The oxidation product of molybdenum cofactor from milk xanthine oxidase

In extracts of acid treated molybdenum cofactor containing xanthine oxidase, fluorescence is maximally developed upon a three hours incubation. Analysis by means of reversed phase HPLC revealed the presence of several fluorescent compounds, the main one being a blue fluorescent compound with an emission maximum of 465 nm when maximal excited at 395 nm at a neutral pH. Definite proof is presented that this compound is the oxidation product of the molybdenum cofactor. The remaining fluorescent products are shown to be pterin-derivatives, yielding predominantly pterin-6-carboxylic acid upon permanganate oxidation. Purified oxidation product of molybdenum cofactor however, didn's yield a fluorescent derivative at all upon treatment with permanganate.     

Extraction and purification of molybdenum cofactor from milk xanthine oxidase

Molybdenum cofactor (mocofactor) is extracted efficiently, free of impurities and in high concentrations, by acid treatment of xanthine oxidase and subsequent incubation of the precipitate with phosphate buffer containing EDTA, molybdate and oxygen. It is suggested that cofactor is bound to the enzyme via hydrophobic forces as well as via an oxygen-sensitive mechanism. Upon extraction, the capability to complement the apo nitrate reductase of Neurospora crassa nit-1 can be conserved only in the total absence of oxygen. Cysteine and glutathione were shown to protect efficiently free mocofactor from oxidation. Two species of active mocofactor, probably a molybdoform and a demolybdoform, could be separated by means of reversed-phase HPLC with a mobile phase of 5 mM sodium citrate at a pH of 6.5. The mode of interaction between either of these species with thiol reagents is discussed.     

A molybdopterin-free form of xanthine oxidase

A previously unidentified fraction lacking xanthine:O2 activity has been isolated during affinity chromatography of bovine milk xanthine oxidase preparations on Sepharose 4B/folate gel. Unlike active, desulfo, or demolybdo forms of xanthine oxidase, this form, which typically comprises about 5% of an unfractionated enzyme solution, passes through the affinity column without binding to it, and is thus easily separated from the other species. The absorption spectrum of this fraction is very similar to that of the active form, but has a 7% lower extinction at 450 nm. Analysis of the fraction has shown that it is a dimer of normal size, but that it does not contain molybdenum or molybdopterin (MPT). The "MPT-free" xanthine oxidase contains 90-96% of the Fe found in active xanthine oxidase, and 100% of the expected sulfide. EPR and absorption difference spectroscopy indicate that the MPT-free fraction is missing approximately half of its Fe/S I centers. The presence of a new EPR signal suggests that an altered Fe/S center may account for the nearly normal Fe and sulfide content. Microwave power saturation parameters for the Fe/S II and Fe/S I centers in the MPT-free fraction are normal, with P1/2 equal to 1000 and 60 mW, respectively. The new EPR signal shows intermediate saturation behavior with a P1/2 = 200 mW. The circular dichroism spectrum of the MPT-free fraction shows distinct differences from that of active enzyme. The NADH:methylene blue activity of the MPT-free fraction is the same as that of active xanthine oxidase which exhibits xanthine:O2 activity, but NADH:cytochrome c and NADH:DCIP activities are diminished by 54 and 37%, respectively.     

Reaction of fluorescein bimercuric acetate with a molybdenum center of xanthine oxidase from milk

