Effects of vanadate on the oxidation of NADH by xanthine oxidase

Vanadate (V(V)) stimulates the oxidation of NADH by xanthine oxidase and superoxide dismutase eliminates the effect of V(V). Paraquat stimulates both the oxidation of NADH by xanthine oxidase and the V(V) enhancement of that oxidation. Xanthine, which is a better substrate for xanthine oxidase than is NADH, causes a V(V)-dependent co-oxidation of NADH which is transient and eliminated by SOD. Urate inhibits the V(V)-stimulated oxidation of NADH by xanthine oxidase or by Rose Bengal plus light. Measurement of rates of both O2- production and V(V)-stimulated NADH oxidation showed that many molecules of NADH were oxidized per O2-. These chain lengths were an inverse function of overall reaction rate. Minimum chain lengths, calculated on the basis of 100% univalent reduction of O2 to O2-, were smaller than measured average chain lengths by a factor of five. All of these results are in accord with the view that V(V) does not directly affect the activity of the enzyme, but rather catalyzes the free radical chain oxidation of NADH by O2-. It was further shown that phosphate was not involved and that the active form of V(V) was orthovanadate, rather than decavanadate.     

Purification and properties of nitroalkane oxidase from Fusarium oxysporum.

A nitroalkane-oxidizing enzyme, which was inducibly formed by addition of nitroethane to the medium was purified to homogeneity from an extract of Fusarium oxysporum (IFO 5942) with an overall yield of about 20%. The enzyme catalyzed the oxidative denitrification of 1-nitropropane as follows: CH2(NO2)CH2CH3 + O2 + H2O leads to OHCCH2CH3 + HNO2 + H2O2. In addition to 1-nitropropane, 3-nitro-2-pentanol, 2-nitropropane, and nitrocyclohexane are good substrates; the enzyme is designated "nitroalkane oxidase" (EC class 1.7.3). The enzyme has a molecular weight of approximately 185,000 and consists of four subunits identical in molecular weight (47,000). Flavin adenine dinucleotide was required for the enzyme activity and could be replaced in part by riboflavin 5'-phosphate. The maximum reactivity was found at about pH 8.0. The enzyme was inhibited significantly by HgCl2, KCN, p-chloromercuribenzoate, and N-ethylmaleimide. The Michaelis constants are as follows: 1-nitropropane, 1.54 mM; 2-nitropropane, 7.40 mM; nitroethane, 1.00 mM; 3-nitro-2-pentanol, 3.08 mM; nitrocyclohexane, 0.90 mM; and flavin adenine dinucleotide, 1.33 micrometer.     

Oxidation of anionic nitroalkanes by flavoenzymes,and participation of superoxide anion in the catalysis

The reactivities of anionic nitroalkanes with 2-nitropropane dioxygenase of Hansenula mrakii, glucose oxidase of Aspergillus niger, and mammalian d-amino acid oxidase have been compared kinetically. 2-Nitropropane dioxygenase is 1200 and 4800 times more active with anionic 2-nitropropane than d-amino acid oxidase and glucose oxidase, respectively. The apparent K_m values for anionic 2-nitropropane are as follows: 2-nitropropane dioxygenase, 1.61 mm; glucose oxidase, 16.7 mm; and d-amino acid oxidase, 11.1 mm. Anionic 2-nitropropane undergoes an oxygenase reaction with 2-nitropropane dioxygenase and glucose oxidase, and an oxidase reaction with d-amino acid oxidase. In contrast, anionic nitroethane is oxidized through an oxygenase reaction by 2-nitropropane dioxygenase, and through an oxidase reaction by glucose oxidase. All nitroalkane oxidations by these three flavoenzymes are inhibited by Cu and Zn-superoxide dismutase of bovine blood, Mn-superoxide dismutases of bacilli, Fe-superoxide dismutase of Serratia marcescens, and other $\text{O}{}_2{}^{\mathrm{?}}$ scavengers such as cytochrome c and NADH, but are not affected by hydroxyl radical scavengers such as mannitol. None of the $\text{O}{}_2{}^{\mathrm{?}}$ scavengers tested affected the inherent substrate oxidation by glucose oxidase and d-amino acid oxidase. Furthermore, the generation of $\text{O}{}_2{}^{\mathrm{?}}$ in the oxidation of anionic 2-nitropropane by 2-nitropropane dioxygenase was revealed by ESR spectroscoy. The ESR spectrum of anionic 2-nitropropane plus 2-nitropropane dioxygenase shows signals at g₁ = 2.007 and g₁₁ = 2.051, which are characteristic of $\text{O}{}_2{}^{\mathrm{?}}$. The $\text{O}{}_2{}^{\mathrm{?}}$ generated is a catalytically essential intermediate in the oxidation of anionic nitroalkanes by the enzymes.     

