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1.
Biosens Bioelectron ; 261: 116486, 2024 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-38861811

RESUMO

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.


Assuntos
Técnicas Biossensoriais , Enzimas Imobilizadas , Ouro , Ressonância de Plasmônio de Superfície , Urato Oxidase , Ácido Úrico , Urato Oxidase/química , Ácido Úrico/química , Ácido Úrico/análise , Ouro/química , Humanos , Enzimas Imobilizadas/química , Técnicas Biossensoriais/métodos , Limite de Detecção , Nanopartículas Metálicas/química , Succinimidas
2.
Int J Biol Macromol ; 269(Pt 1): 131989, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38697425

RESUMO

Uric acid is the end product of purine metabolism in humans due to inactivation of the uricase determined by the mutated uricase gene. Uricase catalyzes the conversion of uric acid into water-soluble allantoin that is easily excreted by the kidneys. Hyperuricemia occurs when the serum concentration of uric acid exceeds its solubility (7 mg/dL). However, modifications to improve the uricase activity is under development for treating the hyperuricemia. Here we designed 7 types of human-porcine chimeric uricase by multiple sequence comparisons and targeted mutagenesis. An optimal human-porcine chimeric uricase mutant (uricase-10) with both high activity (6.33 U/mg) and high homology (91.45 %) was determined by enzyme activity measurement. The engineering uricase was further modified with PEGylation to improve the stability of recombinant protein drugs and reduce immunogenicity, uricase-10 could be more suitable for the treatment of gout and hyperuricemia theoretically.


Assuntos
Polietilenoglicóis , Proteínas Recombinantes de Fusão , Urato Oxidase , Animais , Humanos , Hiperuricemia/tratamento farmacológico , Hiperuricemia/genética , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Polietilenoglicóis/química , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solubilidade , Urato Oxidase/química , Urato Oxidase/genética , Urato Oxidase/metabolismo , Ácido Úrico/metabolismo
3.
Biofabrication ; 16(3)2024 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-38697098

RESUMO

Uricase (EC 1.7.3.3) is an oxidoreductase enzyme that is widely exploited for diagnostic and treatment purposes in medicine. This study focuses on producing recombinant uricase fromE. coliBL21 in a bubble column bioreactor (BCB) and finding the optimal conditions for maximum uricase activity. The three most effective variables on uricase activity were selected through the Plackett-Burman design from eight different variables and were further optimized by the central composite design of the response surface methodology (RSM). The selected variables included the inoculum size (%v/v), isopropylß-d-1-thiogalactopyranoside (IPTG) concentration (mM) and the initial pH of the culture medium. The activity of uricase, the final optical density at 600 nm wavelength (OD600) and the final pH were considered as the responses of this optimization and were modeled. As a result, activity of 5.84 U·ml-1and a final OD600of 3.42 were obtained at optimum conditions of 3% v/v inoculum size, an IPTG concentration of 0.54 mM and a pH of 6.0. By purifying the obtained enzyme using a Ni-NTA agarose affinity chromatography column, 165 ± 1.5 mg uricase was obtained from a 600 ml cell culture. The results of this study show that BCBs can be a highly effective option for large-scale uricase production.


Assuntos
Reatores Biológicos , Urato Oxidase , Urato Oxidase/química , Urato Oxidase/metabolismo , Escherichia coli/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/isolamento & purificação , Concentração de Íons de Hidrogênio
4.
Nano Lett ; 24(22): 6634-6643, 2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38742828

RESUMO

The effect of strong metal-support interaction (SMSI) has never been systematically studied in the field of nanozyme-based catalysis before. Herein, by coupling two different Pd crystal facets with MnO2, i.e., (100) by Pd cube (Pdc) and (111) by Pd icosahedron (Pdi), we observed the reconstruction of Pd atomic structure within the Pd-MnO2 interface, with the reconstructed Pdc (100) facet more disordered than Pdi (111), verifying the existence of SMSI in such coupled system. The rearranged Pd atoms in the interface resulted in enhanced uricase-like catalytic activity, with Pdc@MnO2 demonstrating the best catalytic performance. Theoretical calculations suggested that a more disordered Pd interface led to stronger interactions with intermediates during the uricolytic process. In vitro cell experiments and in vivo therapy results demonstrated excellent biocompatibility, therapeutic effect, and biosafety for their potential hyperuricemia treatment. Our work provides a brand-new perspective for the design of highly efficient uricase-mimic catalysts.


