Narrow pH Range Research Articles

Modeling of formation and prevention of a pure water zone in capillary isoelectric focusing with narrow pH range carrier ampholytes.

This paper comprises a continuation of computer simulation studies dealing with carrier ampholyte based CIEF in presence of narrow pH gradients. With this technique, amphoteric sample components with pI values outside the pH gradient are migrating isotachophoretically toward the cathode or anode whereas components with pI values within the gradient become focused. In order to understand the processes occurring in presence of the electric field, the behavior of both carrier ampholytes and amphoteric sample components is investigated by computer modeling. Characteristics of two pH unit gradients with end components having pI values at or around 7.00 and conditions that lead to the formation of a water zone at neutrality were investigated. Data obtained reveal that a zone of water is formed in focusing with carrier ampholytes when the applied pH range does not cover the neutral region, ends at pH 7.00 or begins at pH 7.00. The presence of additional amphoteric components that cover the neutrality region prevent water zone formation under current flow. This situation is met in experiments with narrow pH gradients that end or begin around neutrality. Simulation data reveal that no water zone evolves when atmospheric carbon dioxide dissolved in the catholyte causes the migration of carbonic acid (in the form of carbonate and/or hydrogen carbonate ions) from the catholyte through the focusing structure. An electrolyte change in the electrode solution does not have an impact on the focusing part but does change the isotachophoretic pattern migrating behind the leading ion.

Main characteristic of N-bromo poly(styrene-co-divinylbenzene) sulphonamide acid: a cation exchanger and redox polymer

N-bromo poly(styrene-co-divinylbenzene) sulphonamide acid with macroporous structure has been obtained from its sodium salt using weak acid solution (0.02 mol L−1 CH3COOH) to transform the functional groups from the Na+ form to the H+ form. This transformation, carried out in a column method, allowed to obtain a new reactive polymer which exists in an aqueous medium only in a narrow pH range and does not have low-molecular weight analogue (N-bromo-p-toluenesulphonic acid is not separable from the aqueous solution and does not occur as a pure chemical compound). It has been demonstrated that the –SO2NBrH groups, stable under certain conditions (in water and in weak acid solutions), showed cation exchange and oxidizing properties. Their main properties, such as the acidity, the formal redox potential, and the stability in aqueous media of different acidities, were determined. It has been explained the difference in behavior of copolymeric Na+ and H+ form sulphonamides. FTIR spectra and TG/DTG charts of the copolymers with –SO2NBrH and –SO2NBrNa groups have been compared.

The chondroitin sulfate/dermatan sulfate 4-O-endosulfatase from marine bacterium Vibrio sp FC509 is a dimeric species: Biophysical characterization of an endosulfatase.

Sulfatases catalyze hydrolysis of sulfate groups. They have a key role in regulating the sulfation states that determine the function of several scaffold molecules. Currently, there are no studies of the conformational stability of endosulfatases. In this work, we describe the structural features and conformational stability of a 4-O-endosulfatase (EndoV) from a marine bacterium, which removes specifically the 4-O-sulfate from chondroitin sulfate/dermatan sulfate. For that purpose, we have used several biophysical techniques, namely, fluorescence, circular dichroism (CD), FTIR spectroscopy, analytical ultracentrifugation (AUC), differential scanning calorimetry (DSC), mass spectrometry (MS), dynamic light scattering (DLS) and size exclusion chromatography (SEC). The protein was a dimer with an elongated shape. EndoV acquired a native-like structure in a narrow pH range (7.0-9.0); it is within this range where the protein shows the maximum of enzymatic activity. The dimerization did not involve the presence of disulphide-bridges as suggested by AUC, SEC and DLS experiments in the presence of β-mercaptoethanol (β-ME). EndoV secondary structure is formed by a mixture of α and β-sheet topology, as judged by deconvolution of CD and FTIR spectra. Thermal and chemical denaturations showed irreversibility and the former indicates that protein did not unfold completely during heating.

