Ampholyte Solution Research Articles

Separation and fractionation of glutamic acid and histidine via origami isoelectric focusing

We demonstrated the fractionation of two amino acids, glutamic acid and histidine, separated via isoelectric focusing (IEF) on filter paper folded and stacked in an origami fashion. Channels for electrophoresis were fabricated as circular zones acquired via wax printing onto the filter paper. An ampholyte solution with amphiphilic samples was deposited on all the circle zones, which was followed by folding to form the electrophoresis channels. IEF was achieved by applying an electrical potential between the anodic and cathodic chambers filled with phosphoric acid and sodium hydroxide solutions, respectively. A pH gradient was formed using either a wide-range ampholyte with a pH of 3 to 10 or a narrow-range version with a pH of 5 to 8, which was confirmed by adding pH indicators to each layer. The origami IEF was used to separate the amino acids, glutamic acid and histidine, by mixing with the ampholytes, which were deposited on the layers. The components in each layer were extracted with water and measured by high-performance liquid chromatography using pre-column derivatization with dansyl chloride. The results indicated that the focus for glutamic acid and that for histidine were at different layers, according to their isoelectric points. The origami isoelectric focusing achieved the fractionation of amino acids in less than 3 min using voltage as low as 30 V.

Generation of H+ and OH− ions in anion-exchange membrane/ampholyte-containing solution systems: A study using electrochemical impedance spectroscopy

Generation of H+ and OH− ions at anion-exchange membranes is a phenomenon, which substantially reduces the performance of a number of electromembrane processes, such as the wastewater treatment, fuel cell energy production and others. We show that the mechanisms of this phenomenon are different, when a Neosepta AMX anion-exchange membrane is bathed in a strong electrolyte solution (NaCl and KCl) or in an ampholyte solution (NaH2PO4, KH2PO4, KHT and KH2Cit). The H+ and OH− ions' generation in the case of strong electrolytes requires a voltage threshold (about 0.3 V of the corrected voltage not including ohmic contribution), which relates to a current close to the limiting current. The process occurs mainly through proton transfer reactions involving the membrane functional groups. In the case of ampholytes, this generation takes place without a threshold; this process is separated in space: the generation of H+ occurs at the depleted solution/membrane interface, that of OH− ions, at the membrane boundary with the enriched solution. Electrochemical impedance spectra (EIS) are used for the determination of the effective rate constants for the H+ and OH− ions’ generation. Their values are compared with the rate constants calculated from the acid dissociation constants (Ka).

Concentration Dependencies of Diffusion Permeability of Anion-Exchange Membranes in Sodium Hydrogen Carbonate, Monosodium Phosphate, and Potassium Hydrogen Tartrate Solutions.

The concentration dependencies of diffusion permeability of homogeneous (AMX-Sb and AX) and heterogeneous (MA-41 and FTAM-EDI) anion-exchange membranes (AEMs) is obtained in solutions of ampholytes (sodium bicarbonate, NaHCO3; monosodium phosphate, NaH2PO4; and potassium hydrogen tartrate, KHT) and a strong electrolyte (sodium chloride, NaCl). It is established that the diffusion permeability of AEMs increases with dilution of the ampholyte solutions, while it decreases in the case of the strong electrolyte solution. The factors causing the unusual form of concentration dependencies of AEMs in the ampholyte solutions are considered: (1) the enrichment of the internal AEM solution with multiply charged counterions and (2) the increase in the pore size of AEMs with dilution of the external solution. The enrichment of the internal solution of AEMs with multiply charged counterions is caused by the Donnan exclusion of protons, which are the products of protolysis reactions. The increase in the pore size is conditioned by the stretching of the elastic polymer matrix due to the penetration of strongly hydrated anions of carbonic, phosphoric, and tartaric acids into the AEMs.

A porous layer open-tubular capillary column with immobilized pH gradient (PLOT-IPG) for isoelectric focusing of amino acids and proteins

A porous layer open-tubular capillary column with immobilized pH gradient (PLOT-IPG) was prepared in two steps. First, a partially filled porous layer open tubular capillary column containing active epoxy groups was synthesized by in situ polymerization of acrylamide, glycidyl methacrylate, and N,N′-methylenebisacrylamide. Then, an aqueous solution of commercial carrier ampholytes (CAs, Pharmalyte) was focused in the prepared polymer column. With the column immobilized by Pharmalyte 4.5–6.0 (narrow pH range) or Pharmalyte 3.0–10.0 (wide pH range), proteins with known pIs were separated and a good linear correlation between pI values and migration times was obtained. Meanwhile, resolution was improved compared to the traditional gradient with a wide pH range. A mixture of low molecular weight amino acids was separated with the narrow pH range PLOT-IPG column; from the obtained results, the resolution of our new column was calculated to be at most 0.09 pH unit. In addition, human serum proteins were analyzed with the newly developed wide and narrow pH range PLOT-IPG columns; its sensitivity and resolution in the narrow pH range one were better than capillary electrophoresis.

