A colorimetric 96-well microtiter plate assay for the determination of urate oxidase activity and its kinetic parameters

Urate oxidase (E.C.1.7.3.3; uricase, urate oxygen oxidoreductase) is an enzyme of the purine breakdown pathway that catalyzes the oxidation of uric acid in the presence of oxygen to allantoin and hydrogen peroxide. A 96-well plate assay measurement of urate oxidase activity based on hydrogen peroxide quantitation was developed. The 96-well plate method included two steps: an incubation step for the urate oxidase reaction followed by a step in which the urate oxidase activity is stopped in the presence of 8-azaxanthine, a competitive inhibitor. Hydrogen peroxide is quantified during the second step by a horseradish peroxidase-dependent system. Under the defined conditions, uric acid, known as a radical scavenger, did not interfere with hydrogen peroxide quantification. The general advantages of such a colorimetric assay performed in microtiter plates, compared to other methods and in particular the classical UV method performed with cuvettes, are easy handling of large amounts of samples at the same time, the possibility of automation, and the need for less material. The method has been applied to the determination of the kinetic parameters of rasburicase, a recombinant therapeutic enzyme.     

The Neutron Structure of Urate Oxidase Resolves a Long-Standing Mechanistic Conundrum and Reveals Unexpected Changes in Protonation

Urate oxidase transforms uric acid to 5-hydroxyisourate without the help of cofactors, but the catalytic mechanism has remained enigmatic, as the protonation state of the substrate could not be reliably deduced. We have determined the neutron structure of urate oxidase, providing unique information on the proton positions. A neutron crystal structure inhibited by a chloride anion at 2.3 Å resolution shows that the substrate is in fact 8-hydroxyxanthine, the enol tautomer of urate. We have also determined the neutron structure of the complex with the inhibitor 8-azaxanthine at 1.9 Å resolution, showing the protonation states of the K10–T57–H256 catalytic triad. Together with X-ray data and quantum chemical calculations, these structures allow us to identify the site of the initial substrate protonation and elucidate why the enzyme is inhibited by a chloride anion.     

Study of pH and temperature-induced transitions in urate oxidase (Uox-EC1.7.3.3) by microcalorimetry (DSC), size exclusion chromatography (SEC) and enzymatic activity experiments

Purified recombinant urate oxidase (urate oxygen oxidoreductase EC 1.7.3.3. re-Uox) has been studied by means of differential scanning calorimetry (DSC) in correlation with enzymatic activity measurements and size exclusion chromatography. Differential scanning calorimetry curves versus pH show two endothermal effects in the pH range 6-10. The first endotherm reveals a maximum stability between pH 7.25 and pH 9.5 corresponding to a temperature of transition T(m1) of 49.0 degrees C and an enthalpy of transition of 326 kJ mol(-1). This value dramatically decreases below pH 7.25. The behavior of the second endotherm is more complex but the temperature of transition T(m2) is constant between pH 9 and 7.25 and a maximum for the corresponding enthalpy is obtained near pH 8 with DeltaH(2)=272 kJ mol(-1). An optimal pH of 8.0 for the stability of the enzymatic activity at elevated temperature was also found which was in good agreement with calorimetric results. Reversibility of the first endotherm is obtained from 20 to 51.5 degrees C. The calorimetric result is correlated to enzymatic activity, purity by size exclusion chromatography (SEC) and protein concentration measurements. In contrast, for the second endotherm, after heating up to 68.9 degrees C, no reversibility was found. Interaction with structural analogues of urate has been studied by DSC. 8-Azahyooxanthine has only a small effect and caffeine has no effect at all. With 8-azaxanthine, a rapid increase of the T(m1) function of the concentration is obtained. At high concentration T(m1) reached the T(m2) value which remained unaffected.     

