Urea Concentration Range Research Articles

Selective determination of urea using urease immobilized on ZnO nanowires

Well-aligned zinc oxide (ZnO) nanowire arrays were fabricated on gold-coated plastic substrates using a low-temperature aqueous chemical growth (ACG) method. The ZnO nanowire arrays with 50–130 nm diameters and ∼1 μm in lengths were used in an enzyme-based urea sensor through immobilization of the enzyme urease that was found to be sensitive to urea concentrations from 0.1 mM to 100 mM. Two linear sensitivity regions were observed when the electrochemical responses (EMF) of the sensors were plotted vs. the logarithmic concentration range of urea from 0.1 mM to 100 mM. The proposed sensor showed a sensitivity of 52.8 mV/decade for 0.1–40 mM urea and a fast response time less than 4 s was achieved with good selectivity, reproducibility and negligible response to common interferents such as ascorbic acid and uric acid, glucose, K + and Na + ions.

Backbone and Side-Chain Contributions in Protein Denaturation by Urea

Urea is a commonly used protein denaturant, and it is of great interest to determine its interaction with various protein groups to elucidate the molecular basis of its effect on protein stability. Using the Trp-cage miniprotein as a model system, we report what we believe to be the first computation of changes in the preferential interaction coefficient of the protein upon urea denaturation from molecular-dynamics simulations and examine the contributions from the backbone and the side-chain groups. The preferential interaction is obtained from reversible folding/unfolding replica exchange molecular-dynamics simulations of Trp-cage in presence of urea, over a wide range of urea concentration. The increase in preferential interaction upon unfolding is dominated by the side-chain contribution, rather than the backbone. Similar trends are observed in simulations using two different force fields, Amber94 and Amber99sb, for the protein. The magnitudes of the side-chain and backbone contributions differ in the two force fields, despite containing identical protein-solvent interaction terms. The differences arise from the unfolded ensembles sampled, with Amber99sb favoring conformations with larger surface area and lower helical content. These results emphasize the importance of the side-chain interactions with urea in protein denaturation, and highlight the dependence of the computed driving forces on the unfolded ensemble sampled.

High-κ Dy2TiO5 Electrolyte-Insulator-Semiconductor Urea Biosensors

In this study, we report on an electrolyte-insulator-semiconductor (EIS) device with a novel high-k Dy2TiO5 sensing membranes deposited on Si substrates through reactive cosputtering method. The effect of thermal annealing (700, 800, and 900°C) on the structural and surface properties of Dy2TiO5 sensing membrane was investigated by x-ray diffraction, x-ray photoelectron spectroscopy, and atomic force microscopy. The EIS device incorporating a Dy2TiO5 sensing film that had been annealed at 800°C exhibited good sensing characteristics, including a high sensitivity (57.59 mV/pH in solutions from pH 2–12), a small hysteresis voltage (0.2 mV in the pH loop 7 → 4 → 7 → 10 → 7), and a low drift rate (0.362 mV/h in the buffer solution at pH 7). This improvement can be attributed to the small number of crystal defects and the large surface roughness. Furthermore, a hybrid configuration of the Dy2TiO5 membrane-EIS with urease-immobilized film was prepared by the entrapment of enzyme molecules in alginate hydrogel and was demonstrated for urea biosensing. Results demonstrated that the Dy2TiO5 EIS biosensor was able to detect urea with good linearity (R2 = 0.99) and reasonable sensitivity of 118.38 mV/pCurea in the urea concentration range of 1–32 mM.

Equilibrium Studies of Protein Denaturation by Urea - Examining Backbone and Sidechain Contributions

