Ion-exchange medium coated with abundant small zwitterions for the purification of soluble proteins.

A new ion-exchange medium was prepared from magnetic particles of ∼1.0 µm through coating with small zwitterions and then functionalizing with ampholytic groups for the isoelectric point of ∼6.4 and denoted MSP-ZEWB. With Meyerozyma guilliermondii uricase (MGU) as the model of soluble proteins, the purification of a protein via ion-exchange was compared between MSP-ZEWB through elution in discontinuous mode and Toyopearl SP-650C as a classical ion-exchange medium through elution in continuous mode. MGU was adsorbed at pH 7.6 or 8.0 and eluted via competitive displacement by NaCl or electrostatic repulsions with an elution buffer at pH 10 to reverse the type of net charges of MGU. From MSP-ZEWB, MGU was eluted more rapidly with the elution percentages higher than those from Toyopearl SP-650C. For yielding a unit of MGU activity, MSP-ZEWB gave the elution solution volumes that were ∼50% of those obtained with Toyopearl SP-650C. The yields of MGU of the highest purity from MSP-ZEWB were higher than those from Toyopearl SP-650C, but the highest purification folds with both media were comparable. MSP-ZEWB regenerated for 16 times still showed the consistent purification efficacy. Therefore, the ion-exchange media bearing small zwitterion coats showed great promise for the purification of soluble proteins.

Structure-based design of a hyperthermostable AgUricase for hyperuricemia and gout therapy.

Arthrobacter globiformis Uricase (AgUricase) is a homotetrameric uricase with the potential for therapeutic use in treating hyperuricemia-related diseases. To achieve sufficient therapeutic effects, it is essential for this enzyme to have high thermostability and long half-life in physiological condition. To improve the thermostability of this enzyme, we introduced a series of cysteine pair mutations into the AgUricase subunits based on its structural model and studied the thermostability of the mutant enzymes with introduced disulfide bridges. Two intersubunit cysteine pair mutations, K12C-E286C and S296C-S296C, were found to markedly increase the melting temperatures of the corresponding mutant enzymes compared with WT AgUricase. The crystal structure of the K12C-E286C mutant at 1.99 Å resolution confirmed the formation of a distinct disulfide bond between the two subunits in the dimer. Structural analysis and biochemical data revealed that the C-terminal loop of AgUricase was flexible, and its interaction with neighboring subunits was required for the stability of the enzyme. We introduced an additional intersubunit K244C-C302 disulfide bond based on the crystal structure of the K12C-E286C mutant and confirmed that this additional disulfide bond further stabilized the flexible C-terminal loop and improved the thermostability of the enzyme. Disulfide cross-linking also protected AgUricase from protease digestion. Our studies suggest that the introduction of disulfide bonds into proteins is a potential strategy for enhancing the thermostability of multimeric proteins for medical applications.

Utilizing intein-mediated protein cleaving for purification of uricase, a multimeric enzyme.

Uric acid, a side product of nucleotide metabolism, should be cleared from blood stream since its accumulation can cause cardiovascular diseases and gout. Uricase (urate oxidase) converts uric acid to 5-hydroxyisourate, but it is absent in human and other higher apes. Yet, the recombinant form of uricase, Rasburicase, is now commercially available to cure tumor lysis syndrome by lowering serum uric acid level. Developing new methods to efficiently purify pharmaceutical proteins like uricase has attracted researchers' attention. Self-cleaving intein mediated single column purification is one of these novel approaches. Self-cleaving inteins are modified forms of natural inteins that can excise and join only at one junction site. In this study, the synthetic gene of Aspergillus flavus uricase, a homotetrameric protein, was cloned into pTXB1 vector as a fusion with the N-terminal of Mxe GyrA intein and chitin-binding domain (CBD) for simple purification. Expression was confirmed by western blot analysis. The fusion protein containing uricase-intein-CBD was purified on a chitin column. The cleavage was induced by adding DTT,1 as a reducing agent to release uricase. The purity of uricase and complete excision of the intein and CBD were confirmed by SDS-PAGE2 while its proper folding was proved by circular dichroism and fluorescent emission studies. Isoelectric focusing further confirmed its homogeneity when a single protein band was observed at the predicted pI value. This is the first report of successful purification of a multimeric therapeutic enzyme by intein-mediated protein cleaving using a well-established and facile system.

