Peroxynitrite formation from the simultaneous reduction of nitrite and oxygen by xanthine oxidase

One electron reductions of oxygen and nitrite by xanthine oxidase form peroxynitrite. The nitrite and oxygen reducing activities of xanthine oxidase are regulated by oxygen with K(oxygen) 26 and 100 microM and K(nitrite) 1.0 and 1.1 mM with xanthine and NADH as donor substrates. Optimal peroxynitrite formation occurs at 70 microM oxygen with purine substrates. Kinetic parameters: V(max) approximately 50 nmol/min/mg and K(m) of 22, 36 and 70 microM for hypoxanthine, pterin and nitrite respectively. Peroxynitrite generation is inhibited by allopurinol, superoxide dismutase and diphenylene iodonium. A role for this enzyme activity can be found in the antibacterial activity of milk and circulating xanthine oxidase activity.     

Substrate kinetics of the Acanthamoeba castellanii alternative oxidase and the effects of GMP

In Acanthamoeba castellanii mitochondria, the apparent affinity values of alternative oxidase for oxygen were much lower than those for cytochrome c oxidase. For unstimulated alternative oxidase, the K(Mox) values were around 4-5 microM both in mitochondria oxidizing 1 mM external NADH or 10 mM succinate. For alternative oxidase fully stimulated by 1 mM GMP, the KK(Mox) values were markedly different when compared to those in the absence of GMP and they varied when different respiratory substrates were oxidized (K(Mox) was around 1.2 microM for succinate and around 11 microM for NADH). Thus, with succinate as a reducing substrate, the activation of alternative oxidase (with GMP) resulted in the oxidation of the ubiquinone pool, and a corresponding decrease in K(Mox). However, when external NADH was oxidized, the ubiquinone pool was further reduced (albeit slightly) with alternative oxidase activation, and the K(Mox) increased dramatically. Thus, the apparent affinity of alternative oxidase for oxygen decreased when the ubiquinone reduction level increased either by changing the activator or the respiratory substrate availability.     

Mitochondrial respiration at low levels of oxygen and cytochrome c

In the intracellular microenvironment of active muscle tissue, high rates of respiration are maintained at near-limiting oxygen concentrations. The respiration of isolated heart mitochondria is a hyperbolic function of oxygen concentration and half-maximal rates were obtained at 0.4 and 0.7 microM O(2) with substrates for the respiratory chain (succinate) and cytochrome c oxidase [N,N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD)+ascorbate] respectively at 30 degrees C and with maximum ADP stimulation (State 3). The respiratory response of cytochrome c-depleted mitoplasts to external cytochrome c was biphasic with TMPD, but showed a monophasic hyperbolic function with succinate. Half-maximal stimulation of respiration was obtained at 0.4 microM cytochrome c, which was nearly identical to the high-affinity K(')(m) for cytochrome c of cytochrome c oxidase supplied with TMPD. The capacity of cytochrome c oxidase in the presence of TMPD was 2-fold higher than the capacity of the respiratory chain with succinate, measured at environmental normoxic levels. This apparent excess capacity, however, is significantly decreased under physiological intracellular oxygen conditions and declines steeply under hypoxic conditions. Similarly, the excess capacity of cytochrome c oxidase declines with progressive cytochrome c depletion. The flux control coefficient of cytochrome c oxidase, therefore, increases as a function of substrate limitation of oxygen and cytochrome c, which suggests a direct functional role for the apparent excess capacity of cytochrome c oxidase in hypoxia and under conditions of intracellular accumulation of cytochrome c after its release from mitochondria.     

Activation of NADPH oxidase in human neutrophils permeabilized with Staphylococcus aureus alpha-toxin. A lower Km when the enzyme is activated in situ.