Urea is a commonly used denaturant in protein folding studies, and it is of great interest to determine its interaction with various protein groups to elucidate the molecular basis of its effect on protein stability. Using the Trp-cage miniprotein as a model system, we report changes in preferential interaction coefficient of the protein upon urea denaturation and examine the contribution from the backbone and sidechain groups. The preferential interaction was obtained from reversible folding/unfolding Replica Exchange MD simulations of Trp-cage in presence of urea, over a broad range of urea concentration. The simulations were carried out using two different forcefields. AMBER94 and AMBER99sb, in conjunction with TIP3P water and KBFF urea models. In a given ensemble, folded or unfolded, we find the sidechains have a larger preferential interaction than that of the backbone. The increase in preferential interaction upon unfolding can be accounted entirely by sidechain contributions in AMBER94 simulations. In AMBER99sb, we find that both backbone and sidechain contribute to the increase in preferential interaction, with the sidechain contribution being slightly larger. The differences between two forcefields can be attributed to the unfolded ensemble sampled, with AMBER99sb favoring conformations with larger surface area and lower helical content. Both forcefields show that the preferential binding is dominated by binding of urea to sidechain atoms. This work has been supported by the National Science Foundation (Grant MCB-0543769).

A compact miniaturized continuous flow system for the determination of urea content in milk

A multicommutation-based flow system with photometric detection was developed, employing an analytical microsystem constructed with low temperature co-fired ceramics (LTCC) technology, a solid-phase reactor containing particles of Canavalia ensiformis DC (urease source) immobilized with glutaraldehyde, and a mini-photometer coupled directly to the microsystem which monolithically integrates a continuous flow cell. The determination of urea in milk was based on the hydrolysis of urea in the solid-phase reactor and the ammonium ions produced were monitored using the Berthelot reaction. The analytical curve was linear in the urea concentration range from 1.0 x 10(-4) to 5.0 x 10(-3) mol L(-1) with a limit of detection of 8.0 x 10(-6) mol L(-1). The relative standard deviation (RSD) for a 2.0 x 10(-3) mol L(-1) urea solution was lower than 0.4% (n = 10) and the sample throughput was 13 h(-1). To check the reproducibility of the flow system, calibration curves were obtained with freshly prepared solutions on different days and the RSD obtained was 4.7% (n = 6). Accuracy was assessed by comparing the results of the proposed method with those from the official procedure and the data are in close agreement, at a 95% confidence level.

Dietary nitrogen reduction enhances urea transport across goat rumen epithelium1

Ruminants are very capable of adapting their N homeostasis to a reduced dietary N intake. However, the limits of this physiological adaptation are still unknown. The aim of the present study was to determine the quantity of dietary N intake at which the needs of the animal are still satisfied. A study was performed in young White Saanen goats under conditions of dietary N reduction. Different semisynthetic diets with 19 to 7% CP were fed. Urea transport rates across the rumen epithelium from the blood into the ruminal fluid were quantified by Ussing chamber experiments. Reduced N intake increased urea transport rates across the mucosa, which could be inhibited by phloretin. The role of parietal urease in driving urea transfer across the epithelium was negligible because its activity was inhibited by antibiotics during in vitro incubations of the epithelium. Concentrations of ammonia in the ruminal fluid were decreased by reducing dietary N intake, accompanied by diminished urease activity at the smallest dietary N intake. Over the range of plasma urea concentrations observed in the different feeding groups, salivary urea concentrations were 73% of plasma urea concentrations. By plotting plasma urea concentrations against serosal to mucosal urea flux rates, a threshold at 1.75 mmol of urea/L of plasma could be assessed, below which urea flux was strongly increased. This indicates that rumen urea transfer could be stimulated by decreased plasma urea concentrations via unknown mechanisms. The physiological relevance of this adaptation of the rumen epithelium is that it is considered a central mechanism in the N homeostasis of growing goats under reduced N intake.

Equilibrium Study of Protein Denaturation by Urea

Though urea is commonly used to denature proteins, the molecular mechanism of its denaturing ability is still a subject of considerable debate. Previous molecular dynamics simulation studies have sought to elucidate the mechanism of urea denaturation by focusing on the pathway of denaturation rather than examining the effect of urea on the folding/unfolding equilibrium, which is commonly measured in experiment. Here we report the reversible folding/unfolding equilibrium of Trp-cage miniprotein in the presence of urea, over a broad range of urea concentrations, using all-atom Replica exchange MD simulations. The simulations capture the experimentally observed linear dependence of unfolding free energy on urea concentration. We find that the denaturation is driven by favorable direct interaction of urea with the protein through both electrostatic and van der Waals forces and quantify their contribution. Though the magnitude of direct electrostatic interaction of urea is larger than van der Waals, the difference between unfolded and folded ensembles is dominated by the van der Waals interaction. We also find that hydrogen bonding of urea to the peptide backbone does not play a dominant role in denaturation. The unfolded ensemble sampled depends on urea concentration, with greater urea concentration favoring conformations with greater solvent exposure. The m-value is predicted to increase with temperature and more strongly so with pressure.