A Numerical Approach for Kinetic Analysis of the Nonexponential Thermoinactivation Process of Uricase.

Prior to the exponential decrease of activity of a uricase from Candida sp. during storage at 37 °C, there was a plateau period of about 4 days at pH 7.4, 12 days at pH 9.2, and about 22 days in the presence of 30 µM oxonate at pH 7.4 or 9.2, but no degradation of polypeptides and no activity of resolved homodimers. To reveal determinants of the plateau period, a dissociation model involving a serial of conformation intermediates of homotetramer were proposed for kinetic analysis of the thermoinactivation process. In the dissociation model, the roles of interior noncovalent interactions essential for homotetramer integrity were reflected by an equivalent number of the artificial weakest noncovalent interaction; to avoid covariance among parameters, the rate constant for disrupting the artificial weakest noncovalent interaction was fixed at the minimum for physical significance of other parameters; among thermoinactivation curves simulated by numerical integration with different sets of parameters, the one for least-squares fitting to an experimental one gave the solution. Results found that the equivalent number of the artificial weakest noncovalent interaction primarily determined the plateau period; kinetics rather than thermodynamics for homotetramer dissociation determined the thermoinactivation process. These findings facilitated designing thermostable uricase mutants.

Economic analysis of uricase production under uncertainty: Contrast of chromatographic purification and aqueous two-phase extraction (with and without PEG recycle).

Uricase is the enzyme responsible for the breakdown of uric acid, the key molecule leading to gout in humans, into allantoin, but it is absent in humans. It has been produced as a PEGylated pharmaceutical where the purification is performed through three sequential chromatographic columns. More recently an aqueous two-phase system (ATPS) was reported that could recover Uricase with high yield and purity. Although the use of ATPS can decrease cost and time, it also generates a large amount of waste. The ability, therefore, to recycle key components of ATPS is of interest. Economic modelling is a powerful tool that allows the bioprocess engineer to compare possible outcomes and find areas where further research or optimization might be required without recourse to extensive experiments and time. This research provides an economic analysis using the commercial software BioSolve of the strategies for Uricase production: chromatographic and ATPS, and includes a third bioprocess that uses material recycling. The key parameters that affect the process the most were located via a sensitivity analysis and evaluated with a Monte Carlo analysis. Results show that ATPS is far less expensive than chromatography, but that there is an area where the cost of production of both bioprocesses overlap. Furthermore, recycling does not impact the cost of production. This study serves to provide a framework for the economic analysis of Uricase production using alternative techniques.

Purification and characterization of an extracellular uricase from a new isolate of Sphingobacterium thalpophilum (VITPCB5).

An extracellular uricase producing bacterium (VITPCB5) was isolated from soil of the duck farm near Chidambaram, Tamilnadu, India and it was identified based on its 16S rRNA as Sphingobacterium thalpophilum. Uric acid was used as an effective inducer. The enzyme kinetics was studied using uric acid as a substrate. The Km and Vmax for the enzyme was found to be 0.28mM and 0.92µM/minml, respectively. Maximum uricase production was observed when lactose was used as a carbon source. Among the nitrogen sources tested, urea gave the maximum uricase production. The enzyme was successfully purified using a weak cation exchange convective interaction media carboxy methyl (CIM-CM) monolith column with a recovery of 79.7%±0.1 and 14.2±1.8-fold purification. The optimal reaction temperature of the enzyme was observed between 25 and 45°C. The pH optimum of the enzyme was 8.0. The enzyme activity was enhanced by copper and partially inhibited by calcium, iron, zinc and nickel ions. Treatment with ethylene diamine tetraacetic acid completely inhibited the enzyme activity. The in-gel trypsin digested peptides of 48-kDa uricase when analyzed using mass spectrometry, gave 32% sequence coverage with the uricase (30-kDa) from Cyberlindnera jadinii.

Characterization of a Thermostable Uricase Isolated from Bacillus firmus DWD-33 and its Application for Uric Acid Quantification in Human Serum.