Assuntos
Hiperuricemia , Compostos de Manganês , Óxidos , Urato Oxidase , Hiperuricemia/tratamento farmacológico , Urato Oxidase/química , Urato Oxidase/uso terapêutico , Urato Oxidase/metabolismo , Óxidos/química , Compostos de Manganês/química , Compostos de Manganês/farmacologia , Humanos , Paládio/química , Paládio/farmacologia , Animais , Catálise , Ácido Úrico/química , Camundongos
5.
Bioelectrochemistry ; 158: 108725, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38714062

RESUMO

An enzymatic amperometric uric acid (UA) biosensor was successfully developed by modifying a screen-printed carbon electrode (SPCE) with Prussian blue-poly(3,4-ethylene dioxythiophene) polystyrene sulfonate composite (PB-PEDOT:PSS). The modified SPCE was coated with gold nanoparticles-graphene oxide-chitosan composite cryogel (AuNPs-GO-CS cry). Uricase (UOx) was directly immobilized via chemisorption on AuNPs. The nanocomposite was characterized by scanning electron microscopy, transmission electron microscopy, ultraviolet-visible spectroscopy, and Fourier transform infrared spectroscopy. The electrochemical characterization of the modified electrode was performed by cyclic voltammetry and electrochemical impedance spectroscopy. UA was determined using amperometric detection based on the reduction current of PB which was correlated with the amount of H2O2 produced during the enzymatic reaction. Under optimal conditions, the fabricated UA biosensor in a flow injection analysis (FIA) system produced a linear range from 5.0 to 300 µmol L-1 with a detection limit of 1.88 µmol L-1. The proposed sensor was stable for up to 221 cycles of detection and analysis was rapid (2 min), with good reproducibility (RSDs < 2.90 %, n = 6), negligible interferences, and recoveries from 94.0 ± 3.9 to 101.1 ± 2.6 %. The results of UA detection in blood plasma were in agreement with the enzymatic colorimetric method (P > 0.05).


Assuntos
Técnicas Biossensoriais , Criogéis , Eletrodos , Ouro , Grafite , Limite de Detecção , Nanopartículas Metálicas , Ácido Úrico , Técnicas Biossensoriais/métodos , Ácido Úrico/sangue , Ácido Úrico/análise , Ouro/química , Grafite/química , Criogéis/química , Nanopartículas Metálicas/química , Carbono/química , Polímeros/química , Porosidade , Análise de Injeção de Fluxo , Compostos Bicíclicos Heterocíclicos com Pontes/química , Quitosana/química , Poliestirenos/química , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Humanos , Urato Oxidase/química , Técnicas Eletroquímicas/métodos , Nanocompostos/química , Ferrocianetos/química
6.
Bioorg Chem ; 147: 107381, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38669781

RESUMO

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.


Assuntos
Relação Dose-Resposta a Droga , Inibidores Enzimáticos , Hiperuricemia , Transportadores de Ânions Orgânicos , Proteínas de Transporte de Cátions Orgânicos , Hiperuricemia/tratamento farmacológico , Humanos , Animais , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos/metabolismo , Relação Estrutura-Atividade , Estrutura Molecular , Proteínas de Transporte de Cátions Orgânicos/antagonistas & inibidores , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Urato Oxidase/química , Descoberta de Drogas , Simulação de Acoplamento Molecular , Camundongos , Masculino , Ácido Gálico/química , Ácido Gálico/farmacologia , Ácido Gálico/análogos & derivados , Ratos Sprague-Dawley
7.
Biomacromolecules ; 25(5): 3200-3211, 2024 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-38591457

