Uricase biofunctionalized plasmonic sensor for uric acid detection with APTES-modified gold nanotopping.

Current uric acid detection methodologies lack the requisite sensitivity and selectivity for point-of-care applications. Plasmonic sensors, while promising, demand refinement for improved performance. This work introduces a biofunctionalized sensor predicated on surface plasmon resonance to quantify uric acid within physiologically relevant concentration ranges. The sensor employs the covalent immobilization of uricase enzyme using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-Hydroxysuccinimide (NHS) crosslinking agents, ensuring the durable adherence of the enzyme onto the sensor probe. Characterization through atomic force microscopy and Fourier transform infrared spectroscopy validate surface alterations. The Langmuir adsorption isotherm model elucidates binding kinetics, revealing a sensor binding affinity of 298.83 (mg/dL)-1, and a maximum adsorption capacity of approximately 1.0751°. The biofunctionalized sensor exhibits a sensitivity of 0.0755°/(mg/dL), a linear correlation coefficient of 0.8313, and a limit of detection of 0.095 mg/dL. Selectivity tests against potentially competing interferents like glucose, ascorbic acid, urea, D-cystine, and creatinine showcase a significant resonance angle shift of 1.1135° for uric acid compared to 0.1853° for interferents at the same concentration. Significantly, at a low uric acid concentration of 0.5 mg/dL, a distinct shift of 0.3706° was observed, setting it apart from the lower values noticed at higher concentrations for all typical interferent samples. The uricase enzyme significantly enhances plasmonic sensors for uric acid detection, showcasing a seamless integration of optical principles and biological recognition elements. These sensors hold promise as vital tools in clinical and point-of-care settings, offering transformative potential in biosensing technologies and the potential to revolutionize healthcare outcomes in biomedicine.

Discovery of digallic acid as XOD/URAT1 dual target inhibitor for the treatment of hyperuricemia.

The development of XOD/URAT1 dual target inhibitors has emerged as a promising therapeutic strategy for the management of hyperuricemia. Here, through virtual screening, we have identified digallic acid as a novel dual target inhibitor of XOD/URAT1 and subsequently evaluated its pharmacological properties, pharmacokinetics, and toxicities. Digallic acid inhibited URAT1 with an IC50 of 5.34 ± 0.65 µM, which is less potent than benzbromarone (2.01 ± 0.36 µM) but more potent than lesinurad (10.36 ± 1.23 µM). Docking and mutation analysis indicated that residues S35, F241 and R477 of URAT1 confer a high affinity for digallic acid. Digallic acid inhibited XOD with an IC50 of 1.04 ± 0.23 µM. Its metabolic product, gallic acid, inhibited XOD with an IC50 of 0.91 ± 0.14 µM. Enzyme kinetic studies indicated that both digallic acid and gallic acid act as mixed-type XOD inhibitors. It shares the same binding mode as digallic acid, and residues E802, R880, F914, T1010, N768 and F1009 contribute to their high affinity. The anion group (carboxyl) of digallic acid contribute significantly to its inhibition activity on both XOD and URAT1 as indicated by docking analysis. Remarkably, at a dosage of 10 mg/kg in vivo, digallic acid exhibited a stronger urate-lowering and uricosuric effect compared to the positive drug benzbromarone and lesinurad. Pharmacokinetic study indicated that digallic acid can be hydrolyzed into gallic acid in vivo and has a t1/2 of 0.77 ± 0.10 h. Further toxicity evaluation indicated that digallic acid exhibited no obvious renal toxicity, as reflected by CCK-8, biochemical analysis (CR and BUN) and HE examination. The findings of our study can provide valuable insights for the development of XOD/URAT1 dual target inhibitors, and digallic acid deserves further investigation as a potential anti-hyperuricemic drug.

An ultra-sensitive uric acid second generation biosensor based on chemical immobilization of uricase on functionalized multiwall carbon nanotube grafted palm oil fiber in the presence of a ferrocene mediator.