Site-specific incorporation of unnatural amino acids into urate oxidase in Escherichia coli

Urate oxidase catalyzes the oxidation of uric acid with poor solubility to produce 5-hydroxyisourate and allantoin. Since allantoin is excreted in vivo, urate oxidase has the potential to be a therapeutic target for the treatment of gout. However, its severe immunogenicity limits its clinical application. Furthermore, studies on the structure-function relationships of urate oxidase have proven difficult. We developed a method for genetically incorporating p-azido-L-phenylalanine into target protein in Escherichia coli in a site-specific manner utilizing a tyrosyl suppressor tRNA/aminoacyl-tRNA synthetase system. We substituted p-azido-L-phenylalanine for Phe(170) or Phe(281) in urate oxidase. The products were purified and their enzyme activities were analyzed. In addition, we optimized the system by adding a "Shine-Dalgarno (SD) sequence" and tandem suppressor tRNA. This method has the benefit of site-specifically modifying urate oxidase with homogeneous glycosyl and PEG derivates, which can provide new insights into structure-function relationships and improve pharmacological properties of urate oxidase.     

Temporal control of urate oxidase activity during development of the third-instar larva of Drosophila: The role of 20-hydroxyecdysone

The tissue-specific enzyme urate oxidase is confined exclusively to the Malpighian tubules of Drosophila melanogaster and expressed only in the third-instar larva and the adult. Shortly before pupariation urate oxidase activity declines precipitously and is not detectable 24 hours later. That 20-hydroxyecdysone is the factor that triggers the disappearance of urate oxidase activity in late third-instar larvae is demonstrated using the temperature sensitive mutant ecd¹ which at the nonpermissive temperature of 29°C fails to accumulate a sufficient concentration of 20-hydroxyecdysone necessary for puparium formation and thus remains a third-instar larva for 1 to 2 weeks before death. Both the life cycle and the temporal profile of urate oxidase activity in ecd¹ larvae at 19°C is identical to that of the wild type. However, at 29°C ecd¹ third-instar larvae retain high urate oxidase activity. A precipitous decline in urate oxidase activity is observed when ecd¹ larvae at 29°C are fed 20-hydroxyecdysone. These data implicate 20-hydroxyecdysone in the process that controls the rapid decline of urate oxidase activity at the time of puparium formation. In whole homogenates of Malpighian tubules, the urate oxidase polypeptide was identified in SDS-polyacrylamide gels by its Rf with respect to homogeneously pure Drosophila urate oxidase and also by immunoprecipitation with rabbit anti-Drosophila urate oxidase IgG. Throughout development the amount of the urate oxidase polypeptide is correlated with the magnitude of urate oxidase activity.     

Urate produced during hypoxia protects heart proteins from peroxynitrite-mediated protein nitration

Tyrosine nitration is a common modification to proteins in vivo, but the reactive nitrogen species responsible for nitration are often studied in vitro using just the amino acid tyrosine in simple phosphate solutions. To investigate which reactive nitrogen species could nitrate proteins in a complex biological system, we exposed rat heart and brain homogenates to peroxynitrite, nitric oxide under aerobic conditions, and other putative nitrating agents. Peroxynitrite was by far the most efficient nitrating agent when alternative targets were available to compete with tyrosine. Curiously, proteins in heart homogenates were substantially more resistant to nitration than brain homogenates. Ultrafiltration to remove low molecular weight compounds made the heart proteins equally susceptible as the brain proteins to nitration. Endogenous ascorbate and free thiols had little effect on nitration by peroxynitrite in either heart or brain. However, accumulation of urate formed by the oxidation of hypoxanthine by xanthine dehydrogenase and oxidase in heart appeared to be the major inhibitor of nitration. Heart homogenates treated with uricase, which converts urate to allantoin, showed equivalent nitration as in brain homogenates. Urate, as assayed by HPLC, was 58 +/- 8 microM in heart but only 4 +/- 2 microM in brain homogenates. Although xanthine dehydrogenase conversion to a free radical-producing oxidase can serve as an important source of superoxide and hydrogen peroxide during ischemia/reperfusion, our results suggest that urate formation by xanthine dehydrogenase may provide a significant antioxidant defense against peroxynitrite and related nitric oxide-derived oxidants.     