RESUMO

Achieving efficient and site-specific conjugation of therapeutic protein to polymer is crucial to augment their applicability in the realms of biomedicine by improving their stability and enzymatic activity. In this study, we exploited tetrazine bioorthogonal chemistry to achieve the site-specific conjugation of bottlebrush polymers to urate oxidase (UOX), a therapeutic protein for gout treatment. An azido-functionalized zwitterionic bottlebrush polymer (N3-ZBP) using a "grafting-from" strategy involving RAFT and ATRP methods was synthesized, and a trans-cyclooctene (TCO) moiety was introduced at the polymer end through the strain-promoted azide-alkyne click (SPAAC) reaction. The subsequent coupling between TCO-incorporated bottlebrush polymer and tetrazine-labeled UOX using a fast and safe bioorthogonal reaction, inverse electron demand Diels-Alder (IEDDA), led to the formation of UOX-ZBP conjugates with a 52% yield. Importantly, the enzymatic activity of UOX remained unaffected following polymer conjugation, suggesting a minimal change in the folded structure of UOX. Moreover, UOX-ZBP conjugates exhibited enhanced proteolytic resistance and reduced antibody binding, compared to UOX-wild type. Overall, the present findings reveal an efficient and straightforward route for synthesizing protein-bottlebrush polymer conjugates without compromising the enzymatic activity while substantially reducing proteolytic degradation and antibody binding.


Assuntos
Química Click , Reação de Cicloadição , Polímeros , Urato Oxidase , Urato Oxidase/química , Química Click/métodos , Polímeros/química , Ciclo-Octanos/química , Humanos , Azidas/química , Alcinos/química
8.
Enzyme Microb Technol ; 178: 110439, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38579423

RESUMO

Mini protein mimicking uricase (mp20) has shown significant potential as a replacement for natural enzymes in the development of uric acid biosensors. However, the design of mp20 has resulted to an inactive form of peptide, causing of loss their catalytic activity. Herein, this paper delineates the impact of various metal cofactors on the catalytic activity of mp20. The metal ion-binding site prediction and docking (MIB) web server was employed to identify the metal ion binding sites and their affinities towards mp20 residues. Among the tested metal ions, Cu2+ displayed the highest docking score, indicating its preference for interaction with Thr16 and Asp17 residues of mp20. To assess the catalytic activity of mp20 in the presence of metal ions, uric acid assays was monitored using a colorimetric method. The presence of Cu2+ in the assays promotes the activation of mp20, resulting in a color change based on quinoid production. Furthermore, the encapsulation of the mp20 within zeolitic imidazolate framework-8 (ZIF-8) notably improved the stability of the biomolecule. In comparison to the naked mp20, the encapsulated ZIFs biocomposite (mp20@ZIF-8) demonstrates superior stability, selectivity and sensitivity. ZIF's porous shells provides excellent protection, broad detection (3-100 µM) with a low limit (4.4 µM), and optimal function across pH (3.4-11.4) and temperature (20-100°C) ranges. Cost-effective and stable mp20@ZIF-8 surpasses native uricase, marking a significant biosensor technology breakthrough. This integration of metal cofactor optimization and robust encapsulation sets new standards for biosensing applications.


Assuntos
Técnicas Biossensoriais , Cobre , Simulação de Acoplamento Molecular , Urato Oxidase , Ácido Úrico , Urato Oxidase/química , Urato Oxidase/metabolismo , Ácido Úrico/metabolismo , Cobre/química , Cobre/metabolismo , Estruturas Metalorgânicas/química , Sítios de Ligação , Zeolitas/química , Estabilidade Enzimática , Imidazóis/química , Colorimetria/métodos
9.
Anal Sci ; 40(5): 951-958, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38598048

RESUMO

Daily monitoring of serum uric acid levels is very important to provide appropriate treatment according to the constitution and lifestyle of individual hyperuricemic patients. We have developed a suspension-based assay to measure uric acid by adding a sample solution to the suspension containing micro-sized particles immobilized on uricase and horseradish peroxidase (HRP). In the proposed method, the mediator reaction of uricase, HRP, and uric acid produces resorufin from Amplex red. This resorufin is adsorbed onto enzyme-immobilized micro-sized particles simultaneously with its production, resulting in the red color of the micro-sized particles. The concentration of resorufin on the small surface area of the microscopic particles achieves a colorimetric analysis of uric acid with superior visibility. In addition, ethanol-induced desorption of resorufin allowed quantitative measurement of uric acid using a 96-well fluorescent microplate reader. The limit of detection (3σ) and RSD (n = 3) were estimated to be 2.2 × 10-2 µg/mL and ≤ 12.1%, respectively. This approach could also be applied to a portable fluorometer.


