Bovine Milk Xanthine Oxidase Research Articles

Oxidation of hypoxanthines, bearing 8-aryl or 8-pyridyl substituents, by bovine milk xanthine oxidase

1. 1.|Hypoxanthines, bearing at position 8 aryl or pyridyl substituents, are converted by bovine milk xanthine oxidase (xanthine: oxygen oxidoreductase, EC 1.2.3.2) into the corresponding xanthines at low rates. Oxidation is accelerated considerably when the 8-pyridyl substituents are quaternised. 2. 2.|In the enzymic oxidation of quaternary 8-pyridylhypoxanthines a lag phase precedes the attainment of a constant, maximal reaction rate. It is assumed that the delay is due to a relatively slow conformational change in the active enzymic center. 3. 3.|In 8-(3′- N-methylpyridinio)xanthine bethane, also the pyridinium moiety is attacked at high pH (9–11) to yield an N-methyl-2-pyridone. The analogous pyridone is the only oxidation product of 1-ethyl-8-(3′- N-methylpyridinio)hypoxanthine betaine, which is not attacked in the pyrimidine ring. 4. 4.|The cationic substrates are attracted to the enzyme by an anionic group, which probably forms an ion pair with a protonated amino group in or near the active center.

Bovine Milk Xanthine Oxidase, Blood Lipids and Coronary Plaques in Rabbits

The effects of prolonged intravenous administration of bovine milk xanthine oxidase (EC 1.2.3.2.) on blood lipids and arterial integrity were measured to determine if the administration of this enzyme produces metabolic changes conducive to plaque formation. New Zealand White rabbits were injected intravenously with bovine milk xanthine oxidase at 4-day intervals during a 13-week test period. At the end of the test period, the rabbits were killed and blood, heart, aorta, liver, and kidneys were collected and evaluated. Rabbits injected with phosphate buffer or acid-denatured xanthine oxidase for the same length of time served as negative controls. Additional rabbits fed a diet containing 3% added cholesterol for the same time period served as positive controls. The administration of xanthine oxidase in large amounts over a prolonged period did not alter serum cholesterol or triglyceride levels and did not reduce plasmalogen levels in the aorta or heart. Xanthine oxidase administration did not induce arterial plaque formation. Cholesterol feeding over the same time period increased serum cholesterol levels, reduced liver xanthine oxidase activity levels and resulted in a marked development of arterial plaques. Althouth xanthine oxidase activity was found in liver from all rabbits, enzyme activity was not detectable in aorta, heart or kidneys from any rabbit. Free or complexed bovine milk xanthine oxidase could not be demonstrated in heart, aorta, liver or kidneys from any of the rabbits with immunodiffusion or with immunofluorescent techniques. The study showed that when large intravenous doses of bovine milk xanthine oxidase were given to rabbits, the enzyme was not deposited in heart, aorta, liver or kidneys. The study also showed that large intravenous doses of xanthine oxidase over prolonged periods did not deplete arterial or coronary tissue plasmalogens, and did not induce arterial plaque formation.

Enzymic oxidation of 3-hydroxyxanthine to 3-hydroxyuric acid

1. 1. Bovine milk xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) oxidises 3-hydroxyxanthine slowly to 3-hydroxyuric acid; the 1-methyl derivative of 3-hydroxyxanthine is attacked about twice as fast. 2. 2. The pH optimum for the reaction of 2-hydroxyxanthine is near 5, i.e. the neutral form of this substrate is attacked much faster than the anion. Probably in the ‘active’ form of the latter, the negative charge is located mainly in the imidazole ring, thus inhibiting nucleophilic attack at C-8.