The NADPH oxidase is a multicomponent enzyme system that produces the reduced oxygen species essential for bacterial killing by polymorphonuclear leukocytes (PMN). Study of the oxidase has typically been carried out in cell-free systems in which Km values of 20-150 microM NADPH have been reported. However, when compared with affinities reported for other flavoprotein dehydrogenases and when considering the cellular concentration of NADPH/NADP+ of approximately 35 microM, the reported affinity of the oxidase for NADPH appears low. To investigate this apparent discrepancy we have studied the kinetics of NADPH oxidase activation in situ in human PMN permeabilized with Staphylococcus aureus alpha-toxin. alpha-Toxin permeabilization of human PMN did not initiate NADPH oxidase activation at physiologic concentrations of NADPH. If permeabilized cells were stimulated with 1 microM formyl-methionyl-leucyl-phenylalanine, 10 microM guanosine 5'-O-(3-thiotriphosphate), 0.5 mM Ca2+, 5 micrograms/ml cytochalasin B in the presence of varying concentrations of NADPH, we were able to demonstrate activation of the oxidase complex as shown by superoxide dismutase-inhibitable reduction of cytochrome c. In this system we determined that the Km for oxidase activation was 4-7 microM NADPH, a 4-10-fold decrease from reported values. The oxidase was the enzyme being studied as shown by the absence of enzymatic activity in patients with chronic granulomatous disease. In addition, if the enzyme was initially activated in permeabilized cells, the cells homogenized, and the Km for the oxidase determined in a cell-free system, the observed Km reverted to previously reported values (36 microM). These results indicate that NADPH oxidase, studied in situ, has a significantly higher substrate affinity than that observed in isolated membranes and, moreover, indicate that substrate affinity is optimal for catalysis at reported concentrations of cytosolic NADPH.     

Lack of oxygen sensing by mitochondria in platelets.

The range over which cells are sensitive to changes in oxygen concentration remains uncertain. Wilson and colleagues [Wilson, D.F. (1994) Med. Sci. Sports Exerc. 26, 37-43] have suggested that cytochrome oxidase is sensitive to oxygen concentrations below about 40 microM, but proposed that this sensitivity is obscured in intact cells because an increase in reduction state of cytochrome c acts to maintain oxygen consumption. We have tested this hypothesis in platelets, which are small cells (2-4 micrometer diameter, < 0.5 micrometer thick) that do not decrease their rate of oxygen consumption until oxygen concentrations fall below 2.5 microM. Contrary to the expectations of the hypothesis, the reduction state of cytochrome c, the concentration of NADH and the rate of glycolytic output are not changed as oxygen concentration declines from 40 microM down to 5 microM. Therefore, we conclude that at least some cell types contain mitochondria that are not capable of sensing oxygen above 5 microM by the mechanism proposed by Wilson and colleagues.     

Amperometric glucose sensor based on catalytic reduction of dissolved oxygen at soluble carbon nanofiber

This work shows excellent catalytic activity of soluble carbon nanofiber (CNF), which was obtained with a simple nitric acid treatment, toward the electroreduction of dissolved oxygen at a low operating potential. Thus the CNF was applied in the construction of amperometric biosensors for oxidase substrates using glucose oxidase as a model. The good dispersion of CNF led to convenient preparation and acceptable repeatability of the proposed sensors. UV-vis spectra, Fourier transform infrared spectra, X-ray photoelectron spectra and titration curves demonstrated that the good dispersion resulted from the large numbers of surface oxygen-rich groups produced in the treatment process. The membrane of CNF showed good stability and provided fast response to dissolved oxygen with a linear range from 0.1 to 78 microM and detection limit of 0.07 microM. The proposed glucose biosensor could monitor glucose ranging from 10 to 350 microM with detection limit of 2.5 microM and sensitivity of 36.3 nA cm(-2) microM(-1). The coefficients of variation for intra-assay were 4.7 and 3.2% at glucose concentrations of 20 and 210 microM, respectively. The use of a low operating potential (-0.3 V) and Nafion membrane produced good selectivity toward the glucose detection. CNF-based biosensors would provide wide range of bioelectrochemical applications in different fields.     

Allopurinol-insensitive oxygen radical formation by milk xanthine oxidase systems.

Oxygen radical generation in the xanthine- and NADH-oxygen reductase reactions by xanthine oxidase, was demonstrated using the ESR spin trap 5,5'-dimethyl-1- pyrroline-N-oxide. No xanthine-dependent oxygen radical formation was observed when allopurinol-treated xanthine oxidase was used. The significant superoxide generation in the NADH-oxygen reductase reaction by the enzyme was increased by the addition of menadione and adriamycin. The NADH-menadione and -adriamycin reductase activities of xanthine oxidase were assessed in terms of NADH oxidation. From Lineweaver-Burk plots, the Km and Vmax of xanthine oxidase were estimated to be respectively 51 microM and 5.5 s-1 for menadione and 12 microM and 0.4 s-1 for adriamycin. Allopurinol-inactivated xanthine oxidase generates superoxide and OH.radicals in the presence of NADH and menadione or adriamycin to the same extent as the native enzyme. Adriamycin radicals were observed when the reactions were carried out under an atmosphere of argon. The effects of superoxide dismutase and catalase revealed that OH.radicals were mainly generated through the direct reaction of H2O2 with semiquinoid forms of menadione and adriamycin.     