Liposome oxidation and erythrocyte lysis by enzymically generated superoxide and hydrogen peroxide.

Xanthine oxidase, acting on acetaldehyde under aerobic conditions, produces a flux of O2- and H2O2 which attacks artificial liposomes and washed human erythrocytes. The liposomes were peroxidized and the erythrocytes suffered oxidation of hemoglobin followed by lysis. The oxidation of hemoglobin followed by lysis. The oxidation of hemoglobin, within the exposed erythrocytes, could be largely prevented by prior conversion to carbon monoxyhemoglobin, without preventing lysis. Hemolysis thus appeared to be a consequence of direct oxidative attack on the cell stroma. The enzyme-generated flux of O2- and of H2O2 also inactivated the xanthine oxidase. Superoxide dismutase or catalase, present in the suspending medium, protected the liposomes against peroxidation, the erythrocytes against lysis, and the xanthine oxidase against inactivation. Scavengers of O2('deltag), such as histidine or 2,5-dimethylfuran, which do not react with O2- or H2O2, also prevented peroxidation of liposomes and lysis of erythrocytes when present at low concentrations. In contrast a scavenger of OH-, such as mannitol was ineffective at low concentrations and provided significant protection only at much higher concentrations. It is proposed that O2- and H2O2 cooperated in producing OH- and O2('deltag), which were the proximate causes of lipid peroxidation and of hemolysis.     

Crystal structure and site-directed mutagenesis of a nitroalkane oxidase from Streptomyces ansochromogenes

Nitroalkane oxidase (NAO) catalyzes neutral nitroalkanes to their corresponding aldehydes or ketones, hydrogen peroxide and nitrite. The crystal structure of NAO from Streptomyces ansochromogenes was determined; it consists of two domains, a TIM barrel domain bound to FMN and C-terminal domain with a novel folding pattern. Site-directed mutagenesis of His¹⁷⁹, which is spatially adjacent to FMN, resulted in the loss of enzyme activity, demonstrating that this amino acid residue is important for catalysis. The crystal structure of mutant H179D-nitroethane was also analyzed. Interestingly, Sa-NAO shows the typical function as nitroalkane oxidase but its structure is similar to that of 2-nitropropane dioxygenase. Overall, these results suggest that Sa-NAO is a novel nitroalkane oxidase with TIM barrel structure.     

Nitroalkane oxidation by streptomycetes.

Crude cell-free extracts of nine strains of Streptomyces tested for nitroalkane-oxidizing activity showed production of nitrous acid from 2-nitropropane, 1-nitropropane, nitroethane, nitromethane, and 3-nitropropionic acid. These substrates were utilized in most strains but to a decreasing extent in the order given, and different strains varied in their relative efficiency of oxidation. p-Nitrobenzoic acid, p-aminobenzoic acid, enteromycin, and omega-nitro-l-arginine were not attacked. d-Amino acid oxidase, glucose oxidase, glutathione S-transferase, and xanthine oxidase, enzymes potentially responsible for the observed oxidations in crude cellfree extracts, were present at concentrations too low to play any significant role. A nitroalkane-oxidizing enzyme from streptozotocin-producing Streptomyces achromogenes subsp. streptozoticus was partially purified and characterized. It catalyzes the oxidative denitrification of 2-nitropropane as follows: 2CH(3)CH(NO(2))CH(3) + O(2) --> 2CH(3)COCH(3) + 2HNO(2). At the optimum pH of 7.5 of the enzyme, 2-nitropropane was as good a substrate as its sodium salt; t-nitrobutane was not a substrate. Whereas Tiron, oxine, and nitroxyl radical acted as potent inhibitors of this enzyme, superoxide dismutase was essentially without effect. Sodium peroxide abolished a lag phase in the progress curve of the enzyme and afforded stimulation, whereas sodium superoxide did not affect the reaction. Reducing agents, such as glutathione, reduced nicotinamide adenine dinucleotide, and nicotinamide adenine dinucleotide phosphate, reduced form, as well as thiol compounds, were strongly inhibitory, but cyanide had no effect. The S. achromogenes enzyme at the present stage of purification is similar in many respects to the enzyme 2-nitropropane dioxygenase from Hansenula mrakii. The possible involvement of the nitroalkane-oxidizing enzyme in the biosynthesis of antibiotics that contain a nitrogen-nitrogen bond is discussed.     