Chitosan-based core-shell nanomaterials for pH-triggered release of anticancer drug and near-infrared bioimaging

As a naturally-abundant biopolymer, chitosan (CS) exhibit pH-sensitive structural transformation within a narrow pH range. Integrating hydrophobic groups to CS molecules gives modified CS polymers with more adjustable pH responsiveness. In this paper, near-infrared (NIR) photoluminescent Ag2S QDs capped by long-chain carboxylic acid were synthesized and then conjugated with CS via esterification reaction. The anticancer drug doxorubicin (DOX) has an affinity for the hydrophobic oleoyl groups and was entrapped by them to produce Ag2S(DOX)@CS nanospheres. A variety of experiments were performed to characterize the nanospheres. In vitro and in vivo experiments showed that the nanospheres can release DOX at lowered pH in tumor cells and have high antitumor efficacy. In addition, the strong NIR signal derived from the encapsulated Ag2S QDs makes real-time monitoring of the nanosphere distribution in a body possible. This study provides a new CS-based nanocomposite drug carrier for efficient cancer therapy.

Structural transition of nanogel star polymers with pH by controlling PEGMA interactions with acid or base copolymers

ABSTRACTWe use small angle X-ray scattering (SAXS) to characterise a class of star diblock polymers with a nanogel core on which the outer block arms are comprised of random copolymers of temperature sensitive PEGMA with pH sensitive basic (PDMAEMA) and acidic (PMAA) monomers. The acquired SAXS data show that many of the nanogel star polymers undergo a sharp structural transition over a narrow range of pH, but with unexpectedly large shifts in the apparent pKa with respect to that of the acidic or basic monomer unit, the linear polymer form or even an alternate star polymer with a tightly cross-linked core chemistry. We have demonstrated a distinct and quantifiable structural response for the nanogel star copolymers by altering the core or by pairing the monomers PDMAEMA–PEGMA and PMAA–PEGMA to achieve structural transitions that have typically been observed in stars through changes in arm length and number.

Multi-responsive polymethacrylamide homopolymers derived from tertiary amine-modified -alanine

Four poly(N-methacryloyl-l-alanine) homopolymers containing different tertiary amine moieties, dimethylaminoethyl, dimethylaminopropyl, diethylaminoethyl, and diethylaminopropyl, were prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization in an effort to use natural amino acids as building blocks to design stimuli-responsive polymers that display pH-tunable lower critical solution temperature (LCST) transitions in the basic pH range. Monomer structure was found to be critical for the corresponding polymer to exhibit desired stimuli-responsive properties in water. While all four polymethacrylamides showed thermosensitive property in water, only poly(N-methacryloyl-l-alanine 2-(diethylamino)ethylamide) (PMAEE) exhibited LCST behavior in a wide pH range, from 9.0 to 13.0. Other polymers' thermoresponsive properties were found either at very high pH values (e.g., ≥13.0) or in a rather narrow pH range. The effects of pH, polymer molecular weight, polymer concentration, presence of NaCl, and end groups on cloud point of PMAEE in water were investigated; the cloud point decreased with the increase of solution pH and polymer concentration, the addition of NaCl, and the introduction of a more hydrophobic end group but varied little with polymer molecular weight. The incorporation of tertiary amine moieties endowed the polymers with a CO2-responsive property; we demonstrated that the thermosensitive property of PMAEE can be reversibly tuned by bubbling its solution alternately with CO2 and N2 gases.