Mitochondria and xanthine oxidase both generate reactive oxygen species in isolated perfused rat liver after hypoxic injury

Hypoxia caused severe damage in isolated perfused livers from fasted male Fischer rats without evidence of the formation of reactive oxygen species during hypoxia. Reoxygenation caused a significant increase in intracellular oxygen species in the injured liver, as indicated by increases in sinusoidal GSSG efflux and tissue GSSG levels. Both parameters were elevated further by addition of KCN (100 microM) or antimycin A (8 microM). Sinusoidal GSSG efflux was suppressed in part by addition of allopurinol (500 microM) and enhanced by hypoxanthine (250 microM). Xanthine oxidase appears to be a partial source, and damaged mitochondria a continuous and quantitatively greater source, of reactive oxygen as a result of liver injury following hypoxia.     

Effects of oxygen on kynurenine-3-monooxygenase activity

Kynurenine-3-monooxygenase (KM), the third enzyme in the kynurenine (KYN) pathway from tryptophan to quinolinic acid (QA), is a monooxygenase requiring oxygen, NADPH and FAD for the catalytic oxidation of L-kynurenine to 3-hydroxykynurenine and water. KM is innately low in the brain and similar in activity to indoleamine oxidase, the rate-limiting pathway enzyme. Accumulation in the CNS of QA, a known excitotoxin, is proposed to cause convulsions in several pathologies. Thus, we theorized that hyperbaric oxygen (HBO) induced convulsions arise from increased QA via oxygen K, effects on this pathway [Brown OR, Draczynska-Lusiak. Oxygen activation and inactivation of quinolinate-producing and iron-requiring 3-hydroxyanthranilic acid oxidase: a role in hyperbaric oxygen-induced convulsions? Redox Report 1995; 1: 383-385]. To complement prior studies on the effects of oxygen on pathway enzymes, in this paper we report the effects of oxygen on KM. Brain and liver KM enzyme are not known to be identical, and some systemically-produced KYN pathway intermediates can permeate the brain and might stimulate the brain pathway. Thus, KM from both brain and liver was assayed at various oxygen substrate concentrations to evaluate, in vitro, the potential effects of increases in oxygen, as would occur in mammals breathing therapeutic and convulsive HBO. In crude tissue extracts, KM was not activated during incubation in HBO up to 6 atm. The effects of oxygen as substrate on brain and liver KM activity was nearly identical: activity was nil at zero oxygen with an apparent oxygen Km of 20-22 microM. Maximum KM activity occurred at about 1000 microM oxygen and decreased slightly to plateau from 2000 to 8000 microM oxygen. This compares to approximately 30-40 microM oxygen typically reported for brain tissue of humans or rats breathing air, and an unknown but surely much lower value (perhaps below 1 microM) intracellularly at the site of KM. Thus HBO, as used therapeutically and at convulsive pressures, likely stimulates flux through the KM-catalyzed step of the KYN pathway in liver and in brain and could increase brain QA, by Km effects on brain KM, or via increased KM pathway intermediates produced systemically (in liver) and transported into the brain.     

The reaction of the electrostatic cytochrome c-cytochrome oxidase complex with oxygen.

The reaction of the electrostatic cytochrome c-cytochrome oxidase complex with oxygen is measured by transient absorption spectroscopy. The oxygen reaction is initiated by photolytic removal of CO from cytochrome oxidase, using a flash-pumped dye laser. The subsequent reaction of the cytochrome c-cytochrome oxidase complex with oxygen is reported at 550, 605, 744, and 830 nm at different cytochrome c:cytochrome oxidase ratios and different oxygen concentrations. In the absence of cytochrome c the time course of the reaction of the oxidase is well described by a triple exponential process at any of the measured wavelengths. The three processes are well resolved at high O2 levels (i.e. greater than 200 microM), where they reach first-order rate limits of 2.4 x 10(4), 7.5 x 10(3), and 650 s-1. When cytochrome c is added the oxidation of cytochrome a and one of the redox active cooper centers (CuA) are interrupted. The maximal effect of cytochrome c on the oxidation of the oxidase occurs at a c:aa3 ratio of 1. Cytochrome c reacts in a biphasic process with rates of up to 7 x 10(3) and 550 s-1 at high oxygen. The fast phase takes up 60% of the process, and this is independent of the cytochrome c:cytochrome oxidase ratio. The results are discussed in the context of a model in which electron entry into cytochrome oxidase from cytochrome c is via CuA, and cytochrome a functions to mediate electron transfer from CuA to the oxygen binding site. The role of CuA as initial electron acceptor in cytochrome c oxidase is related to its physical proximity to cytochrome c is the cytochrome c-cytochrome oxidase complex.     