Urea-Induced Unfolding of Na,K-ATPase As Evaluated by Electron Paramagnetic Resonance Spectroscopy

Urea-induced unfolding of Na,K-ATPase from pig kidney and from shark salt gland was studied by electron paramagnetic resonance (EPR) spectroscopy of a nitroxyl derivative of maleimide covalently attached to sulfhydryl groups which are essential for activity. Urea-induced structural changes lead to the inhibition of Na,K-ATPase activity. Structural changes detected by EPR are reversible over the whole range of urea concentrations (0-8 M), although activity loss is always irreversible. The structure of the cytoplasmic domain is more accessible and more susceptible to perturbations than is the transmembrane sector of the Na,K-ATPase and thus is more sensitive to denaturant. Conformational changes at the active thiol groups of these enzymes indeed take place before unfolding of the enzyme as a whole, together with enzyme inactivation. Na,K-ATPase from pig kidney is more stable not only to thermal denaturation but also to urea-induced denaturation than is the Na,K-ATPase from shark salt gland. Susceptibility of the latter could arise from the nonhomologous regions in the cytoplasmic domain.

Conformational Properties of β-PrP

Prion propagation involves a conformational transition of the cellular form of prion protein (PrPC) to a disease-specific isomer (PrPSc), shifting from a predominantly alpha-helical conformation to one dominated by beta-sheet structure. This conformational transition is of critical importance in understanding the molecular basis for prion disease. Here, we elucidate the conformational properties of a disulfide-reduced fragment of human PrP spanning residues 91-231 under acidic conditions, using a combination of heteronuclear NMR, analytical ultracentrifugation, and circular dichroism. We find that this form of the protein, which similarly to PrPSc, is a potent inhibitor of the 26 S proteasome, assembles into soluble oligomers that have significant beta-sheet content. The monomeric precursor to these oligomers exhibits many of the characteristics of a molten globule intermediate with some helical character in regions that form helices I and III in the PrPC conformation, whereas helix II exhibits little evidence for adopting a helical conformation, suggesting that this region is a likely source of interaction within the initial phases of the transformation to a beta-rich conformation. This precursor state is almost as compact as the folded PrPC structure and, as it assembles, only residues 126-227 are immobilized within the oligomeric structure, leaving the remainder in a mobile, random-coil state.

Fusarium head blight in wheat: Impact of tillage and other agronomic practices under natural infection

Fusarium head blight (FHB) is a destructive disease of the world's wheat-growing areas. In Argentina, the area under reduced tillage has increased due to soil erosion that threatened productivity. The effect of conventional and no tillage systems on FHB combined with other agronomic practices such as nitrogen fertilization, and the influence of the environmental conditions was analysed under natural infection on different cultivars. Wheat trials were conducted in three consecutive years; maize was the previous crop in the first and wheat the previous crop in the subsequent two years of the study. The experimental design was a split plot, with tillage treatments (conventional tillage and no-till) as the main plots. Fertilizer treatments were applied as a range of urea concentrations. Fusarium head blight was observed in all three experimental years. Differences of disease expression among trials were associated with the environmental factors prevailing during the experiments. In the first year, with moderate weather conditions, the only interactions for FHB incidence were between no-till and urea split doses. Seedling emergence, seed health, thousand kernel weight and deoxynivalenol (DON) concentration did not differ suggesting that the fertilizers and genotypes did not affect disease susceptibility. Climatic conditions prevailing in the second year were more conducive to disease development and severe FHB infection was observed. Tillage and fertilization treatments did not affect the variables analysed. Only cultivars showed significant differences and DON values were high. In the third year, unfavourable conditions for disease development resulted in low disease levels and no effect or interaction among variables was observed. The results obtained would suggest that favourable weather conditions are likely to be more important than tillage practice and fertilizer treatments. Since soil conservation practices have been widely adopted in most areas of Argentina, an integrated used of all the available strategies should be considered to decrease FHB damage.

