Full pH Range Research Articles

Full pH-range responsive hyperbranched polyethers: synthesis and responsiveness

In order to impart full pH-range responsiveness within biocompatible hyperbranched polyethers, new amphiphilic polyethers, i.e. HPMHO–Amines and HPMHO–Carboxys, which have a molecular structure similar to hyperbranched PEG, were prepared through ring-opening polymerization and modified by amination or carboxylation.

Mapping the Pharyngeal and Intestinal pH of Caenorhabditis elegans and Real-Time Luminal pH Oscillations Using Extended Dynamic Range pH-Sensitive Nanosensors

Extended dynamic range pH-sensitive ratiometric nanosensors, capable of accurately mapping the full physiological pH range, have been developed and used to characterize the pH of the pharyngeal and intestinal lumen of Caenorhabditis elegans in real-time. Nanosensors, 40 nm in diameter, were prepared by conjugating pH-sensitive fluorophores, carboxyfluorescein (FAM) and Oregon Green (OG) in a 1:1 ratio, and a reference fluorophore, 5-(and-6)-carboxytetramethylrhodamine (TAMRA) to an inert polyacrylamide matrix. Accurate ratiometric pH measurements were calculated through determination of the fluorescence ratio between the pH-sensitive and reference fluorophores. Nanosensors were calibrated with an automated image analysis system and validated to demonstrate a pH measurement resolution of ±0.17 pH units. The motility of C. elegans populations, as an indicator for viability, showed nematodes treated with nanosensors, for concentrations ranging from 50.00 to 3.13 mg/mL, were not statistically different to nematodes not challenged with nanosensors up to a period of 4 days (p < 0.05). The nanosensors were also found to remain in the C. elegans lumen >24 h after nanosensor challenge was removed. The pH of viable C. elegans lumen was found to range from 5.96 ± 0.31 in the anterior pharynx to 3.59 ± 0.09 in the posterior intestine. The pharyngeal pumping rate, which dictates the transfer of ingested material from the pharynx to the intestine, was found to be temperature dependent. Imaging C. elegans at 4 °C reduced the pharyngeal pumping rate to 7 contractions/min and enabled the reconstruction of rhythmic pH oscillations in the intestinal lumen in real-time with fluorescence microscopy.

Electrochemical response of titania, zirconia, and alumina electrodes to phosphate adsorption

Low-range (nanomolar), on-site sensing of phosphate concentrations in water is yet to be available, as current methods trade off sensitivity for processing time. To aid in the development of an electrochemical phosphate sensor that satisfies these needs, metal oxides were tested for sensitivity to the phosphate analyte. Titania (TiO2), zirconia (ZrO2) and alumina (γ-Al2O3) coated electrodes produced via sol–gel methods were compared as sensing materials for this purpose, as these materials strongly adsorb phosphate. Perturbations of cyclic voltammograms and electrochemical impedance spectra indicated the effects of phosphate addition over the pH range of 6.00–8.75. Potential cycling of the titania and zirconia electrodes produced cathodic features that showed competition between the phosphate ion and OH−. Both lower pH and greater phosphate concentrations increased capacitance for all metal oxides tested. Titania and zirconia electrodes produced a lin–log relationship between the phase of impedance and phosphate concentration, and the relationship proved to be definitive for the porous titania electrodes over the full pH range tested. Titania also showed lesser memory effects with respect to the signal coming from phosphate adsorption than did that of zirconia. Overall, the titania-coated electrode is better poised for purposes of developing a sensor for phosphate.

A study on the self-assembly behavior of dark materials from molasses

We have previously demonstrated that dark materials (DM) in acidified molasses are effectively adsorbed to Amberlite XAD7HP resin and are eluted from the resin with 0.1 M sodium hydroxide. In this paper, we have characterized the self-assembly behavior of molasses DM by using dynamic and static light scattering in combination with isoelectric focusing and infrared absorption spectroscopy in order to better understand the resin adsorption mechanism. One of DM derivatives, X-G2, contained carboxyl and hydroxyl groups and had a weight-average molar mass of 9.39 × 10(3) to 4.42 × 10(4) at pH 2.1-11.5. The aggregates retained their spherical shape over the full pH range and the large gyration radius (66.4-80.0 nm) indicated that the inner structure was loosely packed. Furthermore, X-G2 had an isoelectric point of 1.8, and its density increased sharply at pH 5.9 and then approached a nearly constant value under alkaline conditions. In summary, the self-assembly processes of DM are controlled by intermolecular hydrogen-bonding and hydrophobic interactions. The aggregates adsorb to the resin through hydrophobic interactions and are eluted when excess carboxylate anions are generated.