Whey protein–pectin soluble complexes for beverage applications

There is a strong interest in the consumption of beverages containing whey proteins due to implications in health outcomes such as increased satiety and metabolic regulation. However, low thermal stability limits the conditions under which whey protein beverages can be formulated. Studies have shown that at a narrow pH range near the protein isoelectric points, whey proteins and polysaccharides self assemble into soluble complexes (SCs) that exhibit unique functionality. This study investigated the formation and thermal stability of SCs under conditions relevant to beverage applications. Complexes were formed at pH 5 using whey protein isolate (WPI; 1–6% w/w) and high-methoxyl pectin (HMP; 0.125–0.75% w/w) and then heat-set at 85 °C for 25 min. Hydrodynamic properties, particle size distribution, ζ–potential, and dispersion viscosity were evaluated before and after heat-setting. Mean particle diameter ranged from 300 to 715 nm for unheated SCs, and 230 nm to 1 μm for heat-set SCs. Heat-setting SCs led to a significant (p < 0.05) reduction in intrinsic viscosity from 93.6 mL/g to 79.5 mL/g, suggesting conformational changes that favor a smaller hydrodynamic size. Dispersions of SCs exhibited decreased apparent viscosity, consistent with the lower intrinsic viscosity and smaller particle size. Heat-set SCs (4% WPI, 0.5% HMP) remained as sub-micron particles (d = 303–829 nm) after pH adjustment (pH 4–7) and thermal processing (142 °C for 6 s), indicating that WPI and HMP can be heat-set into complexes with enhanced colloidal stability in beverage applications.

A Single Methylene Group in Oligoalkylamine-Based Cationic Polymers and Lipids Promotes Enhanced mRNA Delivery.

The development of chemically modified mRNA holds great promise as a new class of biologic therapeutics. However, the intracellular delivery and endosomal escape of mRNA encapsulated in nanoparticles has not been systematically investigated. Here, we synthesized a diverse set of cationic polymers and lipids from a series of oligoalkylamines and subsequently characterized their mRNA delivery capability. Notably, a structure with an alternating alkyl chain length between amines showed the highest transfection efficiency, which was linked to a high buffering capacity in a narrow range of pH 6.2 to 6.5. Variation in only one methylene group resulted in enhanced mRNA delivery to both the murine liver as well as porcine lungs after systemic or aerosol administration, respectively. These findings reveal a novel fundamental structure-activity relationship for the delivery of mRNA that is independent of the class of mRNA carrier and define a promising new path of exploration in the field of mRNA therapeutics.

A Single Methylene Group in Oligoalkylamine‐Based Cationic Polymers and Lipids Promotes Enhanced mRNA Delivery

AbstractThe development of chemically modified mRNA holds great promise as a new class of biologic therapeutics. However, the intracellular delivery and endosomal escape of mRNA encapsulated in nanoparticles has not been systematically investigated. Here, we synthesized a diverse set of cationic polymers and lipids from a series of oligoalkylamines and subsequently characterized their mRNA delivery capability. Notably, a structure with an alternating alkyl chain length between amines showed the highest transfection efficiency, which was linked to a high buffering capacity in a narrow range of pH 6.2 to 6.5. Variation in only one methylene group resulted in enhanced mRNA delivery to both the murine liver as well as porcine lungs after systemic or aerosol administration, respectively. These findings reveal a novel fundamental structure–activity relationship for the delivery of mRNA that is independent of the class of mRNA carrier and define a promising new path of exploration in the field of mRNA therapeutics.

Water soluble cationic porphyrin showing pH-dependent optical responses to G-quadruplexes: Applications in pH-sensing and DNA logic gate

A water soluble cationic porphyrin derivative 5,10,15,20-tetra-{4-[3-(1-methyl-1-piperidinyl) propoxy]phenyl}porphyrin (TMPipPrOPP), which showed attractive pH-dependent optical responses to G-quadruplexes, was synthesized and characterized. Under molecular crowding conditions, a stable TMPipPrOPP/G-quadruplex complex was formed in acidic pH range. Alkalization of the system to neutral and basic pH caused destruction of the complex due to reduced electrostatic interactions. The formation and destruction of the TMPipPrOPP/G-quadruplex complex was reversibly adjusted by pH, accompanied by repeated on-off switching of the colorimetric and fluorescent responses of TMPipPrOPP to G-quadruplexes. In comparison, no optical responses were observed for single-stranded, duplex and i-motif DNAs in the tested pH range (5.0–8.5). Besides highlighting the importance of designing novel drugs targeting G-quadruplexes in specific genome regions, this work may also provide a useful tool for G-quadruplex-based biosensing and nanomachine construction. One example is the design of colorimetric and fluorescent sensors for ratiometric pH-sensing. The proposed sensing platform can be used not only for highly sensitive pH-sensing in a narrow biological pH range, but also for pH detection in a broad range. Another example is the construction of DNA-based dual-output all-optical molecular logic gates that can perform basic AND, NAND, OR, NOR, INHIBIT and IMPLICATION logic operations.