In this work, palm oil fiber (POF) grafted functionalized multiwall carbon nanotube (FMWCNT) decorated ferrocene (Fc) has been drop coated on a platinum electrode (Pt), in which uricase (UOx) has been chemically immobilized for sensitive and selective biosensing of uric acid (UA). Through the use of EDC/NHS, a stable bioelectrode (UOx/Fc/FMWCNT-POF/Pt) was obtained and characterized by FTIR/ATRIR, XRD, Raman, EA/EDX, TGA, SEM, TEM, CV, EIS, CA, and DPV. Results from DPV showed the rapid response of the developed bioelectrode towards UA (0.185 V) with high sensitivity (41.14 µA mM-1) and good limit of detection (19 µM) in the linear range 10-1000 µM. The low value of Michaelis-Menten constant (km = 31.364 µM) shows high affinity of the UA towards the enzyme at the electrode surface. The developed biosensor demonstrates good reproducibility, repeatability, and stability with a deviation of less than 2.5%, and was successfully applied for human blood sample analysis. The CA study revealed a fast response time (2 s) of the sensor. The work has pioneered a new addition to the class of tailorable chemical species for biosensor development and proven to be a promising new tool for point of care testing (POCT) applications.

Electrochemical enzyme-based blood uric acid biosensor: new insight into the enzyme immobilization on the surface of electrode via poly-histidine tag.

In a new approach, we considered the special affinity between Ni and poly-histidine tags of recombinant urate oxidase to utilize Ni-MOF for immobilizing the enzyme. In this study, a carbon paste electrode (CPE) was modified by histidine-tailed urate oxidase (H-UOX) and nickel-metal-organic framework (Ni-MOF) to construct H-UOX/Ni-MOF/CPE, which is a rapid, sensitive, and simple electrochemical biosensor for UA detection. The use of carboxy-terminal histidine-tailed urate oxidase in the construction of the electrode allows the urate oxidase enzyme to be positioned correctly in the electrode. This, in turn, enhances the efficiency of the biosensor. Characterization was carried out by X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDX), Fourier transform infrared spectroscopy (FTIR), Brunauer-Emmett-Teller (BET), and field emission scanning electron microscopy (FE-SEM). At optimum conditions, the biosensor provided a short response time, linear response within 0.3-10 µM and 10-140 µM for UA with a detection limit of 0.084 µM, repeatability of 3.06%, and reproducibility of 4.9%. Furthermore, the biosensor revealed acceptable stability and selectivity of UA detection in the presence of the commonly coexisted ascorbic acid, dopamine, L-cysteine, urea, and glucose. The detection potential was at 0.4 V vs. Ag/AgCl.

Liquid crystal-based sensitive and selective detection of uric acid and uricase in body fluids.

The abnormal levels of uric acid (UA) in body fluids are associated with gout, type (II) diabetes, leukemia, Lesch-Nyhan syndrome, uremia, kidney damage, and cardiovascular diseases. Also, the presence of uricase (UOx) symbolizes genetic disorders and corresponding complications. Therefore, the detection of UA and UOx in the body fluids is significant for clinical diagnosis. 4-Cyano-4'-pentylbiphenyl (5CB, a nematic liquid crystal (LC)) was doped with octadecyl trimethylammonium bromide (OTAB, a cationic surfactant), which formed a self-assembled monolayer at the aqueous/5CB interface. The UOx-catalyzed oxidation of UA yielded H2O2, releasing the single-strand deoxyribonucleic acid (ssDNA) from the nanoceria/ssDNA complex. The interaction of the released ssDNA with OTAB disrupted the monolayer at the aqueous/5CB interface, which resulted in a dark to bright change when observed through a polarized optical microscope. The LC-based sensor allowed the detection of UA with a linear range of 0.01-10 µM and a limit of detection (LOD) of 0.001 µM. The UA detection was also performed in human urine samples and the results were comparable to that of a standard commercial colorimetric method. Similarly, the detection of UOx was performed, with a noted linear range of 20-140 µg/mL. The LOD was as low as 0.34 µg/mL. The detection of UOx was also demonstrated in human serum samples with excellent performance. This method provides a robust sensing platform for the detection of UA and UOx and has potential for applications in clinical analysis.

Urate oxidase-loaded MOF electrodeposited on boron nanosheet-doxorubicin complex as multifunctional nano-enzyme platform for enzymatic and ratiometric electrochemical biosensing.