Effects of reactive oxygen species on prostacyclin production in perinatal rat lung cells

A differentiation-arrested primary cell culture model was used to examine the role of reactive oxygen species in the control of prostacyclin (PGI2) production in the perinatal rat lung. Coincubation of the lung cells with arachidonic acid (AA) and xanthine (X, 0.25 mM) plus xanthine oxidase (XO, 10 mU/ml) or with AA and glucose (25 mM) plus glucose oxidase (25 mU/ml) augmented the AA-induced PGI2 output. Superoxide dismutase (10 U/ml) did not alter the X + XO effect, whereas catalase (10 U/ml) eliminated both X + XO and glucose plus glucose oxidase effects. H2O2 (1-200 microM) showed a dose-related biphasic augmentation with peak stimulation at 20 microM. Catalase again blocked this effect, but dimethylthiourea, a hydroxyl radical scavenger, did not. A 20-min pretreatment of the cells with X + XO, glucose plus glucose oxidase, or H2O2, however, diminished the capacity of the cells to convert exogenous AA to PGI2. This pretreatment effect was also blocked by catalase. The responses were similar in lung cells obtained from day 20 rat fetuses (term = 22 days) and 1-day-old newborn rats. Lactate dehydrogenase release was not detected during treatment periods but increased significantly after exposure to reactive oxygen species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of oxygen on the metabolism of nitroxide spin labels in cells

The products of the reduction of nitroxides in cells are the corresponding hydroxylamines, which cells can oxidize back to the nitroxides in the presence of oxygen. Both the reduction of nitroxides and the oxidation of hydroxylamines are enzyme-mediated processes. For lipid-soluble nitroxides, the rates of reduction are strongly dependent on the intracellular concentration of oxygen; severely hypoxic cells reduce nitroxides more rapidly than cells supplied with oxygen. In contrast, the rates of oxidation of hydroxylamines increase smoothly with increasing intracellular oxygen concentration up to 150 microM. In order to separate the effects on the rates of metabolism of nitroxides due directly to oxygen from effects due to the redox state of enzymes, we studied the cells under conditions in which each of these variables could be changed independently. Oxygen affects the metabolism of these nitroxides primarily by interacting with cytochrome c oxidase to change the redox state of the enzymes in the respiratory chain. Our results are consistent with the conclusions that in these cells reduction of lipophilic nitroxides occurs at the level of ubiquinone in the respiratory chain in mitochondria, and oxidation of the corresponding hydroxylamines occurs at the level of cytochrome c oxidase.     

Some properties of monoamine oxidase in carp heart

Studies using clorgyline, deprenyl and semicarbazide as inhibitors showed that carp heart homogenate contained a new type of monoamine oxidase (MAO) and a clorgyline- and deprenyl-resistant amine oxidase (CRAO). The deamination of monoamines by carp heart MAO proceeded in two steps by a double-displacement (ping-pong) mechanism. The Km values of the MAO for oxygen (K0 values) with tyramine, 5-hydroxytryptamine (5-HT) and beta-phenylethylamine (PEA) as substrates were identical (59 microM).     

The effect of inhibitors on the oxygen kinetics of terminal oxidases of Acanthamoeba castellanii.