A thermostable uricase identified in Bacillus firmus DWD-33, which was isolated for the first time from soil, with an apparent molecular weight of 33.5 kDa was stable against oxidants and SDS. The highest expression yields were obtained in medium containing 0.8% maltose and 1.2% soybean powder as carbon and nitrogen sources, respectively. Enzyme purification increased the specific activity about 24-fold with 27% recovery. As compared with other microbial uricases, the pure enzyme showed a high thermostability. The Vmax was 387 µmol/L/min, the turnover number (Kcat) was 21.8x10(3) s(-1) and the catalytic efficiency (Kcat /Km) was 2.76x10(8) s(-1)M(-1). The enzyme was stable from pH 7.0 to 10.0 and up to 70 °C and the optimal conditions were 50 °C and pH 8.0. Mg(2+) significantly enhanced the enzymatic activity, while Hg(2+), EDTA, and o-phenanthroline greatly suppressed the activity. Mg(2+) might be the uricase cofactor, as the enzyme activity was restored after its addition to EDTA-chelated enzyme. Inhibition of the enzyme by the copper- chelating agent 2,9-dimethyl-1,10-phenanthroline suggests that this enzyme belongs to the cuprouricase-type. The purified uricase retained 72% and 82% of its original activity after incubation with 0.5% H2O2 and 0.5% SDS for 6 h, respectively. It was possible to determine uric acid in human sera with the enzyme with none of the tested uric acid analogs being a competitive substrate, indicating a high specificity of uricase with respect to uric acid measurement in vitro for uric acid concentration up to 500 µmol/L.

Enhancement of a novel extracellular uricase production by media optimization and partial purification by aqueous three-phase system.

Uricase (urate: oxygen oxidoreductase, EC 1.7.3.3), an enzyme belonging to the class of oxidoreductases, catalyzes the enzymatic oxidation of uric acid to allantoin and finds a wide variety of application as therapeutic and clinical reagent. In this study, uricase production ability of the bacterial strains isolated from deep litter poultry soil is investigated. The strain with maximum extracellular uricase production capability was identified as Xanthomonas fuscans subsp. aurantifolii based on 16S rRNA sequencing. Effect of various carbon and nitrogen sources on uricase productivity was investigated. The uricase production for this strain was optimized using statistically based experimental designs and resulted in uricase activity of 306 U/L, which is 2 times higher than initial uricase activity. Two-step purification, such as ammonium sulfate precipitation and aqueous two-phase system, was carried out and a twofold increase in yield and specific activity was observed.

Isolation of a novel uric-acid-degrading microbe Comamonas sp. BT UA and rapid biosensing of uric acid from extracted uricase enzyme.

Uric-acid-utilizing soil bacteria were isolated, and 16s rRNA sequence was studied for strain identification. The most prominent uricase-producing bacterium was identified as Comamonas sp BT UA. Crude enzyme was extracted, freeze-dried and its Km and Vmax were determined as 40 meu M and 0.047 meu M min-1ml-1 using Line-weaver Burke plot. An activity of 80 U/mg of total protein was observed when cultured at 37 degree C for 84 h at pH 7. The purified enzyme was used to measure uric acid by spectrophotometric method and electrochemical biosensor. In the biosensing system the enzyme was immobilized on the platinum electrode with a biodegradable glutaraldehyde-crosslinked gelatin film having a swelling percentage of 109+/- 3.08, and response was observed by amperometry applying fixed potential. The electrochemical process as obtained by the anodic peak current and scan rate relationship was further configured by electrochemical impedance spectroscopy (EIS). The polymer matrix on the working electrode gave capacitive response for the electrode-electrolyte interaction. The sensitivity of the biosensor was measured as 6.93 meu A meu M -1 with a sensor affinity [Km(app)] of 50 mu M and 95 percent reproducibility after 50 measurements. The spectrophotometric method could be used in the range of 6-1000 mu M, whereas the biosensor generated linear response in the 1.5- 1000 mu M range with a response time of 24 s and limit of detection of 0.56 meu M. Uric acid was estimated in human blood samples by the biosensor and satisfactory results were obtained.

Intersubunit salt bridges with a sulfate anion control subunit dissociation and thermal stabilization of Bacillus sp. TB-90 urate oxidase.