Assuntos
Colorimetria , Enzimas Imobilizadas , Fluorometria , Peroxidase do Rábano Silvestre , Urato Oxidase , Ácido Úrico , Ácido Úrico/sangue , Ácido Úrico/química , Ácido Úrico/análise , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Urato Oxidase/química , Urato Oxidase/metabolismo , Peroxidase do Rábano Silvestre/química , Peroxidase do Rábano Silvestre/metabolismo , Tamanho da Partícula , Humanos , Suspensões , Oxazinas/química
10.
Anal Methods ; 15(20): 2456-2466, 2023 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-37165935

RESUMO

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.


Assuntos
Técnicas Biossensoriais , Nanotubos de Carbono , Humanos , Ácido Úrico/análise , Ácido Úrico/química , Urato Oxidase/química , Nanotubos de Carbono/química , Óleo de Palmeira , Metalocenos , Reprodutibilidade dos Testes , Técnicas Biossensoriais/métodos
11.
Mikrochim Acta ; 190(5): 195, 2023 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-37106244

RESUMO

The synthesis of ultrasmall Ta2O5 nanoparticle anchored Pt atom using aspartic acid-functionalized graphene quantum dot (Asp-GQD) is reported. The Asp-GQD was combined with tantalic acid and chloroplatinic acid to rapidly form water-soluble Ta-Asp-GQD and Pt-Asp-GQD complex. Followed by thermal annealing at 900 °C in N2 to obtain Ta2O5-Asp-GQD-Pt. The study shows that the introduction of Asp-GQD as a chelating agent and p-type semiconductor achieves to the formation of ultrasmall Ta2O5 nanoparticle, PN junction at the interface and Pt single atom anchored on the surface of Ta2O5 nanocrystals. The unique structure realizes ultrahigh uricase activity and catalase activities of Ta2O5-Asp-GQD-Pt. The Ta2O5-Asp-GQD-Pt was used as the bifunctional sensing material for the construction of an electrochemical uric acid sensor. The differential pulse voltammetric current at 0.45 V linearly increases with the increase of uric acid concentration in the range 0.001-5.00 mM with the detection limit of 0.41 µM (S/N = 3). The sensor exhibits a much better sensitivity compared with the reported methods for the detection of uric acid. The proposed analytical method has been applied to the electrochemical detection of uric acid in human serum with a spiked recovery of 95-105%. The study also offers one way to design and synthesize multifunctional sensing materials with high catalytic activity.


Assuntos
Nanopartículas , Pontos Quânticos , Humanos , Ácido Úrico , Urato Oxidase/química , Catalase , Pontos Quânticos/química
12.
Proteins ; 91(7): 967-979, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-36908223

RESUMO

Five mini proteins mimicking uricase comprising 20, 40, 60, 80, and 100 amino acids were designed based on the conserved active site residues within the same dimer, using the crystal structure of tetrameric uricase from Arthrobacter globiformis (PDB ID: 2yzb) as a template. Based on molecular docking analysis, the smallest mini protein, mp20, shared similar residues to that of native uricase that formed hydrogen bonds with uric acid and was chosen for further studies. Although purified recombinant mp20 did not exhibit uricase activity, it showed specific binding towards uric acid and evinced excellent thermotolerance and structural stability at temperatures ranging from 10°C to 100°C, emulating its natural origin. To explore the potential of mp20 as a bioreceptor in uric acid sensing, mp20 was encapsulated within zeolitic imidazolate framework-8 (mp20@ZIF-8) followed by the modification on rGO-screen printed electrode (rGO/SPCE) to maintain the structural stability. An irreversible anodic peak and increased semicircular arcs of the Nyquist plot with an increase of the analyte concentrations were observed by utilizing cyclic voltammetry and electrochemical impedance spectroscopy (EIS), suggesting the detection of uric acid occurred, which is based on substrate-mp20 interaction.