Capillary isoelectric focusing after sample enrichment with immunoaffinity chromatography in a single capillary.

For accurate micro-scale quantification of a specific protein in biological fluids, immunoaffinity chromatography (IAC) and isoelectric focusing (IEF) were combined in a single fused-silica capillary. The inner wall of the capillary was coated with an anti-E-tag antibody at the inlet side to form an IAC column, and polydimethylacrylamide, a neutral polymer, at the outlet side to form the capillary for IEF. After loading a sample, the whole capillary was filled with a carrier ampholyte solution. An anode solution, an acid, was then introduced to fill only the IAC column segment. Focusing was started with a pressure that balances with the electroosmotic flow produced in the acidified IAC column. Fluorescence-labeled recombinant Fab with an E-tag spiked at 16 pM to 10 nM in 50% serum was separated and detected with high precision. The coupling principle allows rapid and high-resolution IEF analysis of a protein in a biological sample without any loss of the immunoaffinity captured protein.

Effect of the treatment of MF-4SC membranes on the cross sensitivity of Donnan potential sensors to cations in the aqueous solutions of organic ampholytes

Methods of directional variation of properties of perfluorinated sulfo cation-exchange membranes were proposed based on the use of the effect of ‘memory’ to changes in their structure under the influence of external factors (heat treatment at various humidity or mechanical effect). The relationship between water uptake, transport properties of MF-4SC membranes, and sensitivity of Donnan potential (DP) sensors to the determined organic cations and interferent hydronium ions was established. Lowering sensitivity of DP-sensors to the interferent hydronium ions at a decrease in diffusion permeability and increase in conductivity of the membranes was reached.

Approximation of weak solution for the problem of a pH-gradient creation in isoelectrofocusing.

The mathematical model describing the stationary natural pH-gradient arising under the action of an electric field in an aqueous solution of ampholytes is constructed and investigated. The model is a part of a more general model of the isoelectrofocusing process. Investigation is based on the approximation of a weak solution by the piecewise continuous non-smooth functions. The method can be used for solving classes of problems for ordinary differential equations with a small parameter at the highest derivatives and the turning points.

Effect of electrolyte pH on CIEF with narrow pH range ampholytes

CIEF of components following sequential injection of ampholytes and the sample zone offers unique advantages for analysis. The most important one of these is the efficient separation of amphoteric compounds having pIs outside the pH range of the ampholytes applied, but the resolution of the components can be increased by an adequate setup in the injection protocol. In this study, the effect of the pH of the anolyte and catholyte on the selectivity and speed of the isoelectric focusing was investigated. Changes in the pH values significantly influenced the resolution and the length of the pH gradient, while changes in the charge state of components were also observed. Three ampholyte solutions (from different suppliers) covering only two pH units were used for the analyses of substituted nitrophenol dyes in uncoated capillary. With appropriate setup, the components, with pIs not covered by the ampholyte pH range, migrated in charged state outside the pH gradient. This phenomenon is preferable for coupling isoelectric focusing to MS detection, by evading the undesirable ion suppression effect of ampholytes.

Sequential injection setup for capillary isoelectric focusing combined with MS detection

Capillary isoelectric focusing in the presence of electroosmosis with sequential injection of carrier ampholytes and sample was found to be suitable for MS detection. The separate injection of the sample and the ampholytes provides good condition to suppress and overcome the undesirable effect of the presence of ampholytes in MS. By the appropriate selection of ampholyte solutions, whose pH range not necessarily covers the pI values of the analytes, the migration of the components can be controlled, and the impact of the ampholytes on MS detection is decreased. The unique applicability of this setup is shown by testing several parameters, such as the application of volatile electrolyte solutions, the type of the ampholytes, the order and the number of the ampholyte and sample zones. Broad and narrow pH range ampholytes were applied in experiments using uncoated capillaries with different lengths for the analyses of substituted nitrophenol dyes to achieve optimal conditions for the MS detection. Although the sample components are not leaving the pH gradient, due to the decrease in the ampholyte concentration at the position of the components, and because the sample components migrate in charged state, the ionisation is more effective for MS detection.