Persistence of Bovine Milk Xanthine Oxidase Activity after Gastric Digestion In Vivo and In Vitro

Seventy rats were fasted for 20h and used in two experiments. In the first experiment fasted rats were intubated with 5ml marketed milk of known xanthine oxidase activity. After dosing, food was withheld, and rats were sacrificed at intervals. The stomach of each rat was excised and its contents assayed polarographically for xanthine oxidase activity. Activity decreased with time. At .5 and 1.5h the recovered activities were 62 and 45%, and in some rats activity persisted up to 8h. In the second experiment, fasted rats received 2ml marketed milk. Enzyme activity dropped faster than in the first experiment. Typical gastric transit times for ingested milk were 30 to 40min. Enzyme activity in milk began to decrease at pH 6.70; below pH 3.90 the enzyme was inactivated completely. Inactivation was partly reversible at pH 2.50 and above by adjusting the pH to 7.3. When simulated gastric juice and milk were combined in ratios of 3:1, 2:1, 1:1, 1:2, and 1:3, pH's were 1.85, 2.08, 3.57, 5.16, and 5.56, and respective activities were 0, 0, 0, 14.2, and 23.8%. In one in vitro experiment, when juice and milk (1:2) were intubated at 37C for 0, .5, 8, and 24h, their pH's were 5.20, 5.26, 5.13, and 5.15 while recovered activities were 19.2, 18.7, 14.9, and 9.9%. When juice and milk (1:2) were incubated for .5h and followed by pancreatin solution for 0, .5, 3.5, and 7.5h, the recovered activities were 13.5, 11.9, 7.2, and 6.1%. Considerable xanthine oxidase in milk is not inactivated in the gastrointestinal tract but is available for absorption.

Influence of 8-substituents on the oxidation of hypoxanthine and 6-thioxopurine by bovine milk xanthine oxidase

1. 1. The influence of 8-substituents was studied on the rate of oxidation of hypoxanthine and 6-thioxopurine by bovine milk xanthine oxidase (EC 1.2.3.2). 2. 2. An 8-methyl group does not alter the rate of oxidation of hypoxanthine materially, but an 8-phenyl substituent reduces it markedly. This is ascribed to inhibition of the tautomerisation process, responsible for substrate activation, prior to oxidation. 3. 3. In contrast, the 8-phenyl group in 3-methyl-8-phenylhypoxanthine enhances the rate, presumably by binding to a hydrophobic site near the enzymic center. 4. 4. An 8-phenyl group in 6-thioxopurine markedly increases the rate of enzymic oxidation. Probably the aromatic substituent diverts anion formation to the imidazole ring. In contrast, ionisation of 8-methyl-6-thioxopurine involves the pyrimidine moiety, thus rendering enzymic attack at position 2 more difficult.

Oxidation of methyl derivatives of pteridin-4-one, lumazine and related pteridines by bovine milk xanthine oxidase

1. 1. Pteridin-4-ones, methylated at nitrogen or carbon, N- methylated lumazines and related oxopteridines were studied as substrates of a highly purified bovine milk xanthine oxidase (xanthine: oxygen oxidoreductase, EC 1.2.3.2). 2. 2. The enzyme can oxidise at high rates both uncharged and anionic substrates. Variation of enzymic activity with pH is mainly due to pH-dependent changes in the active enzymic center. 3. 3. Milk xanthine oxidases at different stages of purification convert pteridin-4-one into the 4,7-dione (compound 13 in this article). 4. 4. Methylation at C-6 in the pyrazine moiety enhances enzymic attack at C-2 in the pyrimidine ring. N- Methylation may increase or reduce rates of oxidation. 5. 5. For oxidation at C-2, the most favorable form of the substrate bears a double bond at C(2)  N(3). Attack at C-7 is enhanced strongly in structures bearing a double bond at C(6)  C(7). 6. 6. In general, pteridines react with xanthine oxidase as non-hydrated molecules. However, oxidation of 8-methyllumazine at C-7 may take place by dehydrogenation of the 7-CHOH group of the covalently hydrated molecule.