Mapping the HLA-DO/HLA-DM complex by FRET and mutagenesis

HLA-DO (DO) is a nonclassic class II heterodimer that inhibits the action of the class II peptide exchange catalyst, HLA-DM (DM), and influences DM localization within late endosomes and exosomes. In addition, DM acts as a chaperone for DO and is required for its egress from the endoplasmic reticulum (ER). These reciprocal functions are based on direct DO/DM binding, but the topology of DO/DM complexes is not known, in part, because of technical limitations stemming from DO instability. We generated two variants of recombinant soluble DO with increased stability [zippered DOαP11A (szDOv) and chimeric sDO-Fc] and confirmed their conformational integrity and ability to inhibit DM. Notably, we found that our constructs, as well as wild-type sDO, are inhibitory in the full pH range where DM is active (4.7 to ∼6.0). To probe the nature of DO/DM complexes, we used intermolecular fluorescence resonance energy transfer (FRET) and mutagenesis and identified a lateral surface spanning the α1 and α2 domains of szDO as the apparent binding site for sDM. We also analyzed several sDM mutants for binding to szDOv and susceptibility to DO inhibition. Results of these assays identified a region of DM important for interaction with DO. Collectively, our data define a putative binding surface and an overall orientation of the szDOv/sDM complex and have implications for the mechanism of DO inhibition of DM.

Meta‐barcoding of ‘dirt’ DNA from soil reflects vertebrate biodiversity

DNA molecules originating from animals and plants can be retrieved directly from sediments and have been used for reconstructing both contemporary and past ecosystems. However, the extent to which such 'dirt' DNA reflects taxonomic richness and structural diversity remains contentious. Here, we couple second generation high-throughput sequencing with 16S mitochondrial DNA (mtDNA) meta-barcoding, to explore the accuracy and sensitivity of 'dirt' DNA as an indicator of vertebrate diversity, from soil sampled at safari parks, zoological gardens and farms with known species compositions. PCR amplification was successful in the full pH range of the investigated soils (6.2 ± 0.2 to 8.3 ± 0.2), but inhibition was detected in extracts from soil of high organic content. DNA movement (leaching) through strata was evident in some sporadic cases and is influenced by soil texture and structure. We find that DNA from the soil surface reflects overall taxonomic richness and relative biomass of individual species. However, one species that was recently introduced was not detected. Furthermore, animal behaviour was shown to influence DNA deposition rates. The approach potentially provides a quick methodological alternative to classical ecological surveys of biodiversity, and most reliable results are obtained with spatial sample replicates, while relative amounts of soil processed per site is of less importance.

Reengineering Rate-Limiting, Millisecond Enzyme Motions by Introduction of an Unnatural Amino Acid

Rate-limiting millisecond motions in wild-type (WT) Ribonuclease A (RNase A) are modulated by histidine 48. Here, we incorporate an unnatural amino acid, thia-methylimidazole, at this site (H48C-4MI) to investigate the effects of a single residue on protein motions over multiple timescales and on enzyme catalytic turnover. Molecular dynamics simulations reveal that H48C-4MI retains some crucial WT-like hydrogen bonding interactions but the extent of protein-wide correlated motions in the nanosecond regime is decreased relative to WT. NMR Carr-Purcell-Meiboom-Gill relaxation dispersion experiments demonstrate that millisecond conformational motions in H48C-4MI are present over a similar pH range compared to WT. Furthermore, incorporation of this nonnatural amino acid allows retention of WT-like catalytic activity over the full pH range. These studies demonstrate that the complexity of the protein energy landscape during the catalytic cycle can be maintained using unnatural amino acids, which may prove useful in enzyme design efforts.