Inhibition of milk xanthine oxidase by fluorescein bimercuriacetate (FMA) allows for the classification of S-containing groups according to their localization and role in the catalytic activity of the enzyme. The enzyme (E) complexes with FMA (E--FMA I and E--FMA II) differing in their activity, stoichiometry and spectral properties were studied at various experimental conditions, reaction time and FMA concentrations. The enzyme molecule contains 5 groups that are reactive towards FMA (E--FMA I) and are localized outside the active center. That these groups have no concern with activity and are subjected to modification irrespective of whether or not the xanthine oxidase molecule has an intact Mo-center. The formation of an inactive E--FMA II complex is associated with an additional (in comparison with E--FMA I) binding of two FMA molecules per molecule of the active enzyme. The stoichiometry of the E--FMA II complex was determined by the X-ray fluorescent method from the amount of the Hg in enzyme. A kinetic scheme of xanthine oxidase inhibition by FMA is proposed, according to which the inhibition is a result of modification of two groups in the enzyme active center, of which only one is essential for the enzyme activity. This scheme also postulates the role of reversible E--FMA complexes in the course of irreversible inhibition. Xanthine oxidase is protected against FMA by the substrate (xanthine), competitive inhibitors (azaxanthine and allopurinol) and acceptor (2,6-dichlorophenolindophenol), i. e., compounds which interact with the Mo-center of the enzyme. The EPR spectra of the dithionite-reduced E--FMA II complex were found to contain a "slow" signal, Mo(V), typical of the Mo-center devoid of labile sulphur. It was assumed that the essential group interacting with FMA in the active center of xanthine oxidase as a terminal sulphur which is a component of the coordination region of Mo.     

EXAFS studies of the molybdenum center of xanthine oxidase.

EXAFS spectra associated with the K-absorption edge of molybdenum in the desulpho and functional forms of xanthine oxidase and some potential synthetic analogues have been obtained. These data indicate that the immediate environment of the molybdenum is different in the two forms of the enzyme and that desulpho xanthine oxidase resembles that in [MoO2(S2CNEt2)2] and [MoO2(ethylcysteine)2]. The cyanolysable sulphur atom of functional xanthine oxidase is suggested to be tightly bound to the molybdenum at a distance of less than or equal to 2.3 A.     

A kinetic study of hypoxanthine oxidation by milk xanthine oxidase.

The course of the reaction sequence hypoxanthine----xanthine----uric acid catalysed by xanthine:oxygen oxidoreductase from milk was investigated on the basis of u.v. spectra taken during the course of hypoxanthine and xanthine oxidations. It was found that xanthine accumulated in the reaction mixture when hypoxanthine was used as a substrate. The time course of the concentrations of hypoxanthine, xanthine intermediate and uric acid product was simulated numerically. The mathematical model takes into account the competition of substrate, intermediate and product and the accumulation of the intermediate at the enzyme. This type of analysis permits the kinetic parameters of the enzyme for hypoxanthine and xanthine to be obtained.     

The composition of milk xanthine oxidase

The composition of milk xanthine oxidase has been reinvestigated. When the enzyme is prepared by methods that include a selective denaturation step in the presence of sodium salicylate the product is obtained very conveniently and in high yield, and is homogeneous in the ultracentrifuge and in recycling gel filtration. It has specific activity higher than previously reported preparations of the enzyme and its composition approximates closely to 2mol of FAD, 2g-atoms of Mo and 8g-atoms of Fe/mol of protein (molecular weight about 275000). In contrast, when purely conventional preparative methods are used the product is also homogeneous by the above criteria but has a lower specific activity and is generally comparable to the crystallized enzyme described previously. Such samples also contain 2mol of FAD/mol of protein but they have lower contents of Mo (e.g. 1.2g-atom/mol). Amino acid compositions for the two types of preparation are indistinguishable. These results confirm the previous conclusion that conventional methods give mixtures of xanthine oxidase with an inactive modification of the enzyme now termed ;de-molybdo-xanthine oxidase', and show that salicylate can selectively denature the latter. The origin of de-molybdo-xanthine oxidase was investigated. FAD/Mo ratios show that it is present not only in enzyme purified by conventional methods but also in ;milk microsomes' (Bailie & Morton, 1958) and in enzyme samples prepared without proteolytic digestion. We conclude that it is secreted by cows together with the active enzyme and we discuss its occurrence in the preparations of other workers. Studies on the milks of individual cows show that nutritional rather than genetic factors determine the relative amounts of xanthine oxidase and de-molybdo-xanthine oxidase. A second inactive modification of the enzyme, now termed ;inactivated xanthine oxidase', causes variability in activity relative to E(450) or to Mo content and formation of it decreases these ratios during storage of enzyme samples including samples free from demolybdo-xanthine oxidase. We conclude that even the best purified xanthine oxidase samples described here and by other workers are contaminated by significant amounts of the inactivated form. This may complicate the interpretation of changes in the enzyme taking place during the slow phase of reduction by substrates. Attempts to remove iron from the enzyme by published methods were not successful.     