In this work, a novel multifunctional nano-enzyme platform was developed and used for enzymatic and ratiometric electrochemical biosensing of uric acid (UA). Boron nanosheets (BNSs) were prepared through ultrasound-assisted liquid-phase exfoliation, followed by the loading of doxorubicin (DOX) to form BNSs-DOX complex. The complex was drop-casted on glassy carbon electrode (GCE) surface to prepare BNSs-DOX/GCE. Cobalt-based metal-organic framework (MOF) with encapsulation of urate oxidase (UOx) was in-situ copolymerized and electrodeposited on the BNSs-DOX surface to construct UOx@MOF/BNSs-DOX nanohybrid-modified GCE. The modified electrode serves as an artificial nano-enzyme sensing platform and presents multifunctional functions, including DOX-loaded BNSs carrier, UOx-enzyme immobilization, enzymatic redox and ratiometric electrochemical sensing of UA. The platform was explored as a new ratiometric electrochemical biosensor to detect UA in the concentration range of 0.1-200 µM, with a low limit of detection of 0.025 µM. Experimental results testify high selectivity, sensitivity and stability toward efficient detection of UA over potential interferents, revealing high detection accuracy and repeatability. The explored biosensor shows superior detection performances in real biological samples, together with high detection recoveries. Excellent properties and functions endow the biosensor with great prospects for precise screening and early diagnosis of UA-relevant malignant diseases in clinic.

Carboxylic acid-tethered polyaniline as a generic immobilization matrix for electrochemical bioassays.

Polyaniline (PANI) was functionalized by thiol-ene click chemistry to obtain carboxylic acid-tethered polyaniline (PCOOH). The versatility of PCOOH as an immobilization matrix was demonstrated by constructing four different biosensors for detection of metabolites and cancer biomarker. Immobilization efficiency of PCOOH was investigated by surface plasmon resonance and fluorescence microscopic analysis which revealed dense immobilization of biomolecules on PCOOH as compared to conventional PANI. A sandwich electrochemical biosensor was constructed using PCOOH for detection of liver cancer biomarker, α-fetoprotein (AFP). The sensor displayed sensitivity of 15.24 µA (ng mL-1)-1 cm-2, with good specificity, reproducibility (RSD 3.4%), wide linear range (0.25-40 ng mL-1) at - 0.1 V (vs. Ag/AgCl), and a low detection limit of 2 pg mL-1. The sensor was validated by estimating AFP in human blood serum samples where the AFP concentrations obtained are consistent with the values estimated using ELISA. Furthermore, utilization of PCOOH for construction of enzymatic biosensor was demonstrated by covalent immobilization of glucose oxidase, uricase, and horseradish peroxidase (HRP) for detection of glucose, uric acid, and H2O2, respectively. The biosensors displayed reasonable sensitivity (50, 148, 127 µA mM-1 cm-2), and linear ranges (0.1-5, 0.1-6, 0.1-7 mM) with a detection limit of 10, 1, and 8 µM for glucose, uric acid, and H2O2, respectively. The present study demonstrates the capability of PCOOH to support and enable oxidation of H2O2 generated by oxidase enzymes as well as HRP enzyme catalyzed reduction of H2O2. Thus, PCOOH offers a great promise as an immobilization matrix for development of high-performance biosensors to quantify a variety of other disease biomarkers. Carboxylic acid-tethered polyaniline synthesized by thiol-ene click chemistry was used as matrix to construct four different electrochemical biosensors for detection of cancer biomarker α-fetoprotein, glucose, uric acid, and H2O2.

Synthesis of carbon quantum dots with iron and nitrogen from Passiflora edulis and their peroxidase-mimicking activity for colorimetric determination of uric acid.

Carbon quantum dots co-doped with iron and nitrogen (Fe@NCDs) were synthesized by using Passiflora edulis Sims (P. edulis) as a precursor. The Fe@NCDs exhibit outstanding peroxidase-mimetic activity owing to successful doping with iron resulting in a behavior similar to that of iron porphyrins. In the presence of H2O2, the Fe@NCDs catalyze the oxidation of the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) with a color change from colorless to blue. The blue oxidation product has a characteristic absorption peaking at 652 nm. A colorimetric assay was worked out for uric acid (UA) that measures the hydrogen peroxide produced during oxidation of UA by uricase. Response is linear in the 2-150 µM UA concentration range, and the limit of detection is 0.64 µM. The method was applied to the determination of UA in (spiked) urine, and recoveries ranged from 92.0 to 103.4%. Graphical abstract Schematic representation of the fabrication of iron and nitrogen co-doped carbon dots (Fe@NCDs) using Passiflora edulis Sims as carbon-based materials. First, uric acid (UA) was oxidized to generate H2O2 by uricase. Then, the Fe@NCDs catalyzed the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to form blue-colored oxidized TMB (oxTMB) in the presence of H2O2. UA can be quantified based on the theory.