A De Novo Designed Coiled-Coil Peptide with a Reversible pH-Induced Oligomerization Switch

Protein conformational switches have many useful applications but are difficult to design rationally. Here we demonstrate how the isoenergetic energy landscape of higher-order coiled coils can enable the formation of an oligomerization switch by insertion of a single destabilizing element into an otherwise stable computationally designed scaffold. We describe a de novo designed peptide that was discovered to switch between a parallel symmetric pentamer at pH 8 and a trimer of antiparallel dimers at pH 6. The transition between pentamer and hexamer is caused by changes in the protonation states of glutamatic acid residues with highly upshifted pKa values in both oligomer forms. The drastic conformational change coupled with the narrow pH range makes the peptide sequence an attractive candidate for introduction of pH sensing into other proteins. The results highlight the remarkable ability of simple-α helices to self-assemble into a vast range of structural states.

A supersaturating delivery system of silibinin exhibiting high payload achieved by amorphous nano-complexation with chitosan

The therapeutic potentials of silibinin – a phytochemical isolated from milk thistle plants – have not been fully realized due to its poor oral bioavailability caused by the low aqueous solubility. Existing solubility enhancement strategies of silibinin by nanonization were limited by their low payload. Herein we developed a supersaturating delivery system of silibinin exhibiting a high payload (≈76%) in the form of amorphous silibinin–chitosan nanoparticle complex (or silibinin nanoplex in short) prepared by self-assembly drug-polysaccharide complexation. The effects of (1) pH and (2) charge ratio of chitosan to silibinin on the nanoplex's physical characteristics (i.e. size, zeta potential, and payload) and preparation efficiency (i.e. silibinin utilization, overall yield) were investigated. The formation of nanoplex (≈240nm) was feasible only in a narrow pH range (5.1–5.8) and favored charge ratio below unity. At the optimal condition (pH 5.8 and charge ratio of 0.30), the nanoplex preparation exhibited 87% silibinin utilization rate and 63% yield signifying its high efficiency. The amorphous state and colloidal stabilities of the nanoplex during storage, and prolonged supersaturation generation (3h) at more than 10× of the saturation solubility were successfully demonstrated.

A New Highly Sensitive and Colorimetric pH Indicator Derived from 2-Amino-5-nitrothiazole and H-acid

A new pH indicator having high sensitivity and significant color change visible to the naked eye from red to blue in a narrow pH range (3.2–4.0) with high molar absorption coefficient was designed ...

Hierarchically Assembled Nanofibers Created by a Layer-by-Layer Self-Assembly.

We report the hierarchically assembled nanofibers created by LbL self-assembly depending on the PSS-PAA fraction in the blend solutions and pH during bulid-up of the PAH/(PSS-PAA) multilayer films. The multilayer [(PEI/blend)/(PAH/blend)4] films with ρPAA (PSS-PAA fraction in the blend solutions) = 0.0 in the blend solution exhibited surface morphologies of randomly isolated globular clusters, while at ρPAA = 0.75, worm-like morphologies were observed. Interestingly, the multilayer [(PEI/blend)/(PAH/blend)4 films with ρPAA = 0.9 exhibited unique fibrous morphologies with the diameter of about 50 nm at narrower pH range from 3.5 to 4.2, but also the fiber diameter distribution was narrower. Based on the thickness from the X-ray reflectivity, the thickness of the one bilayer multilayer film seemed to be 8.6 nm. The 3 bilayers multilayer film seemed to be formed as islands with very large roughness. The crystal sizes of the 3 bilayers and 5 bilayers multilayer films were about 71 nm and 123 nm, respectively. The resultant films were characterized using atomic force microscopy (AFM) and real-time in situ X-ray scattering measurements.