Sensor array-based optical portable instrument for determination of pH

A portable optical instrument is presented that makes it possible to determine pH with a colorimetric sensor array. The use of four membranes containing acid–base indicators makes it possible to cover the full range of pH using the H (hue) coordinate measurements of the HSV colour space. pH sensitive membranes were directly cast onto a plastic support to form a two-dimensional array, located between an OLED display as the programmable light source and a set of digital colour detectors. The resulting microcontroller-based system is immune to optical and electrical interferences. A complete optical and electrical characterization and optimization of the hand-held instrument was carried out. The instrument was used to determine pH using a simple algorithm to select the sensor output that was programmed in the microcontroller. The initial eleven candidate pH membranes were reduced to only four, which permit to obtain reliable pH values. The pH response of the selected four sensing elements was modelled, and calibration curves were applied to a validation set and real samples obtaining positive correlations between the real and predicted data.

Determination of chloramphenicol by differential pulse voltammetry at carbon paste electrodes – The use of sodium sulfite for removal of oxygen from electrode surface

The possibility of determination of chloramphenicol by differential pulse voltammetry at four different carbon paste electrodes, in the full pH range (2–12) of Britton–Robinson (BR) buffer was investigated. Electrodes were prepared by mixing spectroscopic graphite powder or glassy carbon microbeads with mineral oil (Nujol) or tricresyl phosphate. Under optimal conditions (BR buffer pH 12, the electrode prepared from glassy carbon microbeads and tricresyl phosphate), linear calibration graph was obtained only in 10–5 M chloramphenicol concentration range. Determination of lower concentrations of chloramphenicol was complicated by irreproducible peak of oxygen from the carbon paste which overlapped with peak of chloramphenicol. Addition of sodium sulfite removed the oxygen peak without influence on the peak of chloramphenicol. Under optimal conditions (electrode paste made from glassy carbon microbeads, BR buffer pH 10 and 0.5 M sodium sulfite), straight calibration line was obtained in the 10–6 and 10–5 M chloramphenicol concentration range. Limit of determination was 5 × 10–7 mol/l.

Full-range optical pH sensor based on imaging techniques

A new colour-based disposable sensor array for a full pH range (0–14) is described. The pH sensing elements are a set of different pH indicators immobilized in plasticized polymeric membranes working by ion-exchange or co-extraction. The colour changes of the 11 elements of the optical array are obtained from a commercial scanner using the hue or H component of the hue, saturation, value ( HSV) colour space, which provides a robust and precise parameter, as the analytical parameter. Three different approaches for pH prediction from the hue H of the array of sensing elements previously equilibrated with an unknown solution were studied: Linear model, Sigmoid competition model and Sigmoid surface model providing mean square errors (MSE) of 0.1115, 0.0751 and 0.2663, respectively, in the full-range studied (0–14). The performance of the optical disposable sensor was tested for pH measurement, validating the results against a potentiometric reference procedure. The proposed method is quick, inexpensive, selective and sensitive and produces results similar to other more complex optical approaches for broad pH sensing.

Full-range optical pH sensor array based on neural networks

A neural network multivariate calibration is used to predict the pH of a solution in the full-range (0–14) from hue (H) values coming from imaging an optical pH sensor array based on 11 sensing elements with immobilized pH indicators. Different colorimetric acid-base indicators were tested for membrane preparation fulfilling the following conditions: 1) no leaching; 2) change in tonal coordinate by reaction and 3) covering the full pH range with overlapping between their pH responses. The sensor array was imaged after equilibration with a solution using a scanner working in transmission mode. Using software developed by us, the H coordinate of the colour space HSV was calculated from the RGB coordinates of each element. The neural network was trained with the calibration data set using the Levenberg–Marquardt training method. The network structure has 11 input neurons (each one matching the hue of a single element in the sensor array), 1 output (the pH approximation value) and 1 hidden layer with 10 hidden neurons. The network provides an MSE = 0.0098 in the training data, MSE = 0.0183 in the validation data and MSE = 0.0426 in the test data coming from a set of real water samples. The resulting correlation coefficient R obtained in the Pearson correlation test is R = 0.999.