Inactivation of xanthine oxidase by hydrogen peroxide involves site-directed hydroxyl radical formation.

The mechanism of xanthine oxidase (XO) inactivation by hydrogen peroxide (H2O2) and its biologic significance are unclear. We found that addition of increasing concentrations of H2O2 progressively decreased xanthine oxidase activity in the presence but not the absence of xanthine in vitro. Inactivation of XO by H2O2 was also enhanced by anaerobic reduction of XO by xanthine. Inactivation of XO by H2O2 was accompanied by production of hydroxyl radical (.OH), measured as formation of formaldehyde from dimethylsulfoxide (DMSO). In contrast, addition of H2O2 to deflavo XO did not produce .OH. Inactivation of XO by H2O2 was decreased by simultaneous addition of the .OH scavenger, DMSO. However, inactivation of XO by H2O2 and formation of .OH were not decreased following addition of the metal chelator. DETAPAC, and/or the O2 scavenger, superoxide dismutase. The results suggest that inactivation of XO by H2O2 occurs by production of .OH following direct reduction of H2O2 by XO at the flavin site.     

The isolation and purification of a specific "protector" protein which inhibits enzyme inactivation by a thiol/Fe(III)/O2 mixed-function oxidation system

Mixed-function oxidation systems comprised of Fe3+, O2, and electron donors such as thiol compounds, ascorbate, NAD(P)H/NAD(P)H oxidase, and xanthine oxidase/hypoxanthine, catalyze the inactivation of many enzymes. This report describes the isolation and purification of a soluble protein from Saccharomyces cerevisiae, which specifically inhibits the inactivation of various enzymes by a nonenzymatic Fe3+/O2/thiol mixed-function oxidase system. When thiol is replaced with another electron donor (e.g. ascorbate), this specific protein no longer protects against iron (or copper)/O2-dependent radical-induced enzyme inactivation. Purification steps included a polyethylene glycol precipitation followed sequentially by a chromatography on DE52 and high pressure liquid chromatography on phenyl, DEAE, and gel-filtrated columns. The final gel filtration step yielded two protein peaks exhibiting protector activity and possessing a Mr of 500,000 and 90,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these two fractions gave a single band at 27 kDa suggesting that these protein species simply represent different oligomeric structures. The protector protein did not possess catalase, glutathione peroxidase, superoxide dismutase, or iron chelation activities. Since the protection activity reported herein is specific for mixed-function oxidation systems containing thiols, we propose that the protector protein functions as a sulfur radical scavenger.     

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).     

Responses of vascular endothelial oxidant metabolism to lipopolysaccharide and tumor necrosis factor-alpha.

Quantification of intracellular and extracellular levels and production rates of reactive oxygen species is crucial to understanding their contribution to tissue pathophysiology. We measured basal rates of oxidant production and the activity of xanthine oxidase, proposed to be a key source of O2- and H2O2, in endothelial cells. Then we examined the influence of tumor necrosis factor-alpha and lipopolysaccharide on endothelial cell oxidant metabolism, in response to the proposal that these inflammatory mediators initiate vascular injury in part by stimulating endothelial xanthine oxidase-mediated production of O2- and H2O2. We determined a basal intracellular H2O2 concentration of 32.8 +/- 10.7 pM in cultured bovine aortic endothelial cells by kinetic analysis of aminotriazole-mediated inactivation of endogenous catalase. Catalase activity was 5.72 +/- 1.61 U/mg cell protein and glutathione peroxidase activity was much lower, 8.13 +/- 3.79 mU/mg protein. Only 0.48 +/- 0.18% of total glucose metabolism occurred via the pentose phosphate pathway. The rate of extracellular H2O2 release was 75 +/- 12 pmol.min-1.mg cell protein-1. Intracellular xanthine dehydrogenase/oxidase activity determined by pterin oxidation was 2.32 +/- 0.75 microU/mg with 47.1 +/- 11.7% in the oxidase form. Intracellular purine levels of 1.19 +/- 1.04 nmol hypoxanthine/mg protein, 0.13 +/- 0.17 nmol xanthine/mg protein, and undetectable uric acid were consistent with a low activity of xanthine dehydrogenase/oxidase. Exposure of endothelial cells to 1000 U/ml tumor necrosis factor (TNF) or 1 microgram/ml lipopolysaccharide (LPS) for 1-12 h did not alter basal endothelial cell oxidant production or xanthine dehydrogenase/oxidase activity. These results do not support a casual role for H2O2 in the direct endothelial toxicity of TNF and LPS.     