Investigation Into the Role of N‐terminal Acetylation of ESAT‐6 in Pathogenesis of Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent for the disease Tuberculosis in humans, is present as a latent infection in approximately one third of the world population. Mtb has become more resilient over the years, as the vaccine, Mycobacterium bovis bacillus Calmette‐Guerin (BCG), has been shown to lose effectiveness 10 years after initial vaccination. Recent research found 6‐kD early secretory antigenic target (ESAT‐6) and 10‐kD culture filtrate protein (CFP‐10) are secreted as a heterodimer by Mtb and are important for virulence. Additionally, ESAT‐6 has been determined to contain membrane lytic activity, while CFP‐10 has been suggested to be a molecular chaperone. Furthermore, membrane lytic ability from ESAT‐6 only occurs after it has dissociated from CFP‐10. Studies suggest ESAT‐6 dissociates from CFP‐10 at low pH to interact with the phagosomal membrane to translocate Mtb into the cytosol of a macrophage, however the mechanism of heterodimer dissociation is unclear. Previous studies have identified and isolated an N‐terminally acetylated ESAT‐6 protein. Binding affinity between CFP‐10 and ESAT‐6 is greatly reduced after acetylation, suggesting that N‐terminal acetylation could be the culprit for the dissociation of the ESAT‐6 and CFP‐10 heterodimer. In this study, we aim to determine whether N‐terminal acetylation of Threonine 2 on ESAT‐6 is required for dissociation of the heterodimer. To test this, three different residues: alanine, glutamine, and arginine, were used to replace Threonine 2. We hypothesized the acetylation‐mimicking residues, alanine and glutamine would contain similar activity to wild‐type ESAT‐6 while acetylation negative control, arginine, would have less activity. Preliminary data, a liposome leakage assay with the dye/quencher pair, 8‐aminonaphthalene‐1,3,6 trisulfonic acid (ANTS) and p‐xylene‐bis‐pyridinium bromide (DPX), revealed these mutations did not affect pore formation of ESAT‐6 alone, but greatly diminished pore formation of the ESAT‐6 complexed with CFP‐10, suggesting alanine and glutamine failed to function as acetylation‐mimicking residues, highlighting the importance of Thr2. Cytotoxicity analysis of post‐macrophage infection using Mycobacterium marinum also revealed decreased activity in Thr2 mutants. Accordingly, if these mutations disrupt dissociation, then decreased translocation to the cytosol would be expected, as such fluorescence electron resonance transfer (FRET) was employed to test this theory. Furthermore, a novel in vitro method to dissociate the heterodimer complex was applied to test whether acetylation is present in wild type and not in proteins containing mutations of Thr2. The resultant proteins were analyzed using narrow range pH gradient two‐dimensional gel electrophoresis (2‐DE). Additionally, 4‐chloro‐7‐nitrobenzofurazan (NBD‐Cl) reacts with un‐acetylated proteins, and exhibits measurable fluorescence, while N‐terminally acetylated proteins do not, compromising a reliable method for detection of acetylation in these proteins. This study has revealed the physiological importance of N‐alpha‐acetylation of ESAT‐6 in Mtb infection.Support or Funding InformationThis work was supported by the National Institutes of Health Grant 5G12RR008124 from the National Center for Research Resources and Grant 2G12MD007592 from National Institutes on Minority Health Disparities, a component of National Institutes of Health. This Research is also supported by University of Texas at El Paso new faculty start‐up funds.

A Novel Acid-Stable Endo-Polygalacturonase from Penicillium oxalicum CZ1028: Purification, Characterization, and Application in the Beverage Industry.