Digestion and Absorption of Bovine Milk Xanthine Oxidase and its Role as an Aldehyde Oxidase

The effects of acidic and intestinal proteolytic environments on bovine milk xanthine oxidase (XO) activity were determined in order to evaluate the extent to which this enzyme was absorbed in biologically active form. The inhibition of XO by folic acid and the relative affinities of XO for the oxidation of palmitaldehyde, stearaldehyde, and xanthine were compared. The effects of acid and gastric juice on XO activity were measured by incubating purified enzyme, and non-purified enzyme (milk), in buffers ranging in pH from 2 to 9. Fresh gastric juice was also incubated with milk. Increasing amounts of the enzyme were inactivated as the pH of the incubation mixture was reduced below pH 6.5. Below pH 3.5, the enzyme was completely inactivated. Gastric juice, pH juice incubated with milk. Milk XO activity was reduced 36% when mild was incubated with an equal volume of gastric juice. Homogenized milk had 59% less XO activity compared with raw molk. Fresh raw milk XO, homogenized milk XO, and purified XO were equally susceptible to inactivation by acid or gastric juice. After incubation of milk with gastric juice, or gastric juice followed by pancreatin, XO activity was associated with a macromolecule of 300,000 daltons molecular weight and subunits containg activity were not found. It was estimated that 0.00008% of the XO in the intestine was absorbed. Both folic acid and allopurinol inhibited XO activity in vitro. Allopurinol was 3.5 times more potent an inhibitor than folic acid. A large excess of dietary folic acid did not reduce rat liver or intestinal XO activity in vivo. XO had a much greater affinity for xanthine than for palmitaldehyde or stearaldehyde substrates. It was estimated that of 100 mg of XO in fresh raw milk, 41 mg remained after homogenization, 27 mg entered the intestine and only 20 ng were absorbed as intact enzyme.

THE PRESENCE AND SOME PROPERTIES OF XANTHINE OXIDASE IN HUMAN MILK AND COLOSTRUM

ABSTRACTHuman milk and colostrum samples were assayed for xanthine oxidase (XO) activity polarographically and by a radiochemical assay. Activity was found in all 59 samples assayed, it varied widely among individuals, and colostrum showed the highest activity. Enzyme activity decreased in all individuals with advance in the stage of lactation. The pH optimum of XO was 7.2 and nearly all of the enzyme was contained in the cream fraction. In stability experiments, milk samples stored at ‐20°C for up to 3 months showed no loss in activity. The pH optimum of the enzyme and its stability at ‐20°C demonstrate that it differs from bovine milk XO and it is not of bacterial origin.

Oxidation of Selected Pteridine Derivatives by Mammalian Liver Xanthine Oxidase and Aldehyde Oxidase

Considerable information is available concerning the oxidation of pteridine derivatives by bovine milk xanthine oxidase, but few investigations have been carried out on the oxidation of such compounds by mammalian liver xanthine oxidase and the related aldehyde oxidase. Xanthine oxidase, obtained from rat liver, oxidizes a variety of substituted amino‐ and hydroxypteridines in a manner identical to that previously observed for milk xanthine oxidase. For example, 2‐aminopteridine and its 4‐ and 7‐hydroxy derivatives were oxidized efficiently to 2‐amino‐4, 7‐dihydroxypteridine (isoxan‐thopterin) by the rat liver enzyme, and 4‐aminopteridine and its 2‐and 7‐hydroxy derivatives were oxidized to 4‐amino‐2, 7‐dihydroxypteridine. 4‐Hydroxypteridine and the isomeric 2‐ and 7‐hydroxypteridines were oxidized by rat liver xanthine oxidase to 2,4,7‐trihydroxypteridine. Rabbit liver aldehyde oxidase, but not rat liver xanthine oxidase, was able to catalyze the oxidation in position 7 of 2,4‐diaminopteridine and its 6‐methyl and 6‐hydroxymethyl derivatives. 2‐Aminopteridine and 4‐aminopteridine were both oxidized to the corresponding 7‐hydroxy derivatives in the aldehyde oxidase system; 2‐amino‐4‐hydroxypteridine appeared to be a minor product in the oxidation of 2‐aminopteridine by rabbit liver aldehyde oxidase. Both aldehyde oxidase and xanthine oxidase were able to catalyze the oxidation of 2‐amino‐6,7‐disubstituted pteridines to the corresponding 4‐hydroxy derivatives; 4‐hydroxy‐6,7‐disubstituted pteridines were oxidized in position 2 by both enzymes. 4‐Amino‐6,7‐disubstituted pteridines were not oxidized by either enzyme. 2‐Amino‐4‐methylpteridine was oxidized in position 7 by aldehyde oxidase but was not an effective substrate for xanthine oxidase; 2‐hydroxypteridine and 7‐hydroxypteridine were not oxidized to a detectable extent by aldehyde oxidase. All oxidations mediated by xanthine oxidase were strongly inhibited by allopurinol (4‐hydroxy‐pyrazolo[3,4‐d]pyrimidine), and all oxidations mediated by aldehyde oxidase were inhibited by menadione (2‐methyl‐1,4‐naphthoquinone). Rat liver xanthine oxidase and, to a lesser extent, rabbit liver aldehyde oxidase were inhibited by 4‐chloro‐6,7‐dimethylpteridine; 2‐amino‐3‐pyrazinecarboxylic acid inhibited xanthine oxidase but not aldehyde oxidase. The oxidations of 2‐ and 4‐aminopteridines by aldehyde oxidase resulted in concomitant reduction of cytochrome c.