Magnetic interactions in milk xanthine oxidase

The relaxation behavior of the EPR signals of MoV, FAD semiquinone, and the reduced Fe/S I center was measured in the presence and absence of other paramagnetic centers in milk xanthine oxidase. Specific pairs of prosthetic groups were rendered paramagnetic by poising the native enzyme or its desulfo glycol inhibited derivative at appropriate potentials and pH values. Magnetic interactions were found between the following species: Mo--Fe/S I (100-fold increase in microwave power required to saturate the MoV EPR signal at 103 K when Fe/S I is reduced as opposed to oxidized), FAD--Fe/S I and FAD--Fe/S II (70-fold increase in power required to saturate the FADH.EPR signal at 173 K when either Fe/S center is reduced), and Fe/S I--Fe/S II (2.5-fold increase in power to saturate the reduced Fe/S I EPR signal at 20 K when Fe/S II is reduced). The Mo--Fe/S I interaction was also detected as a reduced Fe/S I induced splitting of the MoV EPR spectrum at 30 K. No splittings of the FADH. or Fe/S center spectra were detected. No magnetic interactions were found between FAD and Mo or between Mo and Fe/S II. These results, together with those of Coffman & Buettner [Coffman, R. E., & Buettner, G. R. (1979) J. Phys. Chem. 83, 2392-2400], were used to estimate the following approximate distances between the electron carrying prosthetic groups of milk xamthine oxidase: Mo--Fe/S I, 11 +/- 3 A; Fe/S I-Fe/S II, 15 +/- 4 A; FAD-Fe/S I, 16 +/- 4 A; FAD-Fe/S II, 16 +/- 4 A. A model for the arrangement of these groups within the xanthine oxidase molecule is suggested.     

Magnetic coupling of the molybdenum and iron-sulphur centres in xanthine oxidase and xanthine dehydrogenases.

Magnetic interaction between molybdenum and one of the iron-sulphur centres in milk xanthine oxidase [Lowe, Lynden-Bell & Bray (1972) Biochem. J. 130, 239-249] was studied further, with particular reference to the newly discovered Mo(V) e.p.r.(electron-paramagnetic-resonance) signal, Resting II [Lowe, Barber, Pawlik & Bray (1976) Biochem. J. 155, 81-85]. E.p.r. measurements at 35GHz near to 4.2K showed that the interaction has the same sign at all molybdenum orientations and is ferromagnetic. The predicted splitting of the e.p.r. signal from the reduced iron-sulphur centre, Fe/S I, was observed, Providing positive identification of this as the other interacting species. Chemical modification of the molybdenum environment in xanthine oxidase can change the size of the interaction severalfold, but interaction always remains approximately isotropic. The interaction in turkey liver xanthine dehydrogenase is indistinguishable from that in the oxidase. However, a bacterial xanthine dehydrogenase with different iron-sulphur centres shows rather larger interaction. Guanidinium chloride disturbs the iron-sulphur centres of the oxidase, and when this occurs there is a parallel and relatively small change in the interaction. Removal of flavin from the molecule, or raising the pH to 12.0, changes the interaction slightly without affecting the chromophores themselves. It is concluded that the Fe/S I centre and the Mo are at least 1.0nm and probably nearer 2.5nm apart, and that the conformation of the protein between them is relatively stable up to pH 12.     

Proteolytic activity in bovine milk xanthine oxidase preparations.

Five glycosyl-transferases have been found present in purified hepatocyte nuclei of the rat (mannosyl-, galactosyl-, N-acetyl-glucosaminyl-, N-acetyl-galactosaminyl- and sialyl-transferases); these are capable of fixing specific carbohydrates on to endogenous or exogenous protein acceptors.     