Structural properties and sensing performance of high- k Nd 2TiO 5 thin layer-based electrolyte–insulator–semiconductor for pH detection and urea biosensing

For high sensitive pH sensing, an electrolyte–insulator–semiconductor (EIS) device with Nd 2TiO 5 thin layers fabricated on Si substrates by means of reactive sputtering and the subsequent post-deposition annealing (PDA) treatment was proposed. In this work, the effect of thermal annealing (600, 700, 800, and 900 °C) on the structural characteristics of Nd 2TiO 5 thin layer was investigated by X-ray diffraction, X-ray photoelectron spectroscopy, and atomic force microscopy. The observed structural properties were then correlated with the resulting pH sensing performances. For enzymatic field-effect-transistors-based urea biosensing, a hybrid configuration of the proposed Nd 2TiO 5 thin layer with urease-immobilized alginate film attached was established. Within the experimental conditions investigated, the EIS device with the Nd 2TiO 5 thin layer annealed at 800 °C exhibited a higher pH detection sensitivity of 57.2 mV/pH, a lower hysteresis voltage of 2.33 mV, and a lower drift rate of 1.80 mV/h compared to those at other annealing temperatures. These results are attributed to the formation of a thinner low- k interfacial layer at the oxide/Si interface and the higher surface roughness occurred at this annealing temperature. Furthermore, the presented urea biosensor was also proved to be able to detect urea with good linearity ( R 2 = 0.99) and reasonable sensitivity of 9.52 mV/mM in the urea concentration range of 3–40 mM. As a whole, the present work has provided some fundamental data for the use of Nd 2TiO 5 thin layer for EIS-based pH detection and the extended application for biosensing.

Iron oxide-chitosan nanobiocomposite for urea sensor

Urease (Ur) and glutamate dehydrogenase (GLDH) have been co-immobilized onto superparamegnatic iron oxide (Fe 3O 4) nanoparticles-chitosan (CH) based nanobiocomposite film deposited onto indium-tin-oxide (ITO) coated glass plate via physical adsorption for urea detection. The magnitude of magnetization (60.9 emu/g) of Fe 3O 4 nanoparticles (∼22 nm) estimated using vibrating sample magnetometer (VSM) indicates superparamagnetic behaviour. It is shown that presence of Fe 3O 4 nanoparticles results in increased active surface area of CH-Fe 3O 4 nanobiocomposite for immobilization of enzymes (Ur and GLDH), enhanced electron transfer and increased shelf-life of nanobiocomposite electrode. Differential pulse voltammetry (DPV) studies show that Ur-GLDH/CH-Fe 3O 4/ITO bioelectrode is found to be sensitive in the 5–100 mg/dL urea concentration range and can detect as low as 0.5 mg/dL. A relatively low value of Michaelis–Menten constant ( K m, 0.56 mM) indicates high affinity of enzymes (Ur and GLDH) for urea detection.

Structural properties and sensing performance of high- k Sm 2O 3 membrane-based electrolyte–insulator–semiconductor for pH and urea detection

For high sensitive pH sensing, an electrolyte–insulator–semiconductor (EIS) device with a samarium oxide (Sm 2O 3) sensing membrane fabricated by reactive sputtering and the subsequent post-deposition annealing (PDA) treatment was proposed. In this work, the effect of thermal annealing (700, 800, and 900 °C) on the structural characteristics of Sm 2O 3 membranes was investigated by X-ray diffraction, X-ray photoelectron spectroscopy, and atomic force microscopy. The observed structural properties were then correlated with the resulting pH sensing performances. For enzymatic field-effect-transistors-based urea biosensing, a hybrid configuration of the proposed Sm 2O 3 membrane with urease-immobilized alginate film attached was established. The EIS device with the Sm 2O 3 membrane annealed at 700 °C exhibited a higher detection sensitivity of 56.2 mV/pH, a lower hysteresis voltage of 6.2 mV, and a lower drift rate of 1.29 mV/h compared to those at other annealing temperatures. These results are attributed to the formation of a thinner low- k interfacial layer at the oxide/Si interface and the higher surface roughness. Furthermore, the presented urea biosensor was proved to be able to detect urea with good linearity ( R 2 = 0.99) and reasonable sensitivity of 2.457 mV/mM in the urea concentration range of 5–40 mM.