Capillary isoelectric focusing of individual mitochondria.

Mitochondria are highly heterogeneous organelles that likely have unique isoelectric points (pI), which are related to their surface compositions and could be exploited in their purification and isolation. Previous methods to determine pI of mitochondria report an average pI. This article is the first report of the determination of the isoelectric points of individual mitochondria by capillary isoelectric focusing (cIEF). In this method, mitochondria labeled with the mitochondrial-specific probe 10-N-nonyl acridine orange (NAO) are injected into a fused-silica capillary in a solution of carrier ampholytes at physiological pH and osmolarity, where they are focused then chemically mobilized and detected by laser-induced fluorescence (LIF). Fluorescein-derived pI markers are used as internal standards to assign a pI value to each individually detected mitochondrial event, and a mitochondrial pI distribution is determined. This method provides reproducible distributions of individual mitochondrial pI, accurate determination of the pI of individual mitochondria by the use of internal standards, and resolution of 0.03 pH units between individual mitochondria. This method could also be applied to investigate or design separations of organelle subtypes (e.g., subsarcolemmal and interfibrillar skeletal muscle mitochondria) and to determine the pIs of other biological or nonbiological particles.

A systematic study in CIEF: Defining and optimizing experimental parameters critical to method reproducibility and robustness

A systematic study of two-step CIEF analysis was completed to identify key components that could be optimized to enhance the performance of mAb analysis by CIEF. Resolution between mAb isoforms was increased by selecting a narrow detector aperture, utilizing chemical rather than pressure mobilization, and improving protein solubility by incorporating urea into the carrier ampholyte (CA) solutions. Loss of the extreme pI CAs and sample components by the bidirectional ITP inherent to IEF was avoided by setting the concentration of the phosphoric acid anolyte to 200 mM and sodium hydroxide catholyte to 300 mM and by adding sufficient amounts of an acidic (pI<3) and basic (10<pI) sacrificial ampholyte to the CIEF sample solution. Optimization of the concentrations of the sacrificial ampholytes, iminodiacetic acid and arginine to 1.7 and 40 mM, respectively, yielded a stable focused CA train that spanned the 4<pH<10 range without sacrificing either mAb isoform resolution or sample throughput. Intermediate precision studies performed on the CIEF method with three basic mAbs yielded 0.04 to 0.09% CVs for the estimated pI values and 0.6-3% CVs for isoform group percent composition, indicating that the two-step CIEF method developed meets the rigorous demands of therapeutic mAb analysis.

High Speed Two-Dimensional Protein Separation without Gel by Isoelectric Focusing−Asymmetrical Flow Field Flow Fractionation: Application to Urinary Proteome

An online multilane channel system for isoelectric focusing and asymmetrical flow field-flow fractionation (IEF-AF4) is utilized for the two-dimensional separation (2D: isoelectric point, pI, and hydrodynamic diameter, d(s)) of a human proteome sample followed by the shotgun proteomic analysis using nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS-MS). IEF-AF4 was recently developed to carry out nongel-based high speed two-dimensional protein separation [ Kim , K. , et al. Anal. Chem. 2009, 81 , 1715 ]. In IEF-AF4, proteins are separated according to pI along an IEF channel located at the head of six AF4 channels, and then the fractionated protein bands are directed to multilane AF4 channels for size-based separation. In this report, the original IEF-AF4 system has been modified to avoid the possible adsorption of proteins onto the membrane wall of IEF segments during isoelectric focusing by isolating the IEF channel segments from the multilane AF4 channels. The performance of the modified IEF-AF4 system was tested with protein standards and was further applied for the 2D fractionation of the human urinary proteome sample under two ampholyte solutions with different pH ranges (pH 3-10 and 3-6). The entire 2D separation was achieved in less than 30 min. The collected protein fractions were digested for peptide analysis using nLC-ESI-MS-MS, resulting in the identification of 245 total urinary proteins, including 110 unique proteins that are not yet reported in literature. Our experiments also showed a higher efficiency in the identification of urine proteins using ampholyte solution in the narrower pH range.