The optimal activity of Bacillus sp. TB-90 urate oxidase (BTUO) is 45 °C, but this enzyme is one of the most thermostable urate oxidases. A marked increase (>10 °C) in its thermal stability is induced by high concentrations (0.8­1.2 M) of sodium sulfate. Calorimetric measurements and size exclusion chromatographic analyses suggested that sulfate-induced thermal stabilization is related to the binding of a sulfate anion that repressed the dissociation of BTUO tetramers into dimers. To determine the sulfate binding site, the crystal structure was determined at 1.75 Å resolution. The bound sulfate anion was found at the subunit interface of the symmetrical related subunits and formed a salt bridge with two Arg298 residues in the flexible loop that is involved in subunit assembly. Site-directed mutagenesis of Arg298 to Glu was used to extensively characterize the sulfate binding site at the subunit interface. The network of charged hydrogen bonds via the bound sulfate is suggested to contribute significantly to the thermal stabilization of both subunit dimers and the tetrameric assembly of BTUO. Knowledge of the mechanism of salt-induced stabilization will help to develop new strategies for enhancing protein thermal stabilization.

Isolation, screening and production studies of uricase producing bacteria from poultry sources.

Uricase (urate oxidase EC 1.7.3.3) is a therapeutic enzyme that is widely used to catalyze the enzymatic oxidation of uric acid in the treatment of hyperuricemia and gout diseases. In this study, three bacterial species capable of producing extracellular uricase were isolated from a poultry source and screened based on the size of the clear zone using a uric acid agar plate. The bacterial species capable of producing uricase with the highest uricolytic activity was identified as Bacillus cereus strain DL3 using a 16SrRNA gene sequencing approach. The time-course study of uricase production was performed and the medium was optimized. Carboxymethylcellulose and asparagine were found to be the best carbon and nitrogen sources. Maximum uricolytic activity was observed at pH 7.0 with an inducer concentration of 2.0 g/L. Inoculum size of 5% gave maximum uricolytic activity. The maximum uricolytic activity of 15.43 U/mL was achieved at optimized conditions, which is 1.61 times more than the initial activity. Further, enzymatic stability was determined at different pH and temperature.

Development of a new LC-MS/MS based enzyme activity assay for recombinant urate oxidase in plasma and its application to pharmacokinetics in human.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of recombinant urate oxidase in human plasma. This assay was based on the determination of enzyme reaction product, (15)N-allantoin, and phenacetin was used as an internal standard (IS). Separation was achieved on a C18 column by the mobile phase of 30% water (containing 0.5% formic acid) and 70% methanol. Quantification was done using multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z 161 → m/z 118 for (15)N-allantoin and m/z 180 → m/z 110.1 for IS at positive ionization mode. The calibration curve was established over the range of 2.077-42.06 U/l and the correlation coefficient was larger than 0.99. The intra-day and inter-day relative standard deviations were less than 10.6%. Accuracy determined at three concentrations ranged between 98.6% and 109.2%. This method was successfully applied to a pharmacokinetic study of intravenous recombinant urate oxidase produced from Escherichia coli in Chinese healthy volunteers.

High-yield expression, purification, characterization, and structure determination of tag-free Candida utilis uricase.

We report the successful high-yield expression of Candida utilis uricase in Escherichia coli and the establishment of an efficient three-step protein purification protocol. The purity of the recombinant protein, which was confirmed to be C. utilis uricase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer analysis, was >98% and the specific activity was 38.4 IU/mg. Crystals of C. utilis uricase were grown at 18°C using 25% polyethylene glycol 3350 as precipitant. Diffraction by the crystals extends to 1.93 Å resolution, and the crystals belong to the space group P2(1)2(1)2(1) with unit cell parameters a = 69.16 Å, b = 139.31 Å, c = 256.33 Å, and α = ß = γ = 90°. The crystal structure of C. utilis uricase shares a high similarity with other reported structures of the homologous uricases from other species in protein database, demonstrating that the three-dimensional structure of the protein defines critically to the catalytic activities.

Urate oxidase purification by salting-in crystallization: towards an alternative to chromatography.

BACKGROUND: Rasburicase (Fasturtec® or Elitek®, Sanofi-Aventis), the recombinant form of urate oxidase from Aspergillus flavus, is a therapeutic enzyme used to prevent or decrease the high levels of uric acid in blood that can occur as a result of chemotherapy. It is produced by Sanofi-Aventis and currently purified via several standard steps of chromatography. This work explores the feasibility of replacing one or more chromatography steps in the downstream process by a crystallization step. It compares the efficacy of two crystallization techniques that have proven successful on pure urate oxidase, testing them on impure urate oxidase solutions. METHODOLOGY/PRINCIPAL FINDINGS: Here we investigate the possibility of purifying urate oxidase directly by crystallization from the fermentation broth. Based on attractive interaction potentials which are known to drive urate oxidase crystallization, two crystallization routes are compared: a) by increased polymer concentration, which induces a depletion attraction and b) by decreased salt concentration, which induces attractive interactions via a salting-in effect. We observe that adding polymer, a very efficient way to crystallize pure urate oxidase through the depletion effect, is not an efficient way to grow crystals from impure solution. On the other hand, we show that dialysis, which decreases salt concentration through its strong salting-in effect, makes purification of urate oxidase from the fermentation broth possible. CONCLUSIONS: The aim of this study is to compare purification efficacy of two crystallization methods. Our findings show that crystallization of urate oxidase from the fermentation broth provides purity comparable to what can be achieved with one chromatography step. This suggests that, in the case of urate oxidase, crystallization could be implemented not only for polishing or concentration during the last steps of purification, but also as an initial capture step, with minimal changes to the current process.