Bovine Milk Intake and Xanthine Oxidase Activity in Blood Serum

Xanthine oxidase activity in blood serum was measured by a sensitive radio-enzymatic assay. Pigs receiving 7.6 liters of milk daily for 100 days did not show any detectable enzymatic activity in their blood Xanthine oxidase activity in blood serum of 25 human volunteers had an average of 6.7 milliunits per liter with a range of 0 to 34.6 milliunits per liter. Neither a causal nor statistically significant relationship existed between xanthine oxidase activity in blood and average daily milk consumption, age, or sex.

Antigenicity of bovine milk xanthine oxidase in guinea pigs.

Six female and four male albino guinea pigs were immunized with active purified xanthine oxidase of bovine milk mixed with equal volume of Freund's complete adjuvant. One male and one female were immunized with heat-inactivated xanthine oxidase mixed with equal volume of adjuvant. Two males and four females were controls and received a phosphate buffer mixed with equal volume of adjuvant. The mixtures were administered intradermally and subcutaneously at weekly intervals for 6 consecutive wk and blood samples collected weekly. The enzyme was antigenic by the coated tanned red blood cell method. After the third weekly immunization, precipitating antibodies were in the sera of animals that received the active enzyme. Hemagglutination titers increased during subsequent weeks and reached a maximum after the sixth weekly immunization. Antisera from animals immunized with heat-inactivated xanthine oxidase gave a positive response similar to that with animals immunized with the active enzyme. However, when the same antisera were tested with sheep red blood cells coated with heat-inactivated enzyme, no hemagglutination was observed. Ouchterlony double gel-diffusion tests showed that it may be possible to differentiate between antibodies elicited to active and heat-inactivated xanthine oxidase.

Oxidation of N-methyl substituted hypoxanthines, xanthines, purine-6,8-diones and the corresponding 6-thioxo derivatives by bovine milk xanthine oxidase

1. 1. The oxidation of six series of purines (hypoxanthines, xanthines, purine-6,8-diones and the corresponding 6-thioxo derivatives) by a highly purified bovine milk xanthine oxidase (EC 1.2.3.2) has been studied, using a variety of N-methyl derivatives. 2. 2. N-Methyl substituents can either enhance or reduce enzymic rates. Enhancement is ascribed to blockade of groups which mediate unfavorable modes of binding of substrate to enzyme. Introduction of N-methyl groups can also inhibit enzymic oxidation, either by occluding essential binding groups or by preventing spontaneous or enzyme-induced tautomerisation processes, which create suitable binding sites in the substrates. 3. 3. In all purines which are rapidly attacked by xanthine oxidase, proper attachment to the active center is mediated by the groupings (3)NH, (9)N or (3)N, (9)NH. 4. 4. Reduced rates usually express lowered substrate affinity, which finds its expression in weak competitive inhibition of xanthine oxidation.