Assuntos
Grafite , Ácido Úrico , Ácido Úrico/análise , Ácido Úrico/química , Urato Oxidase/genética , Urato Oxidase/química , Urato Oxidase/metabolismo , Simulação de Acoplamento Molecular
13.
Anal Biochem ; 664: 115045, 2023 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-36657510

RESUMO

Cascade reactions catalyzed by natural uricase and mimic peroxidase (MPOD) have been applied for uric acid (UA) detection. However, the optimal catalytic activity of MPOD is mostly in acidic conditions (pH 2-5), mismatching the optimal catalytic alkaline environment of uricase. In this paper, using CuSO4 and urea as raw materials, a MPOD with high catalytic activity in alkaline environment was synthesized by hydrothermal method. Then, based on coupling reaction of uricase/UA/MPOD/guaiacol (GA) system, a novel spectrophotometric method was established to detect 5-60 µmol/L UA (limit of detection = 3.14 µmol/L (S/N = 3)) and accurately quantified serum UA (275.6 ± 39.9 µmol/L, n = 5) with 95-105% of standard addition recovery. The results were consistent with commercial UA kit (p > 0.05). The MPOD could replace natural POD to reduce the cost of UA detection due to simple preparation and cheap raw materials, and is expected to achieve the specific detection of some substances, like glucose and cholesterol, combined with glucose oxidase and cholesterol oxidase.


Assuntos
Peroxidase , Ácido Úrico , Peroxidase/química , Cobre , Urato Oxidase/química , Peroxidases
14.
Prep Biochem Biotechnol ; 53(7): 816-826, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36398928

RESUMO

A novel uricase producing marine bacterium Priestia flexa alkaAU was isolated and identified. The 16S rDNA and the uricase coding gene were sequenced, analyzed and submitted to GenBank. The uricase from Priestia flexa alkaAU (PFU) was purified, determined to be 58.87 kDa, and conjugated with carboxymethyl chitosan (CMCS) by ionic gelation. CMCS conjugation had no effect on the optimum pH of PFU but decreased the optimum temperature by 10 °C. CMCS conjugation increased the specific activity of PFU by 53% at the human body temperature (37 °C) and small intestine's pH (pH 6.8). Uricase thermostabilizing ability of CMCS was significant in the range of 37-80 °C but not at lower temperatures. For improvement of the pH stability of PFU, CMCS was more effective at pHs 3-5 than pHs 6-11. CMCS increased the half-life of PFU against artificial intestinal fluid by 1.5 folds, which demonstrated the potential capability of CMCS-PFU for oral administration.


Assuntos
Quitosana , Urato Oxidase , Humanos , Urato Oxidase/química , Quitosana/química
15.
Mikrochim Acta ; 189(9): 326, 2022 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-35948696

RESUMO

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.


Assuntos
Técnicas Biossensoriais , Urato Oxidase , Técnicas Biossensoriais/métodos , Carbono/química , Eletrodos , Enzimas Imobilizadas/química , Histidina , Reprodutibilidade dos Testes , Urato Oxidase/química , Ácido Úrico
16.
Talanta ; 244: 123455, 2022 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-35397324

RESUMO

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.


Assuntos
Técnicas Biossensoriais , Líquidos Corporais , Cristais Líquidos , Líquidos Corporais/química , DNA de Cadeia Simples , Humanos , Peróxido de Hidrogênio/química , Urato Oxidase/química , Urato Oxidase/metabolismo , Ácido Úrico , Água
17.
Talanta ; 243: 123359, 2022 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-35248945

RESUMO

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.