Designing and engineering of a site-specific incorporation of a keto group in uricase.

Urate oxidase is a potential therapeutic protein in the prevention and treatment of tumor lysis syndrome and hyperuricemia. However, its severe immunogenicity limits its clinical application. In our work, several strides have been made toward engineering site-specific modifications of keto groups in urate oxidase by using evolved Methanocaldococcus jannaschii aminoacyl-tRNA synthetase(s)/suppressor tRNA pairs to reduce its antigenicity. Our approach, described here, consisted of designing a M. jannaschii tyrosyl-tRNA synthetase library based on the homology modeling and molecular docking model of the species-specific TyrRS-Tyr complex. The active mutation was picked, and pBR-RS series vectors were constructed to define the relationship between the expression of aaRS and the efficiency of the orthogonal amber suppressor tRNA/synthetase system. Two sites based on the 3D structure of the Candida utilis uricase, Lys21 and Lys248, were substituted for p-acetyl-l-phenylalanine, and the yields were optimized. The products were purified, and their enzyme activities and antigenic properties were analyzed. The mutated uricase exhibited decreased antigenic properties, while its catalytic activities remained unchanged. This method imparts new insights into structure-function relationship research and provides a means by which site-specific modifications may be achieved by using PEG derivates to improve pharmacological properties of urate oxidase.

[Construction, expression, purification and characterization of mutant of Aspergillus flavus urate oxidase].

We converted the TGC codon (307-309 bp) of Aspergillus flavus urate oxidase (UOX) gene to a GCC codon by using fusion PCR techniques to produce a C103A mutant. This gene was cloned into expression vector pET-42a (+) and then transformed into Escherichia coli BL21 (DE3). The mutant protein (UOX-Ala103) was expressed in soluble form at high levels after induction with IPTG The expressed rUOX-Ala103 accounted for about 45% of total bacterial proteins, rUOX-Ala103 of up to 98% purity was obtained after purified using hydrophobic interaction and anion exchange. Western blotting showed that the anti-UOX antibody specifically recognized rUOX-Ala103. The mutant protein showed a 60% increased in vitro biological activities compared with native protein, and performed a good activity of degrading the uric acid in vivo.

Cloning, purification, and partial characterization of Bacillus subtilis urate oxidase expressed in Escherichia coli.

Urate oxidase (EC 1.7.3.3) is an enzyme involved in purine metabolism which is used in the treatment of gout and as diagnostic reagent for detection of uric acid. In order to produce this enzyme in large quantities for biotechnological purposes, the gene coding for the Bacillus subtilis urate oxidase was cloned and heterologously expressed in Escherichia coli. Time course induction in E. coli showed an induced protein with an apparent molecular mass of approximately 60 kDa. Soluble recombinant enzyme was purified in a single-step procedure using Ni-NTA column. The enzyme was purified 2.1-fold with a yield of 56% compared to the crude extract. MALDI-TOF analysis revealed an ion with a mass of 58675 Da which is in agreement with the expected mass of the recombinant protein. The purified enzyme showed an optimal pH and temperature of 8.0 and 37 degrees C, respectively, and retained 90% of its activity after 72 hours of incubation at -20 degrees C and 4 degrees C.

Fabrication of dissolved O2 metric uric acid biosensor using uricase epoxy resin biocomposite membrane.