Inactivation of glutathione peroxidase by superoxide radical

The selenium-containing glutathione peroxidase, when in its active reduced form, was inactivated during exposure to the xanthine oxidase reaction. Superoxide dismutase completely prevented this inactivation, whereas catalase, hydroxyl radical scavengers, or chelators did not, indicating that O2 was the responsible agent. Conversion of GSH peroxidase to its oxidized form, by exposure to hydroperoxides, rendered it insensitive toward O2. The oxidized enzyme regained susceptibility toward inactivation by O2 when reduced with GSH. The inactivation by O2 could be reversed by GSH; however, sequential exposure to O2 and then hydroperoxides caused irreversible inactivation. Reactivity toward CN- has been used as a measure of the oxidized form of GSH peroxidase, whereas reactivity toward iodoacetate has been taken as an indicator of the reduced form. By these criteria both O2 and hydroperoxides convert the reduced form to oxidized forms. A mechanism involving oxidation of the selenocysteine residue at the active site has been proposed to account for these observations.     

Identification of an essential tyrosine residue in nitroalkane oxidase by modification with tetranitromethane

The flavoprotein nitroalkane oxidase from Fusarium oxysporum catalyzes the oxidation of nitroalkanes to the respective aldehydes or ketones with production of nitrite and hydrogen peroxide. The enzyme is irreversibly inactivated by incubation with tetranitromethane, a tyrosine-directed reagent, at pH 7.3. The inactivation is time-dependent and shows first-order kinetics for two half-lives of inactivation. Further inactivation can be achieved upon a second addition of tetranitromethane. A saturation kinetic pattern is observed when the rate of inactivation is determined versus the concentration of tetranitromethane, indicating that a reversible enzyme-inhibitor complex is formed before irreversible inactivation occurs. Values of 0.096 +/- 0.013 min(-1) and 12.9 +/- 3.8 mM were determined for the first-order rate constant for inactivation and the dissociation constant for the reversibly formed complex, respectively. The competitive inhibitor valerate protects the enzyme from inactivation by tetranitromethane, suggesting an active-site-directed inactivation. The UV-visible absorbance spectrum of the inactivated enzyme is perturbed with respect to that of the native enzyme, suggesting that treatment with tetranitromethane resulted in nitration of the enzyme. Comparison of tryptic maps of nitroalkane oxidase treated with tetranitromethane in the presence and absence of valerate shows a single peptide differentially labeled in the inactivated enzyme. The spectral properties of the modified peptide are consistent with nitration of a tyrosine residue. The amino acid sequence of the nitrated peptide is L-L-N-E-V-M-C-(NO(2)-Y)-P-L-F-D-G-G-N-I-G-L-R. The possible role of this tyrosine in substrate binding is discussed.     

Vanadate-stimulated NADH oxidation by xanthine oxidase: an intrinsic property

Vanadate-dependent oxidation of NADH by xanthine oxidase does not require the presence of xanthine and therefore is not due to cooxidation. Addition of NADH or xanthine had no effect on the oxidation of the other substrate. Oxidation of NADH was high at acid pH and oxidation of xanthine was high at alkaline pH. The specific activity was relatively very high with NADH. Concentration-dependent oxidation of NADH Concentration-dependent oxidation of NADH was obtained in the presence of the polymeric form of vanadate, but not orthovanadate or metavanadate. Both NADH and NADPH were oxidized, as in the nonenzymatic system. Oxidation of NADH, but not xanthine, was inhibited by KCN, ascorbate, MnCl2, cytochrome c, mannitol, Tris, epinephrine, norepinephrine, and triiodothyronine. Oxidation of NADH was accompanied by uptake of oxygen and generation of H2O2 with a stoichiometry of 1:1:1 for NADH:O2:H2O2. A 240-nm-absorbing species was formed during the reaction which was different from H2O2 or superoxide. A mechanism of NADH oxidation is suggested wherein Vv and O2 receive one electron each successively from NADH followed by VIV giving the second electron to superoxide and reducing it to H2O2.     