Voltammetric Characterization of DNA Intercalators across the Full pH Range: Anthraquinone-2,6-disulfonate and Anthraquinone-2-sulfonate

The use of anthraquinone and its derivatives, notably the sulfonate and disulfonate salts, for the detection of DNA via electrochemical techniques, has been the focus of a number of recent articles. This study provides a quantitative model of the two redox systems of anthraquinone-2,6-disulfonate and anthraquinone-2-sulfonate, over the full aqueous pH range (0-13); the model is based upon the theoretical "scheme of squares" for a 2H(+), 2e(-) system, as first proposed by Jacq (Jacq, J. J. Electroanal. Chem. 1971, 29, 149-180). The effect of pH and ionic strength on the observed cyclic voltammetry was investigated experimentally. The variation of the electrochemical response with proton concentration was modeled through use of the commercially available simulation software, DIGISIM; the system was successfully fitted with attention to voltammetric peak height, position, width, and shape. The model demonstrates how the pK(a) values of the anthraquinone intermediates dominate the observed pH dependence of the voltammetry. At high pH (above pH 12), a simple EE process is found to occur. As the pH decreases, the formation of other protonated species becomes possible; this not only causes a Nernstian shift in the measured electrochemical potential for the redox couple but also results in changes in the mechanistic pathway. At pH 10, an EECC process dominates, as the pH is further lowered into the range 4-7, the overall mechanism is an ECEC process, and finally a CECE mechanism operates at around pH 1 and below. This work provides physical insight into the complex mechanistic pathways involved and will aid the future development of more sophisticated and accurate anthraquinone based DNA sensors.

Solubility of B-Nb2O5 and the Hydrolysis of Niobium(V) in Aqueous Solution as a Function of Temperature and Ionic Strength

B-Nb2O5 was recrystallized from commercially available oxide, and XRD analyses indicated that it is stable in contact with solutions over the pH range 0 to 9, whereas solid polyniobates such as Na8Nb6O19⋅13H2O(s) appear to predominate at pH>9. Solubilities of the crystalline B-Nb2O5 were determined in five NaClO4 solutions (0.1≤Im/mol⋅kg−1≤1.0) over a wide pH range at (25.0±0.1) °C and at 0.1 MPa. A limited number of measurements were also made at Im=6.0 mol⋅kg−1, whereas at Im=1.0 mol⋅kg−1 the full range of pH was also covered at (10, 50 and 70) °C. The pH of these solutions was fixed using either HClO4 (pH≤4) or NaOH (pH≥10) and determined by mass balance, whereas the pH on the molality scale was measured in buffer mixtures of acetic acid + acetate (4≤pH≤6), Bis-Tris (pH≈7), Tris (pH≈8) and boric acid + borate (pH≈9). Treatment of the solubility results indicated the presence of four species, \(\mathrm{Nb(OH)}_{n}^{5-n}\) (where n=4–7), so that the molal solubility quotients were determined according to: $$0\mathrm{.5Nb}_{2}\mathrm{O}_{5}\mathrm{(cr)+0}\mathrm{.5(2}n-5\mathrm{)H}_{2}\mathrm{O(l)}_{\leftarrow}^{\to}\mathrm{Nb(OH)}_{n}^{5-n}+(n-5)\mathrm{H}^{+}\quad (n=4\mbox{--}7)$$ and were fitted empirically as a function of ionic strength and temperature, including the appropriate Debye-Huckel term. A Specific Interaction Theory (SIT) approach was also attempted. The former approach yielded the following values of log 10Ksn (infinite dilution) at 25 °C: −(7.4±0.2) for n=4; −(9.1±0.1) for n=5; −(14.1±0.3) for n=6; and −(23.9±0.6) for n=7. Given the experimental uncertainties (2σ), it is interesting to note that the effect of ionic strength only exceeded the combined uncertainties significantly in the case of log 10Ks6 to Im=1.0 mol⋅kg−1, such that these values may be of use by defining their magnitudes in other media. Values of ΔfGo, ΔfHo, So and \(C_{p}^{\mathrm{o}}\) (298.15 K, 0.1 MPa) for each hydrolysis product were calculated and tabulated.