1. Respiration of growing cultures of Acanthamoeba castellanii is inhibited less than 60% by azide (35 mM); the respiration of early-exponential-phase cultures differs from that of late-exponential-phase cultures in being stimulated by up to 120% by low concentrations (less than 1 mM) of this inhibitor. Azide (0.5 mM) plus 1 mM-salicylhydroxamic acid gives 80% inhibition of respiration in early- or late-exponential-phase cultures. 2. Lineweaver-Burk plots of 1/v against 1/[O2] for growing and stationary-phase cultures give values of less than 1 muM for the apparent Km for oxygen. 3. These values are not significantly altered when determined in the presence of 1 mM-salicylhydroxamic acid. 4. Higher values (greater than 7 muM) for apparent Km values for oxygen were obtained in the presence of azide, which gives non-linear Lineweaver-Burk plots. 5. Competitive inhibition of respiration by CO occurs with Ki 2.4 muM. 6. The results are discussed in terms of the presence of three terminal oxidases in this organism, namely two oxidases with high affinities for oxygen (cytochrome c oxidase of the main phosphorylating electron-transport chain and the salicylhydroxamic acid-sensitive oxidase) and a third oxidase with a low affinity for oxygen, sensitive to inhibition by cyanide but not by azide or salicylhydroxamic acid. The relative contributions to oxygen utilization by these oxidases change during the growth of a batch culture.     

Hypoxic reperfusion of the ischemic heart and oxygen radical generation

Postischemic myocardial contractile dysfunction is in part mediated by the burst of reactive oxygen species (ROS), which occurs with the reintroduction of oxygen. We hypothesized that tissue oxygen tension modulates this ROS burst at reperfusion. After 20 min of global ischemia, isolated rat hearts were reperfused with temperature-controlled (37.4 degrees C) Krebs-Henseleit buffer saturated with one of three different O2 concentrations (95, 20, or 2%) for the first 5 min of reperfusion and then changed to 95% O2. Additional hearts were loaded with 1) allopurinol (1 mM), a xanthine oxidase inhibitor, 2) diphenyleneiodonium (DPI; 1 microM), an NAD(P)H oxidase inhibitor, or 3) Tiron (10 mM), a superoxide scavenger, and were then reperfused with either 95 or 2% O2 for the first 5 min. ROS production and tissue oxygen tension were quantitated using electron paramagnetic resonance spectroscopy. Tissue oxygen tension was significantly higher in the 95% O2 group. However, the largest radical burst occurred in the 2% O2 reperfusion group (P < 0.001). Recovery of left ventricular (LV) contractile function and aconitase activity during reperfusion were inversely related to the burst of radical production and were significantly higher in hearts initially reperfused with 95% O2 (P < 0.001). Allopurinol, DPI, and Tiron reduced the burst of radical formation in the 2% O2 reperfusion groups (P < 0.05). Hypoxic reperfusion generates an increased ROS burst originating from multiple pathways. Recovery of LV function during reperfusion is inversely related to this oxygen radical burst, highlighting the importance of myocardial oxygen tension during initial reperfusion.     

Differential requirements of calcium for oxoglutarate dehydrogenase and mitochondrial nitric-oxide synthase under hypoxia: impact on the regulation of mitochondrial oxygen consumption

Studies with isolated mitochondria are performed at artificially high pO(2) (220 to 250 microM oxygen), although this condition is hyperoxic for these organelles. It was the aim of this study to evaluate the effect of hypoxia (20-30 microM) on the calcium-dependent activation of 2-oxoglutarate dehydrogenase (or 2-ketoglutarate dehydrogenase; OGDH) and mitochondrial nitric-oxide synthase (mtNOS). Mitochondria had a P/O value 15% higher in hypoxia than that in normoxia, indicating that oxidative phosphorylation and electron transfer were more efficiently coupled, whereas the intramitochondrial free calcium concentrations were higher (2-3-fold) at lower pO(2). These increases were abrogated by ruthenium red indicating that the higher uptake via the calcium uniporter was involved in this process. Mitochondria at high calcium concentration microdomains may produce nitric oxide, given the K(0.5) of calcium for OGDH (0.16 microM) and mtNOS (approximately 1 microM). Nitric oxide, by binding to cytochrome oxidase in competition with oxygen, decreases the rate of oxygen consumption. This condition is highly beneficial for the following reasons: i, these mitochondria are still able to produce ATP and support calcium clearance; ii, it prevents the accumulation of ROS by slowing the rate of oxygen consumption (hence ROS production); iii, the onset of anoxia is delayed, allowing oxygen to diffuse back to these sites, thereby ameliorating the oxygen gradient between regions of high and low calcium concentration. In this way, oxygen depletion at the latter sites is prevented. This, in turn, assures continued aerobic metabolism which may involve the activated dehydrogenases.     