Lysosomal enzyme leakage during the hypoxanthine/xanthine oxidase reaction

Impairment of lysosomal stability due to reactive oxygen species generated during the oxidation of hypoxanthine by xanthine oxidase was studied in rat liver lysosomes isolated in a discontinuous Nycodenz gradient. Production of O2.- and H2O2 during the hypoxanthine/xanthine oxidase reaction occurred for at least 5 min, while lysosomal damage, indicated by the release of N-acetyl-beta-glucosaminidase, occurred within 30 s, there being no further damage to these organelles thereafter. The extent of lysosomal enzyme release increased with increasing xanthine oxidase concentration. Superoxide dismutase and catalase did not prevent lysosomal damage during the hypoxanthine/xanthine oxidase reaction. Lysosomes reduced xanthine oxidase activity, as assessed in terms of O2 consumption, only slightly but substantially inhibited in a competitive manner the O2.- -mediated reduction of cytochrome c. This inhibition was almost completely reversed by potassium cyanide, thus pointing to the presence of a cyanide-sensitive superoxide dismutase in the lysosomal fraction. However, potassium cyanide did not affect the hypoxanthine/xanthine oxidase-mediated lysosomal damage, thus suggesting an inability of the lysosomal superoxide dismutase to protect the organelles. Negligible malondialdehyde formation was observed in the lysosomes either during the hypoxanthine/xanthine oxidase reaction or with different selective experimental approaches known to produce lipid peroxidation in other organelles such as microsomes and mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Investigation of the chemical basis of nitroalkane toxicity: tautomerism and decomposition of propane 1- and 2-nitronate under physiological conditions

Unlike primary nitroalkanes, such as 1-nitropropane, the secondary nitroalkane 2-nitropropane is geno- and hepatotoxic. Nitroalkanes exist in equilibrium with alkane nitronates. In order to investigate the relationship between nitroalkane toxicity and generation and stability of nitronates, propane 1- or 2-nitronate (4-6 mM) were incubated in buffer (pH 3.8 -7.4) in the absence or presence of cysteine. Equilibrium formation and degradation were studied by 1H-NMR spectroscopy and ion pair HPLC chromatography. Propane 1-nitronate generated 1-nitropropane rapidly and almost quantitatively. In the case of propane 2-nitronate equilibrium at pH 7.4 was reached within 8 h, when 48% of initial nitronate had tautomerised to 2-nitropropane. The pKa of the reaction 2-nitropropane less than--greater than propane 2-nitronate measured by HPLC was 7.63. Equilibrium formation, hydrolysis and reduction of nitronates were pH-dependent and, in the case of propane 2-nitronate, yielded mainly acetone, nitrite and acetone oxime, apart from 2-nitropropane. Hydrolysis of propane 2-nitronate (4 mM) to nitrite was modulated by cysteine (4 mM) and p-methoxyphenol (0.4 mM). At pH 7.4 they increased nitrite generation by 300 and 28%, respectively, at pH 4.8 they decreased nitrite formation by 91 and 82%, respectively, probably by scavenging radical intermediates. Differences between nitroalkanes in terms of content of nitronate tautomer at equilibrium are probably an important chemical determinant of their toxic potential.     

Iron and xanthine oxidase catalyze formation of an oxidant species distinguishable from OH.: comparison with the Haber-Weiss reaction

O2- was produced by gamma irradiation of formate solutions, by the action of xanthine oxidase on hypoxanthine and O2, and by the action of ferredoxin reductase on NADPH and paraquat in the presence of O2. Its reaction with H2O2 and various iron chelates was studied. Oxidation of deoxyribose to thiobarbituric acid-reactive products that was appropriately inhibited by OH. scavengers, or formate oxidation to CO2, was used to detect OH(.). With each source of O2-, and by these criteria, Fe(EDTA) efficiently catalyzed this (Haber-Weiss) reaction, but little catalysis was detectable with iron bound to DTPA, citrate, ADP, ATP, or pyrophosphate, or without chelator in phosphate buffer. O2- produced from xanthine oxidase, but not from the other sources, underwent another iron-dependent reaction with H2O2, to produce an oxidant that did not behave as free OH(.). It was formed in phosphate or bicarbonate buffer, and caused deoxyribose oxidation that was readily inhibited by mannitol or Tris, but not by benzoate, formate, or dimethyl sulfoxide. It did not oxidize formate to CO2. Addition of EDTA changed the pattern of inhibition to that expected for a reaction of OH(.). The other chelators all inhibited deoxyribose oxidation, provided their concentrations were high enough. The results are compatible with iron bound to xanthine oxidase catalyzing production of a strong oxidant (which is not free OH.) from H2O2 and O2- produced by the enzyme.     