Acidic endo-polygalacturonases are the major part of pectinase preparations and extensively applied in the clarification of fruits juice, vegetables extracts, and wines. However, most of the reported fungal endo-polygalacturonases are active and stable under narrow pH range and low temperatures. In this study, an acidic endo-polygalacturonase (EPG4) was purified and characterized from a mutant strain of Penicillium oxalicum. The N-terminal amino acid sequence of EPG4 (ATTCTFSGSNGAASASKSQT) was different from those of reported endopolygalacturonases. EPG4 displayed optimal pH and temperature at 5.0 and 60-70°C towards polygalacturonic acid (PGA), respectively, and was notably stable at pH 2.2-7.0. When tested against pectins, EPG4 showed enzyme activity over a broad acidic pH range (>15.0% activity at pH 2.2-6.0 towards citrus pectin; and >26.6% activity at pH 2.2-7.0 towards apple pectin). The Km and Vmax values were determined as 1.27 mg/ml and 5,504.6 U/mg, respectively. The enzyme hydrolyzed PGA in endo-manner, releasing oligo-galacturonates from PGA, as determined by TLC. Addition of EPG4 (3.6 U/ml) significantly reduced the viscosity (by 42.4%) and increased the light transmittance (by 29.5%) of the papaya pulp, and increased the recovery (by 24.4%) of the papaya extraction. All of these properties make the enzyme a potential application in the beverage industry.

Two-step ion-exchange chromatographic purification combined with reversed-phase chromatography to isolate C-peptide for mass spectrometric analysis.

A liquid chromatography with mass spectrometry on-line platform that includes the orthogonal techniques of ion exchange and reversed phase chromatography is applied for C-peptide analysis. Additional improvement is achieved by the subsequent application of cation- and anion-exchange purification steps that allow for isolating components that have their isoelectric points in a narrow pH range before final reversed-phase mass spectrometry analysis. The utility of this approach for isolating fractions in the desired "pI window" for profiling complex mixtures is discussed.

A wearable fingernail chemical sensing platform: pH sensing at your fingertips

This article demonstrates an example of a wearable chemical sensor based on a fingernail platform. Fingernails represent an attractive wearable platform, merging beauty products with chemical sensing, to enable monitoring of our surrounding environment. The new colorimetric pH fingernail sensor relies on coating artificial nails with a recognition layer consisted of pH indicators entrapped in a polyvinyl chloride (PVC) matrix. Such color changing fingernails offer fast and reversible response to pH changes, repeated use, and intense color change detected easily with naked eye. The PVC matrix prevents leaching out of the indicator molecules from the fingernail sensor toward such repeated use. The limited narrow working pH range of a single pH indicator has been addressed by multiplexing three different pH indicators: bromothymol blue (pH 6.0–7.6), bromocresol green (pH 3.8–5.4), and cresol red (pH 7.2–8.8), as demonstrated for analyses of real-life samples of acidic, neutral, and basic character. The new concept of an optical wearable chemical sensor on fingernail platforms can be expanded towards diverse analytes for various applications in connection to the judicious design of the recognition layer.

The hydrothermal synthesis, crystal structure and electrochemical properties of MnSb2O4.

Phase pure polycrystalline MnSb2O4 was synthesised under hydrothermal conditions. Impurities formed outside a narrow range of pH and metal stoichiometric ratios. The structure and its temperature dependence was studied based on multi-temperature conventional single crystal X-ray diffraction (SC-XRD) and high resolution synchrotron powder X-ray diffraction (PXRD). At low temperature (100-300 K) the lattice parameters expanded linearly with thermal expansion coefficients of αa,300 K = 8(1) × 10-6 K-1 and αc,300 K = 7.2(2) × 10-6 K-1. At high temperature an irreversible annealing effect was observed. Modelling the atomic displacement parameters based on the single crystal X-ray diffraction data gives a Debye temperature of 267(3) K for MnSb2O4. When heated in an oxygen-rich atmosphere MnSb2O4 oxidises above 800 K and it decomposes at 975 K under inert conditions. Electrochemical measurements displayed similar behaviour to the isostructural CoSb2O4, with reversible alloying-dealloying of LixSb after the first cycle with a reversible capacity of 337 mAh g-1 at the 2nd cycle, which degraded to c. 100 mAh g-1 after 30 cycles.

Stitching of tyrosine and 10H-acridin-9-one: turn-ON fluorescence in the narrow pH range 7.4–8.5 and intracellular labelling of cancer cells

We tailored 10H-acridin-9-one and (S)-tyrosine into 3-(4-hydroxyphenyl)-2-[(9-oxo-9,10-dihydroacridine-4-carbonyl) amino]propionic acid (2).