xanthine oxidase is a simple and effective method for reducing aziridinylbenzoquinones.