Free-flow isoelectric focusing microfluidic device with glass coating by sol–gel methods

A device for microfluidic free-flow isoelectric focusing (μ-FFIEF) was fabricated in a glass-coated PDMS structure. The microfluidic channel was made by a PDMS replica molding and standard soft photolithography technique. For the prefractionation and preconcentration of a protein mixture, we patterned palladium electrodes on the glass substrate and coated with TEOS–sol to enhance the bonding with PDMS. The PDMS microchannel was coated with glass by flushing TEOS–sol and annealing at 100 °C. The optimum focusing condition was obtained by pH indicator with ampholyte solution made with MES. Model proteins, RFP and EGFP, were successfully concentrated around 214 μm and 357 μm from the anode, respectively. The optimum focusing condition in the μ-FFIEF device was an electric potential of 1.5 V/cm and pH 6.0. The focusing time in the channel without glass coating was approximately 30 s, but the breakdown of the electrode by electrolysis started immediately when the electric field was applied. However, the glass-coated device was kept intact under continuous application of voltage giving a stable pH gradient, but with twice the focusing time.

Electrical conductivity of cation-and anion-exchange membranes in ampholyte solutions

Several laws governing ampholyte transport through ion-exchange membranes are established by a comparative analysis of the concentration dependence of electrical conductivity for homogeneous (CMX, AMX) and heterogeneous (MK-40, MA-41) membranes in NaCl, LysHCl, and NaH2PO4 solutions. The increase in the electrical conductivity of membranes in ampholyte solutions as the solutions become more dilute is explained by the increased fraction of divalent ions of the amino acid (cation-exchange membrane) or from phosphoric acid (anion-exchange membrane) in the membrane as a result of Donnan exclusion of hydrolysis products (hydroxide ions or protons, respectively).

PVA‐based tunable buffering membranes for isoelectric trapping separations

PVA-based buffering membranes with tunable pH values were prepared on a PVA substrate by reacting PVA, glycerol-1,3-diglycidyl ether, -NH2 group-containing buffers and -NH2 group-containing titrants in the presence of sodium hydroxide. The pH of the buffering membranes could be tuned in the 3<pH<10 range according to the Hendersson-Hasselbach equation while maintaining buffering capacities in excess of 100 mM. Detailed recipes for the preparation of six families of buffering membranes are tabulated and provided as Supporting Information. The buffering membranes were used to trap and desalt ampholyte solutions and separate proteins having a DeltapI as small as 0.1. The membranes were mechanically and hydrolytically stable and could be stored, even in 10<pH sodium hydroxide solutions, in excess of 6 months.

Isoelectric Focusing in a Microfluidically Defined Electrophoresis Channel

A new form of microchip isoelectric focusing that allows efficient coupling with pretreatment processes is reported. The sample is conveyed in a carrier ampholyte solution to the separation channel that is connected at both ends by two V-shaped lead channels, which supply electrode solutions to the connection point and complete the electrical connection to off-chip electrodes. The relatively high electric conductivity of the electrode solutions compared with that of the pH gradient enables focusing with a 2% loss of applied voltage at the electrodes using the lead channels. A glass microchip was constructed specifically for this configuration. The channel wall was coated with polydimethylacrylamide, and the IEF chip was operated in a chip holder equipped with on-chip connector valves. A plug of fluorescence-labeled peptide p I markers with p I values ranging from 3.64 to 9.56 with carrier ampholyte solution (pH 3-10) was introduced into the separation channel. When the plug reached the channel segment (24 mm in length) between the connection points with the electrolyte lead channels, isoelectric focusing was started after filling the lead channels with electrolyte solutions. The peptide markers were observed using scanning fluorescence detection. The entire range of the pH gradient was established in the segment after approximately 2 min. Isoelectric focusing of three consecutively injected sample plugs containing different p I markers was demonstrated.

Allosteric Activation of Human Glucokinase by Free Polyubiquitin Chains and Its Ubiquitin-dependent Cotranslational Proteasomal Degradation