Procatalytic ligand strain. Ionization and perturbation of 8-nitroxanthine at the urate oxidase active site

The binding of the inhibitor 8-nitroxanthine to urate oxidase has been investigated by Raman and UV-visible absorption spectroscopy. The absorption maximum of 8-nitroxanthine shifts from 380 to 400 nm upon binding to the enzyme, demonstrating that the electronic structure of the ligand is perturbed. It has been proposed that oxidation of the substrate urate by urate oxidase is facilitated by formation of the substrate dianion at the enzyme active site, and Raman spectra of urate oxidase-bound 8-nitroxanthine suggest that both the dianionic and monoanionic forms of the ligand are bound to the enzyme under conditions where in solution the monoanion is present exclusively. The C4-C5 stretching frequency appears as a relatively isolated vibrational mode in 8-nitroxanthine whose frequency shifts according to the protonation state of the purine ring. Identification of the C4-C5 stretching mode was confirmed using [4-(13)C]-8-nitroxanthine and ab initio calculation of the vibrational modes. Two peaks corresponding to the C4-C5 stretching mode were evident in spectra of enzyme-bound 8-nitroxanthine, at 1541 and 1486 cm(-)(1). The higher frequency peak was assigned to monoanionic 8-nitroxanthine, and the low-frequency peak was assigned to dianionic 8-nitroxanthine. The C4-C5 stretching frequency for free monoanionic 8-nitroxanthine was at 1545 cm(-)(1), indicating that the enzyme polarizes that bond when the ligand is bound. The C4-C5 stretching frequency in dianionic 8-nitroxanthine is also shifted by 4 cm(-)(1) to lower frequency upon binding. For 8-nitroxanthine free in solution, the C4-C5 stretching frequency shifts to lower frequency upon deprotonation, and the absorption maximum in the UV-visible spectrum shifts to higher wavelength. The spectral shifts observed upon binding of 8-nitroxanthine to urate oxidase are consistent with increased anionic character of the ligand, which is expected to promote catalysis in the reaction with the natural substrate urate. In the Raman spectra of 8-nitroxanthine bound to the F179A, F179Y, and K9M mutant proteins, the C4-C5 stretching frequency was not perturbed from its position for the unbound ligand. Both V(max) and V/K were decreased in the mutant enzymes, demonstrating a correlation between the interaction that perturbs the C4-C5 stretching frequency and the catalytic activity of the enzyme. It is suggested that hydrogen-bonding interactions that lead to precise positioning and deprotonation of the substrate are perturbed by the mutations.     

Urate oxidase of Chlamydomonas reinhardii

Urate oxidase (EC 1.7.3.3) of Chlamydomonas reinhardii cells grown on purines and purine derivatives has been partially characterized. Crude enzyme preparations have a pH optimum of 9.0, require O₂ for activity, have an apparent K_m of 12 μ M for urate, and are inhibited by high concentrations of this substrate. Enzyme activity was particularly sensitive to metal ion chelating agents like cyanide, cupferron, diethyldithiocarbamate and o -phenanthroline, and to structural analogues of urate like hypoxanthine and xanthine. Chlamydomonas cells grow phototrophically on adenine, guanine, hypoxanthine, xanthine, urate, allantoin or allantoate as sole nitrogen source, indicating that in this alga the standard pathway of aerobic degradation of purines of higher plants, animals and many microorganisms operates. As deduced from experiments in vivo , urate oxidase from Chlamydomonas is repressed in the presence of ammonia or nitrate.     

Degradation of peroxisomal catalase and urate oxidase of rat liver.

Urate oxidase and catalase were purified from rat liver peroxisomes, and respective antibodies were prepared from rabbits by the administration of these enzymes. Although urate oxidase generally precipitates in immunoprecipitation-possible pH ranges (pH 4.5--9.5), the enzyme remained soluble in 50 mM glycine buffer (pH 9.5) containing 50% glycerol up to concentration of 0.3 mg/ml. Anti-urate oxidase reacted with purified urate oxidase as well as with the crude preparation. After [3H]leucine was injected to rats, urate oxidase and catalase were purified from rat liver at certain intervals, and further precipitated by respective antibodies. The half-life of the catalase was 39 h and that of urate oxidase, 20 h. When the sonicated light mitochondrial fraction was incubated at 37 degrees C and at pH 7.0 or 5.6, inactivation of catalase did not seem to differ between these pH values, and approximately 80% of the catalase activity remained even after 8 h. Urate oxidase was inactivated very rapidly at pH 5.6; only 30% of its activity survived incubation for 6 h. This inactivation was found to occur by some proteolytic process. From these findings, the turnover rate of urate oxidase was found to be different from that of catalase, and this distinction seemed to be due to different sensitivity to some degradative enzymes.     