Polymeric Nanosensors for Measuring the Full Dynamic pH Range of Endosomes and Lysosomes in Mammalian Cells

Polymer nanoparticle sensors have been constructed for studying pH in the endocytic pathway in mammalian cells. The pH sensors for fluorescence ratiometric measurements were prepared using inverse microemulsion polymerization with rhodamine as reference fluorophor and fluorescein and oregon green as pH sensitive dyes, which gave a dynamic pH measurement range from 4.1-7.5. Thus, the sensors cover the pH range of almost all intracellular compartments in mammalian cells. Both neutral and cationic polyacrylamide particles were synthesized where (3-acrylamidopropyl) trimethylammonium chloride was used to introduce a net positive charge in the cationic particles. It was found that the positively charged particle sensors were internalized spontaneously by HepG2 cancer cells. These new pH nanosensors are potential tools in time resolved quantification of pH in the endocytic pathway of living cells.

Modular coenzyme specificity: A domain‐swopped chimera of glutamate dehydrogenase

Domain-swopped chimeras of the glutamate dehydrogenases from Clostridium symbiosum (CsGDH) (NAD(+)-specific) and Escherichia coli (EcGDH) (NADP(+)-specific) have been produced, with the aim of testing the localization of determinants of coenzyme specificity. An active chimera consisting of the substrate-binding domain (Domain I) of CsGDH and the coenzyme-binding domain (Domain II) of EcGDH has been purified to homogeneity, and a thorough kinetic analysis has been carried out. Results indicate that selectivity for the phosphorylated coenzyme does indeed reside solely in Domain II; the chimera utilizes NAD(+) at 0.8% of the rate observed with NADP(+), similar to the 0.5% ratio for EcGDH. Positive cooperativity toward L-glutamate, characteristic of CsGDH, has been retained with Domain I. An unforeseen feature of this chimera, however, is that, although glutamate cooperativity occurs only at higher pH values in the parent CsGDH, the chimeric protein shows it over the full pH range explored. Also surprising is that the chimera is capable of catalysing severalfold higher reaction rates (V(max)) in both directions than either of the parent enzymes from which it is constructed.

Mutations alter the sodium versus proton use of a Bacillus clausii flagellar motor and confer dual ion use on Bacillus subtilis motors

Bacterial flagella contain membrane-embedded stators, Mot complexes, that harness the energy of either transmembrane proton or sodium ion gradients to power motility. Use of sodium ion gradients is associated with elevated pH and sodium concentrations. The Mot complexes studied to date contain channels that use either protons or sodium ions, with some bacteria having only one type and others having two distinct Mot types with different ion-coupling. Here, alkaliphilic Bacillus clausii KSM-K16 was shown to be motile in a pH range from 7 to 11 although its genome encodes only one Mot (BCl-MotAB). Assays of swimming as a function of pH, sodium concentration, and ion-selective motility inhibitors showed that BCl-MotAB couples motility to sodium at the high end of its pH range but uses protons at lower pH. This pattern was confirmed in swimming assays of a statorless Bacillus subtilis mutant expressing either BCl-MotAB or one of the two B. subtilis stators, sodium-coupled Bs-MotPS or proton-coupled Bs-MotAB. Pairs of mutations in BCl-MotB were identified that converted the naturally bifunctional BCl-MotAB to stators that preferentially use either protons or sodium ions across the full pH range. We then identified trios of mutations that added a capacity for dual-ion coupling on the distinct B. subtilis Bs-MotAB and Bs-MotPS motors. Determinants that alter the specificity of bifunctional and single-coupled flagellar stators add to insights from studies of other ion-translocating transporters that use both protons and sodium ions.

Residue-Specific pKa Determination of Lysine and Arginine Side Chains by Indirect 15N and 13C NMR Spectroscopy: Application to apo Calmodulin

Electrostatic interactions in proteins can be probed experimentally through determination of residue-specific acidity constants. We describe here triple-resonance NMR techniques for direct determination of lysine and arginine side-chain protonation states in proteins. The experiments are based on detection of nonexchangeable protons over the full range of pH and temperature and therefore are well suited for pKa determination of individual amino acid side chains. The experiments follow the side-chain 15Nzeta (lysine) and 15Nepsilon or 13Czeta (arginine) chemical shift, which changes due to sizable changes in the heteronuclear electron distribution upon (de)protonation. Since heteronuclear chemical shifts are overwhelmed by the charge state of the amino acid side chain itself, these methods supersede 1H-based NMR in terms of accuracy, sensitivity, and selectivity. Moreover, the 15Nzeta and 15Nepsilon nuclei may be used to probe changes in the local electrostatic environment. Applications to three proteins are described: apo calmodulin, calbindin D9k, and FKBP12. For apo calmodulin, residue-specific pKa values of lysine side chains were determined to fall between 10.7 and 11.2 as a result of the high net negative charge on the protein surface. Ideal two-state titration behavior observed for all lysines indicates the absence of significant direct charge interactions between the basic residues. These results are compared with earlier studies based on chemical modification.