Rapid-Quench Mixing and Use of Fast Footprinting to Characterize DNA Opening in the Late Steps of Open Complex Formation at λPR by E. coli RNA Polymerase

The precise mechanism by which multi-subunit, DNA-dependent RNA polymerases (RNAP) recognize promoter DNA and form stable, transcriptionally-competent open complexes (RPo) remains elusive. Chemical and enzymatic footprints of early intermediates and RPo provide detailed pictures of the DNA in these initial and final complexes in transcription initiation. We have recently discovered that the start site for transcription (+1) remains double-stranded when it is first loaded into the active site channel of E. coli RNAP at the λ phage PR promoter (Davis et al. PNAS, 2007). These experiments reveal unambiguously that opening of the transcription bubble occurs subsequently in the “jaws” of RNAP as RNAP actively destabilizes the duplex. To probe whether opening of ∼14 bp of DNA occurs in stages or is “all or none” we are using the perturbants urea and KCl to destabilize RPo and populate late transcription initiation intermediates. Analysis of the dissociation kinetics over a range of urea and KCl concentrations provides evidence for two transient intermediates in the DNA opening process. Using KMnO4 footprinting we are probing the extent of DNA opening in these intermediates under these conditions. Results from these novel rapid (millisecond) footprinting experiments and the implications for the mechanism of promoter opening will be presented.

On the role of some conserved and nonconserved amino acid residues in the transitional state and intermediate of apomyoglobin folding

The effect of some amino acid residues in A, B, G, and H helices on the folding nucleus and folding intermediate state formation was estimated. For four apomyoglobin mutant forms with point replacements of hydrophobic amino acid residues by Ala, the influence of the substitutions on the stability of native (N) protein and its folding intermediate state (I) was studied, as well as on the protein folding/unfolding rates. Equilibrium and kinetic studies on mutant proteins over a wide range of urea concentrations have shown that the protein native state was strongly destabilized in comparison with that of the wild type protein. At the same time, stability of the intermediate state changed insignificantly. It was shown that amino acid residues of A, G, and H helices make a small contribution to apomyoglobin folding nucleus stabilization in the rate-limiting I reversible N transition, which occurred after the intermediate state was formed. But the amino acid residue of B-helix was very important for the folding nucleus stabilization in the transition state upon the I reversible N transition.

Urease immobilization on biotinylated polypyrrole coated ChemFEC devices for urea biosensor development

In this report, we describe a novel strategy for the design of a clinical urea biosensor using a process based on assembled multilayer systems. Biotinylated enzyme (urease–subiotin) was immobilized on the biotinylated polypyrrole coated Chemical field effect capacitance (ChemFEC) devices using the high avidin–biotin affinity. The immobilized enzyme activity was checked by its catalytic conversion of urea into carbon dioxide and ammonia. Electrochemical response of the bridge system constructed on Si/SiO 2/Si 3N 4 electrodes to urea addition was evaluated using the capacity–potential measurements. In addition, contact-angle measurements were carried out to control the change of surface energy and their components before and after each layer formation. The developed urea biosensor demonstrates high performances: a good sensitivity of 50 mV/pUrea in the linear urea concentration range from 10 −4 to 10 −1 M and an excellent operational stability after three weeks of continuous use. The immobilized urease was characterised through its apparent Michaelis–Menten constant (5 mM) and the activation energy of the enzymatic reaction (20 kJ mol −1). It was also shown that capacitive measurements can be used to quantify the interaction between molecular systems, based on avidin and biotin molecules.