Peroxisomes of soybean (Glycine max) root nodule vascular parenchyma cells contain a "nodule—specific" urate oxidase

The cortex of soybean ( Glycine max L. cv. Centennial) nodules contain an organellerich layer of vascular parenchyma tissue, which encircles the elaborate vascular tissue of the nodule. Peroxisomes with small, electron-opaque nucleoids are found in the vascular parenchyma cells. Positive cytochemical staining for catalase (EC 1.11.1.6) confirms their morphological identification as peroxisomes. Activities of both glycolate oxidase (EC 1.1.3.1) and urate oxidase (EC 1.7.3.3) were detected cytochemically in these peroxisomes. Nodule-specific urate oxidase was localized principally in the nucleoid region of these vascular parenchyma peroxisomes, as indicated by immunogold labelling using antibodies against nodulin-35, the nodule-specific urate oxidase. The density of urate oxidase immunogold labelling in the vascular parenchyma peroxisome nucleoid is similar to that of the more well-characterized interstitial cell peroxisomes of the infected zone. These results show that the induction of nodule-specific urate oxidase may be induced in tissue outside of the infected zone.     

Degradation of peroxisomal catalase and urate oxidase of rat liver

Urate oxidase and catalase were purified from rat liver peroxisomes, and respective antibodies were prepared from rabbits by the administration of these enzymes. Although urate oxidase generally precipitates in immunoprecipitation-possible pH ranges (pH 4.5–9.5), the enzyme remained soluble in 50 mM glycine buffer (pH 9.5) containing 50% glycerol up to concentration of 0.3 mg/ml. Anti-urate oxidase reacted with purified urate oxidase as well as with the crude preparation.After [³H]leucine was injected to rats, urate oxidase and catalase were purified from rat liver at certain intervals, and further precipitated by respective antibodies. The half-life of the catalase was 39 h and that of urate oxidase, 20 h. When the sonicated light mitochondrial fraction was incubated at 37°C and at pH 7.0 or 5.6, inactivation of catalase did not seem to differ between these pH values, and approximately 80% of the catalase activity remained even after 8 h. Urate oxidase was inactivated very rapidly at pH 5.6; only 30% of its activity survived incubation for 6 h. This inactivation was found to occur by some proteolytic process.From these findings, the turnover rate of urate oxidase was found to be different from that of catalase, and this distinction seemed to be due to different sensitivity to some degradative enzymes.     

Immunocytochemical localization of urate oxidase, fatty acyl-CoA oxidase, and catalase in bovine kidney peroxisomes

We investigated the localization of urate oxidase, peroxisomal fatty acyl-CoA oxidase, and catalase in bovine kidney by immunoblot analysis and protein A-gold immunocytochemistry, using the respective polyclonal monospecific antibodies raised against the enzymes purified from rat liver. By immunoblot analysis, these three proteins were detected in bovine kidney and bovine liver homogenates. Subcellular localization of these three enzymes in kidney was ascertained by protein A-gold immunocytochemical staining of Lowicryl K4M-embedded tissue. Peroxisomes in bovine kidney cortical epithelium possessed crystalloid cores or nucleoids, which were found to be the exclusive sites of urate oxidase localization. The limiting membrane, the marginal plate, and the matrix of renal peroxisomes were negative for urate oxidase staining. In contrast, catalase and fatty acyl-CoA oxidase were found in the peroxisome matrix. These results demonstrate that, unlike rat kidney peroxisomes which lack urate oxidase, peroxisomes of bovine kidney contain this enzyme as well as peroxisomal fatty acyl-CoA oxidase.     

Glucocorticoid regulation of rat liver urate oxidase

Urate oxidase, an enzyme involved in purine catabolism, comprises the crystalline core of rat liver peroxisomes. An affinity-purified monospecific antibody was developed to study the expression of urate oxidase protein levels. Immunoreactive urate oxidase was not detectable in prenatal liver; however, it is present at low levels after birth until approximately day 15 (postnatal age); expression sharply increases just prior to day 20, after which the enzyme is maintained at adult levels. This pattern of expression was similar to that of another peroxisomal enzyme, catalase; these developmental increases reflect the increase in peroxisomal number. Administration of exogenous glucocorticoid hormone to 10-day-old rats resulted in a precocious rise (2.5-fold) in urate oxidase levels. Adrenalectomy at 10 days of age did not cause decreased levels in the fourth week of life. In adult animals, while exogenous glucocorticoid administration did not influence urate oxidase levels, adrenalectomy at 60 days of age decreased urate oxidase levels to 40 percent of control levels. Subsequent administration of exogenous glucocorticoid hormone restored urate oxidase to normal levels. Parallel studies of catalase levels indicate that this glucocorticoid-sensitive response is not generalized for all peroxisomal proteins. Our results suggest that peroxisomes proliferate during early postnatal development, but after this process is complete, the biogenesis of individual peroxisomal proteins may be independently regulated.     