A ''naked-eye'' colorimetric and ratiometric fluorescence probe for uric acid based on Ti3C2 MXene quantum dots.

In this work, we developed a ''naked-eye'' colorimetric and ratiometric fluorescence probe for a very important biomarker of uric acid (UA). The method was based on the oxidation of UA by uricase to allantoin and hydrogen peroxide, and then o-Phenylenediamine (OPD) was oxidized to the yellow-colored 2,3-diaminophenazine (oxOPD) in the presence of horseradish peroxidase (HRP) and hydrogen peroxide. The fluorescence emission of glutathione functionalized Ti3C2 MQDs (GSH-Ti3C2 MQDs) centered at 430 nm overlaps with the UV absorption of oxOPD at 425 nm to a large extent, which facilitates fluorescence resonance energy transfer (FRET) between GSH-Ti3C2 MQDs and oxOPD. With the increase of the UA concentration, the emission at 430 nm of GSH-Ti3C2 MQDs is progressively quenched and the emission at 568 nm of oxOPD was gradually increased. Moreover, the probe we designed is easier to distinguish with color change by naked eye for the detection of UA. This is the first report about the determination of UA by a ''naked-eye'' colorimetric and ratiometric fluorescence method combining GSH-Ti3C2 MQDs and uricase/HRP enzymes. This work enables assays to perform fluorescence and visual detection of biomarker in biological fluids based on Ti3C2 MQDs.

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.

Nanocapsules of therapeutic proteins with enhanced stability and long blood circulation for hyperuricemia management.

Among a broad spectrum of medical treatments, protein therapeutics holds tremendous opportunities for the treatment of metabolic disorders, cancer, autoimmune diseases and etc. Broad adaption of protein therapeutics, however, still remain challenging, not only because of poor protein stability, but they also experience fast clearance after administrated and elicit immune responses, resulting in undesirable biodistribution and short blood residence time. In this study, we demonstrate a novel protein delivery method via encapsulating therapeutic proteins within thin shells of poly(N-vinylpyrrolidone) (PVP), which leads to significantly improved protein stability, reduced macrophage uptake, prolonged circulation time and reduced immunogenicity. Exemplified with urate oxidase (UOx), the enzyme used for hyperuricemia treatment, as-formed UOx nanocapsules, n(UOx), exhibits enhanced stability, more significant therapeutic effects, and a more than 10-fold improvement in circulation time when compared with native UOx. This technology not only demonstrates the use of UOx nanocapsules for hyperuricemia management, but also provides a general approach for a broad spectrum of therapeutic proteins for in vivo applications.

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.

Modification with polysialic acid-PEG copolymer as a new method for improving the therapeutic efficacy of proteins.

A new protein derivatization method was developed with a block copolymer to reduce the immunogenicity of therapeutic proteins. The block copolymer consisted of polyethylene glycol (PEG) and polysialic acid (PSA), a nonimmunogenic and biodegradable biopolymer. Uricase was used as a model protein. Molecular weight analysis results indicated that the uricase-PEG-PSA conjugate was linked with 2.5 copolymers for each uricase unit. The residual enzyme activity of the uricase with modification by the PEG-PSA copolymer was 72.4%. The tolerance and stability to heat, acid, alkaline, and trypsin treatments significantly improved compared with the native uricase. The immunogenicity of uricase modified with PEG-PSA copolymer was remarkably reduced. The transmission electron microscopy results of the uricase-PEG-PSA conjugate showed a spherical hydrated shell with a larger particle size. These findings proved that the PSA-PEG-protein conjugate is a formulation that can potentially be used to deliver the protein and peptide-based drugs.

Hyperstabilization of Tetrameric Bacillus sp. TB-90 Urate Oxidase by Introducing Disulfide Bonds through Structural Plasticity.