Uricase purified from 20-day-old leaves of cowpea was immobilized on to epoxy resin membrane with 80% retention of initial activity of free enzyme and a conjugation yield of 0.056 mg/cm(2). The uricase epoxy resin bioconjugate membrane was mounted over the sensing part of the combined electrode of 'Aqualytic' dissolved O(2) (DO) meter to construct a uric acid biosensor. The biosensor measures the depletion of dissolved O(2) during the oxidation of uric acid by immobilized uricase, which is directly proportional to uric acid concentration. The biosensor showed optimum response within 10-12s at a pH 8.5 and 35 degrees C. A linear relationship was found between uric acid concentration from 0.025 to 0.1 mM and O(2) (mg/l) consumed. The biosensor was employed for measurement of uric acid in serum. The mean value of uric acid in serum was 4.92 mg/dl in apparently healthy males and 3.11 mg/dl in apparently healthy females. The mean analytic recoveries of added uric acid in reaction mixture (8.9 and 9.8 mg/dl) were 93.6 +/- 2.34 and 87.18 +/- 3.17% respectively. The within and between batch CVs were < 6.5 and < 5.0%, respectively. The serum uric acid values obtained by present method and standard enzymic colorimetric method, showed a good correlation (r - 0.996) and regression equation being y - 0.984x + 0.0674. Among the various metabolites tested only, glucose (11%), urea (38%), NaCl (25%) and cholesterol (13%) and ascorbic acid (56%) caused decrease, while, MgSO(4) and CaCl(2) had no effect on immobilized enzyme. The enzyme electrode showed only 32% decrease during its use for 100 times over a period of 60 days at 4 degrees C.

[Expression in Escherichia coli, purification and enzymatic properties of porcine urate oxidase].

The aims of this research were to construct prokaryotic expression vector containing the gene of porcine urate oxidase (pUOX), optimize the conditions of the expression of pUOX in recombinant Escherichia coli BL21(DE3), and analyze the in vitro activity and the enzymological properties of pUOX. The pUOX gene was amplified by RT-PCR from the extracted total RNA of porcine liver, and was inserted into the prokaryotic expression vector pET30a(+) to construct a recombinant expression vector pET30a(+)/pUOX. We identified the recombinant vector by endonuclease digestion and sequence analysis. The pUOX gene was amplified and cloned into the vector pET30a(+) successfully. And then the recombinant vector was transformed into E. coli BL21(DE3). The expression of pUOX with a molecular of approximately 41 kD was induced by IPTG. We also optimized the expression conditions of the recombinant protein. The recombinant protein was mostly located in the cytoplasm and it was insoluble. After the inclusion body was solved in 8 mol/L urea and refolding in 2 mol/L urea, the recombinant protein was collected and purified by Ni2+-NTA column. This recombinant protein had a specific activity of 50.61 IU/mg and showed similar properties of optimum temperature and thermal stability, base on the enzymatic assay and analysis of enzymological properties. These results would help to analyze the in vivo activity by testing animal.

Characterization of a uricase from Bacillus fastidious A.T.C.C. 26904 and its application to serum uric acid assay by a patented kinetic uricase method.

An intracellular uricase from Bacillus fastidious A.T.C.C. 26904 was characterized and evaluated for serum uric acid assay by a patented kinetic uricase method. The active uricase was 151 kDa by gel filtration through Sephadex G-200. Both SDS/PAGE and matrix-assisted laser-desorption ionization-time-of-flight MS resolved a single polypeptide with a molecular mass of approx. 36.0 kDa. The N-terminal sequence was AERTMFYGKGDV. The optimum pH for this uricase ranged from 9.0 to 10.5. At pH 9.2, the Km (Michaelis-Menten constant) was 204+/-14 micromol/l (n=8) and the Ki (inhibition constant) for xanthine was 41+/-7 micromol/l (n=5). By analysing the data monitored within 5 min at 0.03 unit/ml uricase, this kinetic uricase method gave linear response to uric acid in reaction solution from 1.3 to 60 micromol/l. Aside from other common errors, 30 micromol/l xanthine in the reaction solution caused no error in this kinetic uricase method, while it caused negative error in the indirect equilibrium method by peroxidase-coupled assay of H2O2. Uric acid in clinical sera by this kinetic uricase method (Ck) closely and positively correlated with that from the indirect equilibrium method (Ce) (Ck = 0.008+1.081 x Ce, r>0.990, n=99). However, Bland-Altman analysis suggested inconsistency between Ck and Ce. These results indicated that this kinetic uricase method using this uricase was reliable for serum uric acid assay with enhanced resistance to xanthine besides other common errors.