A radiochemical-high-performance liquid chromatographic assay for urate oxidase in human plasma

Polyethylene glycol-modified urate oxidase (PEG-uricase) holds promise as a hypouricemic agent for treating gout and as an adjunct to cytolytic therapy of hematologic malignancies. Spectrophotometric assays of urate oxidase are not sensitive enough for pharmacokinetic evaluation of PEG-uricase in clinical trials. We have therefore developed a more sensitive radiochemical-HPLC assay for urate oxidase activity in untreated plasma, in which 14C in urate and in the reaction product, allantoin, is monitored in the uv detector effluent with a flow-through scintillation counter. The assay is linear with amount of enzyme and time of incubation and can detect less than 1 x 10(-5) U/ml uricase in plasma. The assay accounts for plasma samples of widely differing urate content.     

Contributions of the peroxisome and β-oxidation cycle to biotin synthesis in fungi

The first step in the synthesis of the bicyclic rings of D-biotin is mediated by 8-amino-7-oxononanoate (AON) synthase, which catalyzes the decarboxylative condensation of l-alanine and pimelate thioester. We found that the Aspergillus nidulans AON synthase, encoded by the bioF gene, is a peroxisomal enzyme with a type 1 peroxisomal targeting sequence (PTS1). Localization of AON to the peroxisome was essential for biotin synthesis because expression of a cytosolic AON variant or deletion of pexE, encoding the PTS1 receptor, rendered A. nidulans a biotin auxotroph. AON synthases with PTS1 are found throughout the fungal kingdom, in ascomycetes, basidiomycetes, and members of basal fungal lineages but not in representatives of the Saccharomyces species complex, including Saccharomyces cerevisiae. A. nidulans mutants defective in the peroxisomal acyl-CoA oxidase AoxA or the multifunctional protein FoxA showed a strong decrease in colonial growth rate in biotin-deficient medium, whereas partial growth recovery occurred with pimelic acid supplementation. These results indicate that pimeloyl-CoA is the in vivo substrate of AON synthase and that it is generated in the peroxisome via the β-oxidation cycle in A. nidulans and probably in a broad range of fungi. However, the β-oxidation cycle is not essential for biotin synthesis in S. cerevisiae or Escherichia coli. These results suggest that alternative pathways for synthesis of the pimelate intermediate exist in bacteria and eukaryotes and that Saccharomyces species use a pathway different from that used by the majority of fungi.     

Recapture of [S]-allantoin, the product of the two-step degradation of uric acid, by urate oxidase

Urate oxidase from Aspergillus flavus catalyzes the degradation of uric acid to [S]-allantoin through 5-hydroxyisourate as a metastable intermediate. The second degradation step is thought either catalyzed by another specific enzyme, or spontaneous. The structure of the enzyme was known at high resolution by X-ray diffraction of I222 crystals complexed with a purine-type inhibitor (8-azaxanthin). Analyzing the X-ray structure of urate oxidase treated with an excess of urate, the natural substrate, shows unexpectedly that the active site recaptures [S]-allantoin from the racemic end product of a second degradation step.     

A new colorimetric determination of D-amino acid oxidase and urate oxidase activity.

A new method of colorimetric determination of d-amino acid oxidase and urate oxidase using catalase and 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole is reported. This method is based on the combination of two steps of enzyme reactions and colorimetric procedure. The values obtained by this method are satisfactorily correlated with those obtained by the dinitrophenylhydrazine method for d-amino acid oxidase activity and the ultraviolet method for urate oxidase activity and showed good reproducibility and accuracy. It is considered that the method can be useful as a method of activity determination for studying enzyme kinetics and the reaction mechanism.