Xanthine oxidases

The xanthine oxidase for the hydroxylation was obtained by procedures published earlier. [Pg.339]

Xanthine oxidase is a flavoenzyme that also contains molybdenum and iron. It shows activity toward a variety of substrates including purines, pyrimidines, aldehydes, and NADH. In this assay, isoxanthopterin is produced from 2-amino-4-hydroxypteridine. [Pg.339]

Isoxanthopterin was separated from 2-amino-4-hydroxypteridine by chromatography on a Unisil, ODS column (4.6 mm x 50 mm, 3 /u.m). The mobile phase was 0.1 M phosphate buffer (pH 2.0). A fluorescence detector was used, with excitation and detection wavelengths 340 and 410 nm, respectively. [Pg.339]

The standard reaction mixture contained in a final volume of 250 /xL 50 fiL of 0.5 M sodium phosphate buffer (pH 7.0), 25 fiL of 200 yM AHP solution, 25 fiL of 10 yM 2,6-dichlorophenolindolphenol sodium, 10 fiL of enzyme sample, and 10 fiL of 10 mg/mL bovine serum albumin. After a 10-minute incubation at 37°C, the reaction was stopped by adding 25 y.L of 60% perchloric acid and cooling in an ice bath for 10 minutes. The mixture was centrifuged before injection of 10 fiL of the supernate into the HPLC system. [Pg.339]

The assay was linear for up to 30 minutes and with up to 40 /xg of the soluble fraction of rat liver. [Pg.340]

The molybdoenzyme, xanthine oxidoreductase is a homodimer of 150 kDa subunits and exists in two interconvertible forms, dehydrogenase (EC 1.1.1.204) and xanthine oxidase (EC 1.1.3.22). Reduction of oxygen by either form of the enzyme yields superoxide radical anion and hydrogen peroxide with xanthine or hypoxanthine as substrates. Xanthine dehydrogenase preferentially reduces NAD whereas xanthine oxidase does not reduce NAD preferring molecular oxygen. [Pg.89]

The molybdenum in these enzymes is bound by a special organic pterin cofactor, and is not held directly by the sidechains of proteins. The pterin cofactor actually is a dithiolate complex. The molybdenum in the enzymes is not re-oxidised directly by molecular dioxygen and the ancillary flavin and Fe/ S centres have to do with the way in which dioxygen is activated oxygen transfer by molybdenum enzymes is of oxygen atoms from water and not from dioxygen. [Pg.89]

Xanthine dehydrogenase from Eubacterium bar-keri has a mass of 530 kDa and three types of subunits. It contains molybdopterin as the molybde-num-complexing cofactor and 1 mol of selenium in a non-selenocysteine form per mol of native enzyme (Schrader et al. 1999). [Pg.89]

Xanthine oxidase from buttermilk produced 02 and H2O2 in a ratio of 25 75, while a microbial xanthine oxidase was unable to produce any 02 (WiP-piCH et al. 2001). [Pg.89]

Xanthine oxidoreductase is asymmetrically localised not only in the cytoplasm but also on the outer surface of human endothelial (umbilical vein endothelial cells and EA-hy-926 permanent endothelial cell line) and epithelial (HB4a, a mammary epithelial cell line conditionally immortalised by transfection with SV40 virus) cells in culture (Rouquette et al. 1998). [Pg.90]

A pyranopterin cofactor, consisting of a bicyclic pterin fused to a monocyclic pyran ring, is known to directly coordinate the molybdenum of xanthine oxidase through a dithiolene side chain. The pyranopterin cofactor probably does not directly participate in the catalytic sequence of molybdenum hydroxylases but has been implicated in mediating electron transfer to other redox-active cofactors and/or modulating the reduction potential of the molybdenum. [Pg.246]

Following isolation, the molybdenum cofactor in most molybdenum-containing enzymes is in the Mo(VI) state. The Mo(V) species are usually unstable and intermediate forms during catalysis hence they are often given the label rapid . [Pg.246]