Decavanadate induces mitochondrial membrane depolarization and inhibits oxygen consumption

Decavanadate induced rat liver mitochondrial depolarization at very low concentrations, half-depolarization with 39 nM decavanadate, while it was needed a 130-fold higher concentration of monomeric vanadate (5 microM) to induce the same effect. Decavanadate also inhibits mitochondrial repolarization induced by reduced glutathione in vitro, with an inhibition constant of 1 microM, whereas no effect was observed up to 100 microM of monomeric vanadate. The oxygen consumption by mitochondria is also inhibited by lower decavanadate than monomeric vanadate concentrations, i.e. 50% inhibition is attained with 99 M decavanadate and 10 microM monomeric vanadate. Thus, decavanadate is stronger as mitochondrial depolarization agent than as inhibitor of mitochondrial oxygen consumption. Up to 5 microM, decavanadate does not alter mitochondrial NADH levels nor inhibit neither F(O)F(1)-ATPase nor cytochrome c oxidase activity, but it induces changes in the redox steady-state of mitochondrial b-type cytochromes (complex III). NMR spectra showed that decameric vanadate is the predominant vanadate species in decavanadate solutions. It is concluded that decavanadate is much more potent mitochondrial depolarization agent and a more potent inhibitor of mitochondrial oxygen consumption than monomeric vanadate, pointing out the importance to take into account the contribution of higher oligomeric species of vanadium for the biological effects of vanadate solutions.     

Effects of platinum and palladium ions on the production and reactivity of neutrophil-derived reactive oxygen species

This study was designed to investigate the effects of platinum, as hydrogen hexachloroplatinate (Pt; 0.0025-25 microM), on the production of reactive oxygen species (ROS) by human neutrophils in vitro. ROS were measured by lucigenin-enhanced chemiluminescence (LECL). Addition of Pt to neutrophils was accompanied by a lag phase of about 1 min, followed by a linear dose-related increase in LECL, which peaked at around 4 min and achieved statistical significance at concentrations of 0.025 microM Pt and higher. Interestingly, Pt-mediated enhancement of LECL was not associated with meaningful alterations in neutrophil oxygen consumption, assembly of NADPH oxidase, or cytosolic Ca2+ and was completely attenuated by superoxide dismutase and inhibitors of NADPH oxidase, but not by catalase or scavengers of hydroxyl radical, and was undetectable with cells from individuals with chronic granulomatous disease. Exposure of alpha1-proteinase inhibitor to Pt-treated neutrophils resulted in inactivation of elastase-inhibitory capacity, underscoring the potential toxicity of neutrophil/Pt interactions. The pro-oxidative actions of Pt were mimicked by palladium (Pd), but not by cisplatin or rhodium. These observations demonstrate that Pt and Pd potentiate the reactivity, as opposed to the generation of neutrophil-derived oxidants, an activity that may contribute to airway inflammation in occupationally and possibly environmentally exposed individuals.     

Urate oxidase electrode based on dissolved oxygen probe for urine uric acid determination

A biosensor for the specific determination of uric acid in urine was developed using urate oxidase (EC 1.7.3.3) in combination with a dissolved oxygen probe. Urate oxidase was immobilized with gelatin by means of glutaraldehyde and fixed on a pretreated teflon membrane to serve as enzyme electrode. The electrode response was maximum when 50 mM glycine buffer was used at pH 9.2 and 35 degrees C. The enzyme electrode response depends linearly on uric acid concentration between 5-40 microM with a response time of 5 min. The enzyme electrode is stable for more than 2 weeks and during this period over 35 assays were performed.     

Application of the theory of oxygen transfer: determination of the Michaelis constant of glucose oxidase with respect to oxygen]

The oxidation of beta-D-glucose with glucose oxidase generally requires oxygen, which, under normal conditions is present at low concentrations in the reaction medium. Experiments show that glucose oxidase is no longer saturated by oxygen at enzyme concentrations greater than 0.4 mg.ml1. This is due to the decrease in the oxygen concentration of the solution. The value of the oxygen mass transfer coefficients and dissolved oxygen concentrations are determined. These dissolved oxygen concentrations are found to correlate with direct measurements with an oxygen electrode. From this, the Michaelis constant of glucose oxidase for oxygen is calculated. These experiments also show that oxygen is a limiting factor for this reaction.