Subunit structure of bovine milk xanthine oxidase. Effect of limited cleavage by proteolytic enzymes on activity and structure

Bovine milk xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) has been purified by a modified method without the use of proteases, and its structure has been analyzed by polyacrylamide gel electrophoresis. Native xanthine oxidase is found to consist of only two polypeptide chains A with molecular weights of 150 000 each. These chains have NH 2-terminal methionine. Limited proteolysis with trypsin, chymotrypsin, or subtilisin at pH 8 did not affect molecular weight and activities of the enzyme while each of the A chains was cleaved under these conditions to three fragments C, E, and F with molecular weights of 92 000, 42 000 and 20 000, respectively. These fragments remained bound to each other and were relatively resistant to subsequent proteolysis. The isolation of xanthine oxidase in the presence of pancreatin as described by Hart et al. (1970, Biochem.J. 116, 851) gives partially digested enzyme composed mainly of chains C, E ( M r 35 000 ) and a small component ( M r approx. 15 000 ). The action of subtilisin on xanthine oxidase at pH 11 resulted in complete digestion of E chains, FAD separation, and total loss of xanthine:oxygen oxidoreductase activity while xanthine:indophenol oxidoreductase activity was relatively little affected. The residual enzyme has a molecular weight of about 200 000, is composed mainly of two C chains (and may probably contain F and/or proteolytic fragments of low molecular weight), contains molybdenum, and does not contain FAD.

Proteolytic activity in bovine milk xanthine oxidase preparations

Bovine milk xanthine oxidase isolated in the presence of pancreatin was found to copurify with proteases from pancreatin. The effect of these proteases was observed by incubating azo-albumin with different xanthine oxidase preparations and by the observation of polypeptide degradation products with sodium dodecyl sulfate polyacrylamide gel electrophoreses. A commercial preparation of xanthine oxidase exhibited significant proteolytic activity.

A new purification procedure for bovine milk xanthine oxidase: Effect of proteolysis on the subunit structure

A new purification procedure for bovine milk xanthine oxidase is reported. The enzyme so obtained is of the highest purity and shows little evidence of degradation. The enzyme displays a single protein band on either polyacrylamide gels or on sodium dodecyl sulfate-urea polyacrylamide gels. Sedimentation equilibrium studies indicate a native molecular weight of 303,000 and a subunit molecular weight of approximately 150,000. The latter value is in good agreement with the minimum molecular weight of 157,000 calculated from dry weight determination and flavin analysis. In contrast, purification of xanthine oxidase from pancreatin-treated cream yields a protein which displays two subunits corresponding to molecular weights of 92,000 and 39,000 as determined by dodecyl sulfate-urea polyacrylamide gel electrophoresis. Pancreatinized enzyme has a greater mobility than unproteolyzed enzyme on polyacrylamide gels. Exposure of milk xanthine oxidase to pancreatin before isolation or after purification yields the same result.

Quinazolines as Inhibitors of Xanthe Oxidase

A series of 23 quinazolines and 5,6,7,8-tetrahydroquinazolines was evaluated for inhibition of bovine milk xanthine oxidase. The synthesis of four of these compounds has not been reported previously. Several 2-amino-4-hydroxyquinazolines, as well as 2-amino-4-mercaptoquinazoline, were effective inhibitors of this enzyme. 2-Amino-4-hydroxy-6-hydroxymethylquinazoline was a threefold better inhibitor than its pteridine counterpart, while 2-amino-4-hydroxyquinazoline-6-carboxaldehyde was the most potent compound studied.

Effect of Tungsten and Vanadium on the in Vitro Assembly of Assimilatory Nitrate Reductase Utilizing Neurospora Mutant nit-1