CCD camera full range pH sensor array

Changes in colors of an array of optical sensors that responds in full pH range were recorded using a CCD camera. The data of the camera were transferred to the computer through a capture card. Simple software was written to read the specific color of each sensor. In order to associate sensor array responses with pH values, a number of different mathematics and chemometrics methods were investigated and compared. The results show that the use of “Microsoft Excel's Solver” provides results which are in very good agreement with those obtained with chemometric methods such as artificial neural network (ANN) and partial least square (PLS) methods.

MODELING THE BEHAVIOR OF LISTERIA MONOCYTOGENES IN pH‐MODIFIED CHICKEN SALAD DURING COLD STORAGE AND TEMPERATURE ABUSE CONDITIONS

ABSTRACTListeria monocytogenes grows at refrigeration temperatures (5C or below) and tolerates various environmental stressors. The Food and Drug Administration specifies a zero tolerance for this pathogen in certain ready‐to‐eat processed foods. Modeling its dynamic behavior to fluctuation in temperature at various pH levels is critical to the safety of food. This study presents linear and nonlinear models to predict the behavior of L. monocytogenes in pH‐modified chicken salad at various cold storage and temperature abuse conditions. A linear model of the kinetics accounting for simple and interactive effects of storage time, temperature and pH was developed. Predictions of the linear model were inconsistent with laboratory observations. The limitations of the linear model were reflected in the poor correlation of model predictions to the observed values (r2 = 0.58). A proposed nonlinear model was therefore used to model the observed data. The four model parameters (N(0), Cc(0), kmax and Nres ) were optimized for each of the nine treatments. Correlation coefficient (r2) values ranged from 0.70 (pH 5.2, 7.2C) to 0.99 (pH 4.0, 21.1C), indicating an improved accuracy. Developing a functional and validated microbial predictive model for chicken salad requires further analyses and collection of data at additional pH and temperature values to determine a single set of parameter values that would represent the microbial behavior at the full range of pH and temperatures observed under storage conditions. Future experiments should address the adaptive nature of L. monocytogenes, as the response to environmental stressors affects the survival of the organism in food systems.

Location of the Coenzyme Binding Site in the Porcine Mitochondrial NADP-dependent Isocitrate Dehydrogenase

The structure of crystalline porcine mitochondrial NADP-dependent isocitrate dehydrogenase (IDH) has been determined in complex with Mn2+-isocitrate. Based on structural alignment between this porcine enzyme and seven determined crystal structures of complexes of NADP with bacterial IDHs, Arg83, Thr311, and Asn328 were chosen as targets for site-directed mutagenesis of porcine IDH. The circular dichroism spectra of purified wild-type and mutant enzymes are similar. The mutant enzymes exhibit little change in Km for isocitrate or Mn2+, showing that these residues are not involved in substrate binding. In contrast, the Arg83 mutants, Asn328 mutants, and T311A exhibit 3-20-fold increase in the Km(NADP). We propose that Arg83 enhances NADP affinity by hydrogen bonding with the 3'-OH of the nicotinamide ribose, whereas Asn328 hydrogen bonds with N1 of adenine. The pH dependence of Vmax for Arg83 and Asn328 mutants is similar to that of wild-type enzyme, but for all the Thr311 mutants, pK(es) is increased from 5.2 in the wild type to approximately 6.0. We have previously attributed the pH dependence of Vmax to the deprotonation of the metal-bound hydroxyl of isocitrate in the enzyme-substrate complex, prior to the transfer of a hydride from isocitrate to NADP's nicotinamide moiety. Thr311 interacts with the nicotinamide ribose and is the closest of the target amino acids to the nicotinamide ring. Distortion of the nicotinamide by Thr311 mutation will likely be transmitted to Mn2+-isocitrate resulting in an altered pK(es). Because porcine and human mitochondrial NADP-IDH have 95% sequence identity, these results should be applicable to the human enzyme.