Luminol chemiluminescence using xanthine and hypoxanthine as xanthine oxidase substrates

Luminol chemiluminescence induced by the xanthine or hypoxanthine-O2-xanthine oxidase system is analyzed and compared. Characteristics of the light emission curves were examined considering the conventional reaction scheme for the oxidation of both substrates in the presence of xanthine oxidase. The ratio of the areas of the rate of superoxide production during substrate oxidation to uric acid. The O2-. to uric acid ratio for each substrate can account for differences in xanthine and hypoxanthine-supported light emission, since uric acid is a strong inhibitor of O2-.-dependent luminol chemiluminescence. These results are consistent with a free radical scavenging role for uric acid. A similar but weaker scavenging effect of xanthine may also contribute to the observed differences in chemiluminescent yields between both substrates.     

Hydroxyl radical is not a product of the reaction of xanthine oxidase and xanthine. The confounding problem of adventitious iron bound to xanthine oxidase

The reaction of xanthine and xanthine oxidase generates superoxide and hydrogen peroxide. In contrast to earlier works, recent spin trapping data (Kuppusamy, P., and Zweier, J.L. (1989) J. Biol. Chem. 264, 9880-9884) suggested that hydroxyl radical may also be a product of this reaction. Determining if hydroxyl radical results directly from the xanthine/xanthine oxidase reaction is important for 1) interpreting experimental data in which this reaction is used as a model of oxidant stress, and 2) understanding the pathogenesis of ischemia/reperfusion injury. Consequently, we evaluated the conditions required for hydroxyl radical generation during the oxidation of xanthine by xanthine oxidase. Following the addition of some, but not all, commercial preparations of xanthine oxidase to a mixture of xanthine, deferoxamine, and either 5,5-dimethyl-1-pyrroline-N-oxide or a combination of alpha-phenyl-N-tert-butyl-nitrone and dimethyl sulfoxide, hydroxyl radical-derived spin adducts were detected. With other preparations, no evidence of hydroxyl radical formation was noted. Xanthine oxidase preparations that generated hydroxyl radical had greater iron associated with them, suggesting that adventitious iron was a possible contributing factor. Consistent with this hypothesis, addition of H2O2, in the absence of xanthine, to "high iron" xanthine oxidase preparations generated hydroxyl radical. Substitution of a different iron chelator, diethylenetriaminepentaacetic acid for deferoxamine, or preincubation of high iron xanthine oxidase preparations with chelating resin, or overnight dialysis of the enzyme against deferoxamine decreased or eliminated hydroxyl radical generation without altering the rate of superoxide production. Therefore, hydroxyl radical does not appear to be a product of the oxidation of xanthine by xanthine oxidase. However, commercial xanthine oxidase preparations may contain adventitious iron bound to the enzyme, which can catalyze hydroxyl radical formation from hydrogen peroxide.     

Mechanisms of the regulation of the conversion of xanthine oxidase in enterocytes exposed to x-irradiation

X-Irradiation of rats has been shown to change electron acceptor properties of xanthine oxidase of enterocytes the tropism towards NAD being lost and that towards O2 increased. The transformation of xanthine oxidase forms depends on the oxidation level of glutathione. Excess concentrations of hypoxanthine are responsible for the xanthine oxidase O-form functioning.     

Inactivation of Rabbit Muscle Creatine Kinase by Hydrogen Peroxide

The effects of xanthine + xanthine oxidase-generated reactive oxygen species (ROS) on rabbit muscle creatine kinase (CK) were studied. Xanthine (0.1 mM) + xanthine oxidase (30 mU/ml) inhibited activity of rabbit muscle CK (1.2mU/ml). Catalase (100/ml), but not SOD (100 U/ml), deferoxamine (100μM) or mannitol (20 mM), protected CK from inactivation; suggesting that H₂O₂ was responsible for inactivation. These results were different from previously reported findings on bovine heart CK that superoxide radicals inactivate the enzyme. Thus, enzymes with homologous structures may have different reactivities to different ROS. H₂O₂-induced inactivation of rabbit muscle CK was accompanied by a decrease in its thiol group content, whereas no significant changes in the protein structure were detected by SDS-PAGE or carbonyl content. These results suggest that oxidation of -SH groups by H₂O₂ seems to be a major mechanism of activation of rabbit muscle CK by xanthine + xanthine oxidase. Such inactivation of CK by H₂O₂ may be important in ROS-induced pathology.