Bacillus sp. TB-90 urate oxidase (BTUO) is one of the most thermostable homotetrameric enzymes. We previously reported [Hibi, T., et al. (2014) Biochemistry 53, 3879-3888] that specific binding of a sulfate anion induced thermostabilization of the enzyme, because the bound sulfate formed a salt bridge with two Arg298 residues, which stabilized the packing between two ß-barrel dimers. To extensively characterize the sulfate-binding site, Arg298 was substituted with cysteine by site-directed mutagenesis. This substitution markedly increased the protein melting temperature by ∼ 20 °C compared with that of the wild-type enzyme, which was canceled by reduction with dithiothreitol. Calorimetric analysis of the thermal denaturation suggested that the hyperstabilization resulted from suppression of the dissociation of the tetramer into the two homodimers. The crystal structure of R298C at 2.05 Å resolution revealed distinct disulfide bond formation between the symmetrically related subunits via Cys298, although the Cß distance between Arg298 residues of the wild-type enzyme (5.4 Å apart) was too large to predict stable formation of an engineered disulfide cross-link. Disulfide bonding was associated with local disordering of interface loop II (residues 277-300), which suggested that the structural plasticity of the loop allowed hyperstabilization by disulfide formation. Another conformational change in the C-terminal region led to intersubunit hydrogen bonding between Arg7 and Asp312, which probably promoted mutant thermostability. Knowledge of the disulfide linkage of flexible loops at the subunit interface will help in the development of new strategies for enhancing the thermostabilization of multimeric proteins.

Zwitterionic gel encapsulation promotes protein stability, enhances pharmacokinetics, and reduces immunogenicity.

Advances in protein therapy are hindered by the poor stability, inadequate pharmacokinetic (PK) profiles, and immunogenicity of many therapeutic proteins. Polyethylene glycol conjugation (PEGylation) is the most successful strategy to date to overcome these shortcomings, and more than 10 PEGylated proteins have been brought to market. However, anti-PEG antibodies induced by treatment raise serious concerns about the future of PEGylated therapeutics. Here, we demonstrate a zwitterionic polymer network encapsulation technology that effectively enhances protein stability and PK while mitigating the immune response. Uricase modified with a comprehensive zwitterionic polycarboxybetaine (PCB) network exhibited exceptional stability and a greatly prolonged circulation half-life. More importantly, the PK behavior was unchanged, and neither anti-uricase nor anti-PCB antibodies were detected after three weekly injections in a rat model. This technology is applicable to a variety of proteins and unlocks the possibility of adopting highly immunogenic proteins for therapeutic or protective applications.

Cloning, Expression, and Purification of Recombinant Uricase Enzyme from Pseudomonas aeruginosa Ps43 Using Escherichia coli.

Uricase is an important microbial enzyme that can be used in the clinical treatment of gout, hyperuricemia, and tumor lysis syndrome. A total of 127 clinical isolates of Pseudomonas aeruginosa were tested for uricase production. A Pseudomonas strain named Ps43 showed the highest level of native uricase enzyme expression. The open reading frame of the uricase enzyme was amplified from Ps43 and cloned into the expression vector pRSET-B. Uricase was expressed using E. coli BL21 (DE3). The ORF was sequenced and assigned GenBank Accession No. KJ718888. The nucleotide sequence analysis was identical to the coding sequence of uricase gene puuD of P. aeruginosa PAO1. We report the successful expression of P. aeruginosa uricase in Escherichia coli. E. coli showed an induced protein with a molecular mass of about 58 kDa that was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. We also established efficient protein purification using the Ni-Sepharose column with activity of the purified enzyme of 2.16 IU and a 2-fold increase in the specific activity of the pure enzyme compared with the crude enzyme.

Comparison of modification of a bacterial uricase with N-hydroxysuccinimide esters of succinate and carbonate of monomethoxyl poly(ethylene glycol).