Assuntos
Técnicas Biossensoriais , Estruturas Metalorgânicas , Técnicas Biossensoriais/métodos , Boro , Doxorrubicina , Técnicas Eletroquímicas/métodos , Eletrodos , Limite de Detecção , Estruturas Metalorgânicas/química , Enzimas Multifuncionais , Urato Oxidase/química
18.
Acta Crystallogr D Struct Biol ; 78(Pt 2): 162-173, 2022 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-35102882

RESUMO

The stability of the tetrameric enzyme urate oxidase in complex with excess of 8-azaxanthine was investigated either under high hydrostatic pressure per se or under a high pressure of argon. The active site is located at the interface of two subunits, and the catalytic activity is directly related to the integrity of the tetramer. This study demonstrates that applying pressure to a protein-ligand complex drives the thermodynamic equilibrium towards ligand saturation of the complex, revealing a new binding site. A transient dimeric intermediate that occurs during the pressure-induced dissociation process was characterized under argon pressure and excited substates of the enzyme that occur during the catalytic cycle can be trapped by pressure. Comparison of the different structures under pressure infers an allosteric role of the internal hydrophobic cavity in which argon is bound, since this cavity provides the necessary flexibility for the active site to function.


Assuntos
Urato Oxidase , Argônio , Sítios de Ligação , Pressão Hidrostática , Ligantes , Urato Oxidase/química , Urato Oxidase/metabolismo
19.
J Mater Chem B ; 10(3): 358-363, 2022 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-35005767

RESUMO

We report enzyme-powered upconversion-nanoparticle-functionalized Janus micromotors, which are prepared by immobilizing uricase asymmetrically onto the surface of silicon particles, to actively and rapidly detect uric acid. The asymmetric distribution of uricase on silicon particles allows the Janus micromotors to display efficient motion in urine under the propulsion of biocatalytic decomposition of uric acid and simultaneously detect uric acid based on the luminescence quenching effect of the UCNPs modified on the other side of SiO2. The efficient motion of the motors greatly enhances the interaction between UCNPs and the quenching substrate and improves the uric acid detection efficiency. Overall, such a platform using uric acid simultaneously as the detected substrate and motion fuel offers considerable promise for developing multifunctional micro/nanomotors for a variety of bioassay and biomedical applications.


Assuntos
Nanopartículas Metálicas/química , Dióxido de Silício/química , Ácido Úrico/urina , Armoracia/enzimologia , Enzimas Imobilizadas/química , Fluoretos/química , Fluoretos/efeitos da radiação , Peroxidase do Rábano Silvestre/química , Luz , Limite de Detecção , Nanopartículas Metálicas/efeitos da radiação , Movimento (Física) , Fenilenodiaminas/química , Espectrofotometria , Túlio/química , Túlio/efeitos da radiação , Urato Oxidase/química , Ácido Úrico/química , Itérbio/química , Itérbio/efeitos da radiação , Ítrio/química , Ítrio/efeitos da radiação
20.
J Biomol Struct Dyn ; 40(4): 1461-1471, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-33000700

RESUMO

Urate oxidase (EC 1.7.3.3) is a key enzyme in the purine metabolism which is applied in the treatment of gout and also, as a diagnostic reagent for uric acid detection. In the current study, the trehalose (TRE) effects as an additive on the structural stability and function of uricase were investigated. For recombinant expression of UOX in E. coli BL21 cells, firstly the coding sequence was subcloned into the pET-28a vector and after induction with IPTG, the recombinant UOX was purified by affinity chromatography using a Ni-NTA agarose column. To specify the trehalose effects on the urate oxidase (UOX) structure, optimum pH, optimum temperature, kinetic and thermodynamic parameters and also, the intrinsic fluorescence of UOX in the absence and presence of trehalose were examined. The UOX half-life is 24.32 min at 40 °C, whereas the UOX-TRE has a higher half-life (32.09 min) at this temperature. Generally, our findings confirm that trehalose has a protective effect on the enzyme structure. Optimum pH and temperature were 9 and 25 °C, respectively for both the naked and treated enzymes and their activity retained 42.18 and 64.80%, respectively after 48 h of incubation at room temperature. Also, theoretical results indicate that the random coil of the enzyme was converted to α-helix and ß-sheet in the presence of trehalose which may preserve the integrity of the active site of the enzyme and increased the enzymatic activity. The MD simulation results indicated greater stability of the uricase structure in the presence of trehalose.Communicated by Ramaswamy H. Sarma.


Assuntos
Trealose , Urato Oxidase , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Trealose/química , Urato Oxidase/química , Ácido Úrico/química
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