Multy-state protein: Determination of carbonic anhydrase free-energy landscape

Studies of the folding pathway of large proteins whose kinetics is complicated due to the formation of several intermediate states are most frequently impeded or totally impossible because of rapid folding phase occurring during instrument dead time. In this paper the obtaining of energy characteristics of one of such proteins—carbonic anhydrase B—is reported. Tryptophan fluorescence and absorption methods have been used to measure the folding and unfolding kinetics of carbonic anhydrase B at different urea concentrations. In spite of the fact that the formation of the initial intermediate state of this protein takes place during the instrument dead time, the population of this state has been estimated in a wide range of urea concentrations. The use of the population of the rapidly formed intermediate state and the effective rates of slow phases of the protein folding/unfolding permitted us to calculate free energies of all the protein states and the height of energy barriers between them. It has been shown that folding of carbonic anhydrase B can be described by a consecutive reaction scheme. The possibility to obtain energy characteristics of carbonic anhydrase would allow studying structural characteristics of both intermediate and transition states via site-directed mutations.

Differential type solid-state urea biosensors based on ion-selective electrodes

Abstract This work presents a differential solid-state urea biosensor based on an ion-selective membrane. The solid-state urea biosensor comprises three parts—a tin oxide (SnO 2 ) pH electrode, a nonactin-based ammonium ion-selective electrode (ISE) and an ammonium ion-selective membrane-based urea electrode. The enzyme was immobilized by the entrapment method onto an ion-selective membrane. For simplicity of fabrication, a SnO 2 /indium tin oxide (ITO) glass device was adopted as a basic substrate. Solid-state urea biosensors based on ammonium ion-selective membrane respond rapidly, stably and linearly in the range of urea concentrations between 0.013 mM and 10 mM. Additionally, the voltage response, selectivity, reproducibility and stability are considered. Experimental results indicate that the effect of the interference ions of a solid-state urea biosensor can be improved using the proposed differential urea sensing structure. Additionally, the biosensors exhibit good operational and storage stability.

Urea-glycerol system: Liquid associated structure studied by dielectric spectroscopy

Dielectric measurements of urea-glycerol mixtures were taken over a wide range of urea concentrations (up to $7.6M$) and temperatures (from $20\phantom{\rule{0.3em}{0ex}}\text{to}\phantom{\rule{0.3em}{0ex}}80\phantom{\rule{0.2em}{0ex}}\ifmmode^\circ\else\textdegree\fi{}\mathrm{C}$). The dielectric properties begin to change as the urea passes a critical molar fraction, which is independent of temperature and corresponds to a ratio of two urea molecules to seven of glycerol. Our dielectric data support a cluster containing nine molecules of glycerol, which is consistent with neutron-scattering data from the literature. Upon addition of urea, two of the glycerol molecules are substituted with urea to form urea-glycerol coclusters.

Unfolding and Refolding of Bovine α-Crystallin in Urea and Its Chaperone Activity

We undertook an unfolding and refolding study of alpha(L)-crystallin in presence of urea to explore the breakdown and formation of various levels of structure and to find out whether the breakdown of various levels of structure occurs simultaneously or in a hierarchal manner. We used various techniques such as circular dichroism, fluorescence spectroscopy, light scattering, polarization to determine the changes in secondary, tertiary, and quaternary structure. Unfolding and refolding occurred through a number of intermediates. The results showed that all levels of structure in alpha(L)-crystallin collapsed or reformed simultaneously. The intermediates that occurred in the 2-4 M urea concentration range during unfolding and refolding differed from each other in terms of the polarity of the tryptophan environment. The ANS binding experiments revealed that refolded alpha(L)-crystallin had higher number of hydrophobic pockets compared to native one. On the other hand, polarity of these pockets remained same as that of the native protein. Both light scattering and polarization measurements showed smaller oligomeric size of refolded alpha(L)-crystallin. Thus, although the secondary structural changes were almost reversible, the tertiary and quaternary structural changes were not. The refolded alpha(L)-crystallin had more exposed hydrophobic sites with increased binding affinity. The refolded form also showed higher chaperone activity than native one. Since the refolded form was smaller in oligomeric size, some buried hydrophobic sites were available. The higher chaperone activity of lower sized oligomer of alpha(L)-crystallin again revealed that chaperone activity was dependent on hydrophobicity and not on oligomeric size.