Uricase after modification with monomethoxy poly(ethylene glycol) (mPEG) is currently the sole agent to treat refractory gout. For formulating Bacillus fastidious uricase, succinimidyl carbonate of mPEG-5000 (SC-mPEG5k) and succinimidyl succinate of mPEG-5000 (SS-mPEG5k) were compared. SC-mPEG5k possessed higher purity, comparable reaction rate constant with glycine but lower hydrolysis rate, and stronger effectiveness to modify amino groups. The uricase possessed two types of amino groups bearing a 25-fold difference in reactivity with SC-mPEG5k or SS-mPEG5k at pH 9.2. Oxonate and xanthine concentration-dependently protected the bacterial uricase from inactivation during PEGylation. With SC-mPEG5k at a molar ratio of 200 to uricase subunits and oxonate of 50 µM, the PEGylated uricase (1) retained about 73% of the original activity, (2) displayed about 10% reactivity to rabbit anti-sera recognizing the native uricase, (3) elicited IgG in rats accounting for about 5% of that by the native uricase, (4) exhibited circulation half-life time of about 25 H in cock plasma in vivo, and (5) concurrently maintained uric acid at lowered levels for over 20 H. Hence, PEGylation with SC-mPEG under the protection of a competitive inhibitor was a practical approach to formulation of the bacterial uricase; protection of enzymes by competitive inhibitors during PEGylation may have universal significance.

Evolutionary history and metabolic insights of ancient mammalian uricases.

Uricase is an enzyme involved in purine catabolism and is found in all three domains of life. Curiously, uricase is not functional in some organisms despite its role in converting highly insoluble uric acid into 5-hydroxyisourate. Of particular interest is the observation that apes, including humans, cannot oxidize uric acid, and it appears that multiple, independent evolutionary events led to the silencing or pseudogenization of the uricase gene in ancestral apes. Various arguments have been made to suggest why natural selection would allow the accumulation of uric acid despite the physiological consequences of crystallized monosodium urate acutely causing liver/kidney damage or chronically causing gout. We have applied evolutionary models to understand the history of primate uricases by resurrecting ancestral mammalian intermediates before the pseudogenization events of this gene family. Resurrected proteins reveal that ancestral uricases have steadily decreased in activity since the last common ancestor of mammals gave rise to descendent primate lineages. We were also able to determine the 3D distribution of amino acid replacements as they accumulated during evolutionary history by crystallizing a mammalian uricase protein. Further, ancient and modern uricases were stably transfected into HepG2 liver cells to test one hypothesis that uricase pseudogenization allowed ancient frugivorous apes to rapidly convert fructose into fat. Finally, pharmacokinetics of an ancient uricase injected in rodents suggest that our integrated approach provides the foundation for an evolutionarily-engineered enzyme capable of treating gout and preventing tumor lysis syndrome in human patients.

A biohybrid hydrogel for the urate-responsive release of urate oxidase.

Functional biomaterials that detect and correct pathological parameters hold high promises for biomedical application. In this study we describe a biohybrid hydrogel that detects elevated concentrations of uric acid and responds by dissolution and the release of uric acid-degrading urate oxidase. This material was synthesized by incorporating PEG-stabilized urate oxidase into a polyacrylamide hydrogel that was crosslinked by the uric acid-sensitive interaction between the uric acid transcription factor HucR and its operator hucO. We characterize the uric acid responsiveness of the material and demonstrate that it can effectively be applied to counteract flares of uric acid in a mouse model. This approach might be a first step towards a biomedical device autonomously managing uric acid burst associated to gouty arthritis and the tumor lysis syndrome.

Preparing a new biosensor for hypoxanthine determination by immobilization of xanthine oxidase and uricase in polypyrrole-polyvinyl sulphonate film.

In this study, a new amperometric biosensor for the determination of hypoxanthine was developed. To this aim, polypyrrole-polyvinyl sulphonate films were prepared on the platinum electrode by the electropolymerization of pyrrole in the presence of polyvinyl sulphonate. Xanthine oxidase and uricase enzymes were immobilized in polypyrrole-polyvinyl sulphonate via the entrapment method. Optimum conditions of enzyme electrode were determined. Hypoxanthine detection is based on the oxidation of hydrogen peroxide at +400 mV produced by the enzymatic reaction on the enzyme electrode surface. The linear working range of biosensor for hypoxanthine was determined. The effects of pH and temperature on the response of the hypoxanthine biosensor were investigated. Optimum pH and temperature were measured as 8 and 30°C, respectively. Operational and storage stability of the biosensor were determined. After 20 assays, the biosensor sustained 74.5% of its initial performance. After 33 days, the biosensor lost 36% of its initial performance. The performance of the biosensor was tested in real samples.