Canne and co-workers have presented EPR studies of three prokaryotic enzymes of the xanthine oxidase family, namely quinoline 2-oxidoreductase, quinaldine 4-oxidase, and isoquinoline l-oxidoreductaseJ In quinoline 2-oxidoreductase a neutral flavin radical was observed, while in quinaldine 4-oxidase an anionic radical was detected. The rapid Mo(V) signal was observed in all three enzymes with only small differences in magnetic parameters. From spectra simulations of Mo (/ = 5/2) substituted quinoline 2-oxidoreductase, a deviation of 25° between the maximal g and Mo-hfc tensor component was derived. The Mo(V) species was detected in small amounts upon reduction with substrates in quinoline 2-oxidoreductase and quinaldine 4-oxidase, but showed a different kinetic behaviour with an intense EPR signal in isoquinoline 1-oxidoreductase. The two [2Fe-2S] clusters produced different EPR signals in all three enzymes and, in isoquinoline 1-oxidoreductase, revealed a dipolar interaction, from which a maximum distance of 15 A was estimated. [Pg.247]

Chemiluminescence and bioluminescence are also used in immunoassays to detect conventional enzyme labels (eg, alkaline phosphatase, P-galactosidase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, microperoxidase, xanthine oxidase). The enhanced chemiluminescence assay for horseradish peroxidase (luminol-peroxide-4-iodophenol detection reagent) and various chemiluminescence adamantyl 1,2-dioxetane aryl phosphate substrates, eg, (11) and (15) for alkaline phosphatase labels are in routine use in immunoassay analyzers and in Western blotting kits (261—266). [Pg.275]

Molybdenum. Molybdenum is a component of the metaHoen2ymes xanthine oxidase, aldehyde oxidase, and sulfite oxidase in mammals (130). Two other molybdenum metaHoen2ymes present in nitrifying bacteria have been characteri2ed nitrogenase and nitrate reductase (131). The molybdenum in the oxidases, is involved in redox reactions. The heme iron in sulfite oxidase also is involved in electron transfer (132). [Pg.387]

Xanthine oxidase, mol wt ca 275,000, present in milk, Hver, and intestinal mucosa (131), is required in the cataboHsm of nucleotides. The free bases guanine and hypoxanthine from the nucleotides are converted to uric acid and xanthine in the intermediate. Xanthine oxidase cataly2es oxidation of hypoxanthine to xanthine and xanthine to uric acid. In these processes and in the oxidations cataly2ed by aldehyde oxidase, molecular oxygen is reduced to H2O2 (133). Xanthine oxidase is also involved in iron metaboHsm. Release of iron from ferritin requires reduction of Fe " to Fe " and reduced xanthine oxidase participates in this conversion (133). [Pg.387]

Several other biosensors have been developed usiag this oxygen-quenched fluorescence approach. Target species iaclude ethanol [64-17-5] hydrogen peroxide [7722-84-17, H2O2, lactate, and xanthine [69-89-6] C H4N402, usiag alcohol oxidase, catalase [9001-05-2] lactate oxidase, and xanthine oxidase, respectively. An additional technique for biocatalytic biosensors iavolves the firefly chemiluminescent reaction (17) ... [Pg.110]

Allomaltol, methyl — see Pyran-4-one, 5-methoxy-2-methyl-Allopurinol applications, 5, 343 metabolism, 1, 237 synthesis, 5, 316, 340 tautomerism, 5, 308 xanthine oxidase inhibition by, 1, 173 Allopurinol, oxy-applications, 5, 343 synthesis, 5, 316 Alloxan... [Pg.514]

Boroxazothienopyridines, 4, 1029-1032 Borsche synthesis, 3, 44 Botrydiplodin mass spectrometry, 4, 585 Boulton-Katritzky rearrangement, 5, 288 Bovine milk xanthine oxidase substrates, 1, 234 Bradsher reaction... [Pg.571]

Xanthine anions structure, 5, 509 Xanthine-8-carboxylic acid synthesis, 3, 308, 322 Xanthine oxidase... [Pg.924]

At the present time, the greatest importance of covalent hydration in biology seems to lie in the direction of understanding the action of enzymes. In this connection, the enzyme known as xanthine oxidase has been extensively investigated.This enzyme catalyzes the oxidation of aldehydes to acids, purines to hydroxypurines, and pteridines to hydroxypteridines. The only structural feature which these three substituents have in common is a secondary alcoholic group present in the covalently hydrated forms. Therefore it was logical to conceive of this group as the point of attack by the enzyme. [Pg.40]