Abstract Tungsten or vanadium in a nutrient medium containing nitrate, but not ammonia, as the sole nitrogen source inhibited the formation of assimilatory NADPH-nitrate reductase (but not nitrate-inducible NADPH-cytochrome c reductase) and growth of wild type Neurospora crassa. Inhibition was prevented by high levels of molybdate in the nutrient medium but was not reversed by adding molybdate to subsequently prepared cell-free enzyme preparations. Neither tungsten nor vanadium was inhibitory when added to partially purified wild type Neurospora NADPH-nitrate reductase. Cell-free preparations of uninduced wild type grown in increasing concentrations of tungsten or vanadium showed progressive inhibition of ability to assemble nitrate reductase in vitro (with extracts of nitrate-induced Neurospora mutant nit-1) which was partially or entirely restored by subsequent addition of molybdate to the extracts of the tunsgten- or vanadium-grown uninduced wild type. Tungsten or vanadium added to acid-treated bovine milk xanthine oxidase or to extracts of uninduced wild type also inhibited in vitro enzyme assembly, with added molybdate partially preventing inhibition. The tungsten or vanadium analogue of nitrate reductase formed in vivo or by in vitro assembly is considerably more labile than its molybdenum counterpart as indicated by the complete loss of 185W or 48V from the enzyme as a result of trichloroacetic acid precipitation or heat treatment. Sucrose density gradient profiles of the Neurospora NADPH-nitrate reductase formed in vitro with 185W- or 48V-grown uninduced wild type or induced nit-1 showed, as in the case of 99Mo, that uninduced wild type and not nit-1 can be the source of tungsten or vanadium in the in vitro assembly of the enzyme. Exogenous 185W added to cell-free extracts of uninduced wild type or acid treated bovine milk xanthine oxidase prior to in vitro assembly of nitrate reductase using nit-1 failed to appear in the subsequently formed enzyme, whereas 48V experienced a small but consistent incorporation only with acid-treated xanthine oxidase. There was no in vitro exchange or incorporation of 185W or 48V into the already formed wild type enzyme. The data implicate tungsten and vanadium as competitive inhibitors of molybdenum in nitrate reductase structure and function.

Involvement of Molybdenum and Iron in the in Vitro Assembly of Assimilatory Nitrate Reductase Utilizing Neurospora Mutant nit-1

Abstract Addition of high levels of sodium molybdate (10-2 m, final concentration) to acid-treated purified bovine milk xanthine oxidase prior to incubation with an extract of the nitrate-induced nitrate reductase Neurospora crassa mutant nit-1 resulted in a significant enhancement of the subsequent in vitro assembly of wild type N. crassa NADPH-nitrate reductase (EC 1.6.6.2). Added molybdate had no effect on the wild type enzyme itself. The enhancement is highly specific for molybdenum, is independent of added phosphate, and also occurs with cell-free preparations of uninduced wild type and with other known acid-treated molybdenum enzymes including chicken liver xanthine dehydrogenase, rabbit liver aldehyde oxidase, bovine sulfite oxidase, and the nitrogenases of Azotobacter and Clostridium. Preincubation at 23° of acid-treated xanthine oxidase preparations resulted in a considerable loss of ability to form nitrate reductase and in a more striking enhancement by subsequently added molybdate. Prior addition of molybdate during the above 23° preincubation prevented in part the loss of the ability of the enzyme to form nitrate reductase, suggesting that the enhancement by molybdate is a restoration effect rather than a stimulatory one. Inclusion of molybdate in the buffer used for homogenization of the uninduced wild type also gave a significant increase in the subsequent in vitro assembly of the enzyme. Various forms and derivatives of molybdenum enhanced to varying degrees the ability of acid-treated xanthine oxidase to form nitrate reductase. Aqueous solutions of several complexes of Mo(V) with cysteine or glutathione under anaerobic conditions failed to substitute for the presumed molybdenum cofactor in the in vitro assembly of nitrate reductase. Sucrose density gradient profiles of NADPH-nitrate reductase formed in vitro by incubation of induced nit-1 extract with a 99Mo-labeled fraction of uninduced wild type (grown on 99MoO4=-containing medium) coincided with that of radioactivity. The addition of 99MoO4= to a cell-free preparation of unlabeled uninduced wild type or to acid-treated xanthine oxidase prior to its incubation with an extract of induced nit-1 gave similar results. Reciprocal experiments using an unlabeled fraction of uninduced wild type and an extract of nit-1 grown and induced in the presence of 99MoO4= showed no incorporation of radioactivity in the in vitro assembled enzyme. Nor was there in vitro exchange or incorporation of 99Mo into the already formed wild type enzyme. Comparable experiments with 55Fe showed that nit-1 and not uninduced wild type or xanthine oxidase is the source of iron in the in vitro assembly of Neurospora NADPH-nitrate reductase.