Inhibition of milk xanthine oxidase by fluorodinitrobenzene

Milk xanthine oxidase reacted with fluorodinitrobenzene resulting in the modification of two lysine residues with a 6-fold decrease in catalytic activity. Continued reaction with fluorodinitrobenzene up to a total of 11 dinitrophenyl residues/equivalent of enzyme-bound FAD resulted in no further decrease in activity. Stopped flow studies revealed that the modification perturbed the reduction of the enzyme by xanthine; this was 6-fold lower with modified than with native enzyme. The reaction of the reduced modified enzyme with oxygen was qualitatively and quantitatively the same as with native enzyme. One nitro group of each dinitrophenyl lysine residue is slowly reduced by xanthine; reduction of both nitro groups is achieved by dithionite. The two dinitrophenyl lysine reduces can be distinguished on the basis of their kinetics of reduction. One appears to be located on the protein surface and is reduced in an intermolecular reaction, while the other appears to be located in a pocket of the enzyme and is reduced in a slow intramolecular reaction.     

The isolation of demolybdo xanthine oxidase from bovine milk.

It was deduced many years ago from indirect evidence that demolybdo xanthine oxidase is present in normal bovine milk. This has now been confirmed by isolation of this enzyme form by a method based on the folate-gel affinity-chromatography procedure described Nishino & Tsushima [(1986) J. Biol. Chem. 261, 11242-11246]. Enzymic and spectroscopic properties of demolybdo xanthine oxidase, which retains flavin and iron-sulphur centres, are generally in accordance with expectations. Like the normal enzyme, it yields on denaturation material fluorescing at 460 nm. Molybdenum cofactor activity measured by the Neurospora crassa nit-1 assay in the presence of added molybdate was 33% of that of the normal enzyme. The absorption spectrum in the near-u.v. region differs slightly, but significantly, from that of the active and desulpho forms of the enzyme. It is concluded that the molybdenum cofactor site contains a pterin-like material not identical with that in the normal enzyme. The significance of the occurrence of demolybdo xanthine oxidase in milk is discussed, and evidence in the literature for demolybdo forms of other molybdoenzymes is briefly reviewed. Additional studies on the use of the affinity procedure for large-scale preparation of high-activity xanthine oxidase are described. In agreement with our ability to isolate the demolybdo enzyme, the procedure appears less effective in eliminating the demolybdo than the desulpho enzyme.     

Tryptophan-containing milk protein-derived dipeptides inhibit xanthine oxidase

Of twelve dipeptides tested, only the Trp containing peptides Val-Trp and its reverse peptide Trp-Val showed a xanthine oxidase (XO) inhibitory activity. Studies with Val and Trp revealed that XO inhibition was mainly attributed to the Trp residue. No significant difference (P ≥ 0.05) was found for the XO inhibitory potency (IC₅₀) values for Trp, Val-Trp and Trp-Val, which were about 200 times higher than that for Allopurinol. Lineweaver and Burke analysis demonstrated that Trp, Val-Trp and Trp-Val were non-competitive inhibitors while Allopurinol was a competitive inhibitor. Of the different milk-protein substrates hydrolyzed with gastro-intestinal enzyme activities, only lactoferrin (LF) hydrolyzates displayed XO inhibition. Peptides present in a LF hydrolyzate (GLF-240 min) were adsorbed onto activated carbon followed by subsequent desorption with stepwise elution using acetonitrile (ACN). Separation and detection of Trp containing peptides within the different fractions were achieved using RP-HPLC coupled with fluorescence detection. The desorbed fractions displayed different XO inhibitory properties, with no inhibition in the unbound fraction and highest inhibition in fractions eluted with 30, 40 and 70% ACN. The fraction eluting at 40% ACN was significantly more potent (19.1 ± 2.3% inhibition at 1.25 mg mL⁻¹) than the GLF-240 min hydrolyzate (13.4 ± 0.4% inhibition at 1.25 mg mL⁻¹), showing the potential for enrichment of the bioactive peptides on fractionation with activated carbon.     

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.     

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.