The situation in the pteridine series is somewhat more complex. Pteridine, 2-, 4-, and 7-hydroxypteridine, and some of the dihydroxy-pteridines are oxidized, stepwise and quantitatively, in the presence of xanthine oxidase to a single substance, 2,4,7-trihydroxypteridine. Notably, 6-hydroxypteridine, which readily forms a covalent hydrate, is not attacked. [Pg.41]

Bergmann has suggested that oxidation is ruled out at positions (where hydration occurs readily) which are not accessible to the enzyme after the pteridine is adsorbed on it. Alternatively, the destruction of co-planarity by hydration may prevent adsorption of the pteridine on the enzyme. The case of xanthopterin (2-amino-4,6-dihydroxypteridine) may be relevant. The neutral species of this substance exists as an equilibrium mixture of approximately equal parts of the anhydrous and 7,8-hydrated forms (in neutral aqueous solution at 20°). Xanthine oxidase cataljrzes the oxidation of the anhydrous form in the 7-position but leaves the hydrated form unaffected and about two hours is required to re-establish the former equilibrium. [Pg.41]

As an inhibitor of xanthine oxidase, allopurinol also markedly decreases oxidation of both hypoxanthine and xanthine itself to the sole source of uric acid (19) in man. This metabolic block thus removes the source of uric acid that in gout causes the painful crystalline deposits in the joints. It is of interest that allopurinol itself is oxidized to the somewhat less effective drug, oxypurinol (21), by xanthine oxidase. [Pg.426]

Therapeutic Function Xanthine oxidase inhibitor gout therapy Chemical Name 1 H-pyra2olo[3,4-d] pyrimidin4-ol Common Name —... [Pg.42]

Table 9.6 Examination of Superoxide Involvement in the Chemiluminescence of PM-2 Elicited by the Fe2+-H2C>2 System and the Xanthine-Xanthine Oxidase System3 (Shimomura, 1991b)...

B) Xanthine-xanthine oxidase system (50 pM xanthine, 5 pg xanthine oxidase) ... [Pg.288]

Storch, J., and Ferber, E. (1988). Detergent-amplified chemiluminescence of lucigenin for determination of superoxide anion production by NADPH oxidase and xanthine oxidase. Anal. Biochem. 169 262-267. [Pg.440]

Anti-gout Drugs. Figure 1 Xanthine oxidase-catalyzed reactions. Xanthine oxidase converts hypoxanthine to xanthine and xanthine to uric acid, respectively. Hypoxanthine and xanthine are more soluble than uric acid. Xanthine oxidase also converts the uricostatic drug allopurinol to alloxanthine. Allopurinol and hypoxanthine are isomers that differ from each other in the substitution of positions 7 and 8 of the purine ring system. Although allopurinol is converted to alloxanthine by xanthine oxidase, allopurinol is also a xanthine oxidase inhibitor. Specifically, at low concentrations, allopurinol acts as a competitive inhibitor, and at high concentrations it acts as a noncompetitive inhibitor. Alloxanthine is a noncompetitive xanthine oxidase inhibitor. XOD xanthine oxidase. [Pg.135]

Schumacher HR Jr (2005) Febuxostat a non-purine, selective inhibitor of xanthine oxidase for the management of hyperaricaemia in patients with gout. Expert Opin Investig Drugs 14 893-903... [Pg.139]

Azathiopurine, mercaptopurine Allopurinol Xanthine oxidase Azathiopurine/mercaptopurine toxicity... [Pg.448]

Uricostatic drugs inhibit the production of uric acid through the inhibition of xanthine oxidase. Allopurinol is the only therapeutically used uricostatic drug. [Pg.1268]

Xanthine oxidase (XOD) is the key enzyme in purine catabolism. XOD catalyses the conversion ofhypoxan-thine to xanthine and of xanthine to uric acid, respectively. The uricostatic drug allopurinol and its major metabolite alloxanthine (oxypurinol) inhibit xanthine oxidase. [Pg.1323]

Allopurinol 1 mM Xanthine oxidase inhibitor, suppresses oxygen free radical production... [Pg.394]

Complexes of molybdenum and tungsten with bidentate sulfur ligands have been investigated extensively. In recent years, the work in this field has been escalated by the impetus of designing models of such bioinorganic enzymes as nitrogenase and xanthine oxidase (125). The early work reviewed by Coucouvanis (1) dealt exclusively with the isolation of oxomolybdenum(V) and -(VI) species. [Pg.224]

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