On the Interaction of the Electron Acceptor 2,6-Dichlorophenolindophenol with Bovine Milk Xanthine Oxidase

Abstract The electron acceptor 2,6-dichlorophenolindophenol (DCI) forms a spectrophotometrically and kinetically detectable complex with bovine milk xanthine oxidase. The visible absorption spectrum of complexed DCI exhibits an absorption maximum at 660 nm at pH 7.8 (e = 34.2 x 103 m-1 cm-1), a marked bathochromic shift from the visible absorption maximum at 608 nm characteristic of the free dye (e = 21.0 x 103 m-1 cm-1. The bound dye can be partially removed from the enzyme by prolonged dialysis (24 hours), and totally removed by gel filtration, indicating a reversible rather than a covalent interaction. From the DCI concentration dependence of the spectral change, the stoichiometry of the interaction is 2.08 DCI-binding sites per molecule of xanthine oxidase (assuming a molar extinction coefficient for the enzyme of 66 x 103 m-1 cm-1 at 450 nm); and the dissociation constant of the DCI-enzyme complex is 1.3 x 10-6 m. The spectral shift characteristic of DCI-xanthine oxidase interaction is blocked by 2 mm arsenite, by 2 mm p-hydroxymercuribenzoate, by the competitive inhibitor 2,4-diamino-6-hydroxy-s-triazine, 1.5 mm, and by the stoichiometric xanthine oxidase inhibitor, 4,6-dihydroxypyrazolo[3,4-d]pyrimidine (4,6-diHPP), at levels equimolar with those of the enzyme. With the latter inhibitor, the DCI spectral shift, although not present initially, develops concomitantly with reoxidation of the reduced enzyme molybdenum of the 4,6-diHPP-xanthine oxidase complex. In contrast to the results seen with the other xanthine oxidase inhibitors, the DCI-xanthine oxidase interaction is not blocked by 16 mm sodium cyanide. In kinetic studies, DCI, in addition to acting as an electron acceptor, is also a noncompetitive inhibitor of the enzyme, with a Ki of 2.8 x 10-6 m. Prior incubation with DCI protects the enzyme from inhibition by arsenite, but not from inhibition by cyanide. On the basis of the spectrophotometric and kinetic data, it is proposed that the DCI-binding site is the molybdenum or molybdenum-sulfur component of the internal electron transport chain of the enzyme.

Stoichiometric Inhibition of Reduced Xanthine Oxidase by Hydroxypyrazolo[3,4-d]pyrimidines

Abstract The inhibition of reduced bovine milk xanthine oxidase by 4-hydroxypyrazolo[3,4-d]pyrimidine (4-HPP) and 4,6-dihydroxypyrazolo[3,4-d]pyrimidine (4,6-diHPP) is stoichiometric or titrating in type, i.e. the inhibition is virtually complete (g90%) at concentrations of inhibitor equimolar with that of the enzyme. The dissociation constant of the reduced enzyme-4,6-diHPP complex, determined by the method of Easson and Stedman (Proc. Roy. Soc. Ser. B Biol. Sci., 121, 142 (1936)), is 5.4 x 10-10 m. No decrease in the fractional inhibition is detectable on increasing the substrate concentration from 0.04 to 0.33 mm, or on 10-fold dilution of the enzyme-inhibitor complex. The inhibitory properties of 4-HPP are virtually identical with those of 4,6-diHPP; this identity in behavior is the result of the conversion of 4-HPP to 4,6-diHPP by the enzyme. The rate of development of 4,6-diHPP inhibition is markedly temperature-dependent, more than 2 hours being required to reach maximal inhibition at 2°, but less than 1 min at 40°. The time required to reach maximal inhibition is not decreased by increasing the 4,6-diHPP level 100-fold, from 28 mµm to 2.8 µm; the inhibition appears, therefore, to be caused, not by the enzyme-inhibitor complex initially formed, but to a subsequent internal rearrangement of the latter to a less readily dissociable form.