Human glucokinase (hGK) is a monomeric enzyme highly regulated in pancreatic beta-cells (isoform 1) and hepatocytes (isoforms 2 and 3). Although certain cellular proteins are known to either stimulate or inhibit its activity, little is known about post-translational modifications of this enzyme and their possible regulatory functions. In this study, we have identified isoforms 1 and 2 of hGK as novel substrates for the ubiquitin-conjugating enzyme system of the rabbit reticulocyte lysate. Both isoforms were polyubiquitinated on at least two lysine residues, and mutation analysis indicated that multiple lysine residues functioned as redundant acceptor sites. Deletion of its C-terminal alpha-helix, as part of a ubiquitin-interacting motif, affected the polyubiquitination at one of the sites and resulted in a completely inactive enzyme. Evidence is presented that poly/multiubiquitination of hGK in vitro serves as a signal for proteasomal degradation of the newly synthesized protein. Moreover, the recombinant hGK was found to interact with and to be allosterically activated up to approximately 1.4-fold by purified free pentaubiquitin chains at approximately 100 nm (with an apparent EC(50) of 93 nm), and possibly also by unidentified polyubiquitinated proteins assigned to their equilibrium binding to the ubiquitin-interacting motif site. The affinity of pentaubiquitin binding to hGK is regulated by the ligand (d-glucose)-dependent conformational state of the site. Both ubiquitination of hGK and its activation by polyubiquitin chains potentially represent physiological regulatory mechanisms for glucokinase-dependent insulin secretion in pancreatic beta-cells.

Anomalous dielectrophoretic behaviour of barium titanate microparticles in concentrated solutions of ampholytes

The dielectrophoretic behaviour of barium titanate (BaTiO3) particles with a mean grain size of 3 µm was studied. Suspensions of the powdered ceramic in the concentration range 0.01–1.60% (w/v) were prepared in dilute aqueous solutions of NaCl and concentrated aqueous solutions of the amphoteric molecules HEPES (N-[2-hydroxyethyl] piperazine-N′4-[2-ethanesulfonic acid] and EACA (ε -aminocaproic acid). When suspended in water without ampholytes, the particles showed positive dielectrophoresis (DEP) over the whole frequency range (1 kHz–20 MHz), independent of the medium conductivity or applied voltage. When amphoteric molecules were added at a final concentration of up to 0.57 M, the particles showed positive DEP at all frequencies. When the concentration of ampholytes was increased to 0.71 M, the particles showed positive DEP at frequencies up to 100 kHz and voltages lower than 12 Vpk–pk at all electrode sizes. However, at 100 kHz, when the amplitude was increased to over 12 Vpk–pk, the particles started to display negative DEP at the smallest electrode size (20 µm) and moved away from the microelectrodes, accumulating in the gap between the electrodes. At the highest voltages used (16–20 Vpk–pk), the particles were seen moving upwards and remained stably levitated above the array. For frequencies larger than 100 kHz, the particles showed positive DEP only. It is shown that such behaviour cannot be expected on the basis of the dielectric properties of barium titanate and the suspending medium, and it is suggested that this behaviour may be caused by the fact that at high amphotere concentration and voltages the electric field across the particles surpasses the dielectric strength of the BaTiO3 particles, resulting in a sudden drop in the particle's permittivity. The fact that not all particles showed negative DEP suggests a spread in the dielectric properties of barium titanate particles. Physical separation of barium titanate particles with presumably different dielectric properties was shown to be possible using a flow-through device.

Development of Non-Gel-Based Two-Dimensional Separation of Intact Proteins by an On-Line Hyphenation of Capillary Isoelectric Focusing and Hollow Fiber Flow Field-Flow Fractionation

A rapid, non-gel-based, on-line, two-dimensional separation method is introduced for proteome analysis. Protein fractionation was carried out by first exploiting the differences in their respective isoelectric points (pI) in a Teflon capillary using isoelectric focusing (IEF), followed by a molecular weight (MW)-based separation in a hollow fiber by flow field-flow fractionation (FlFFF). The method developed here (CIEF-HFFlFFF) may be a powerful alternative to two-dimensional polyacrylamide gel electrophoresis, which is currently used for the separation and purification of proteins. In CIEF-HFFlFFF, proteins can be collected as a fraction of a certain pI and MW interval without being denatured. Additionally, the ampholyte solution is simultaneously removed during separation in the hollow fiber, and the overall process time is significantly reduced. This method was applied to a human urinary proteome sample, leading to the identification of 114 proteins with the subsequent off-line use of nanoflow liquid chromatography-tandem mass spectrometry after the tryptic digestion of each collected protein fraction.

Hydrolytically stable, diaminocarboxylic acid‐based membranes buffering in the pH range from 6 to 8.5 for isoelectric trapping separations

Diaminocarboxylic acid carrier ampholytes, such as L-histidine, 2,3-diaminopropionic acid, L-ornithine, and L-lysine, were reacted with glycerol-1,3-diglycidyl ether (GDGE) and poly(vinyl alcohol) (PVA) in the presence of sodium hydroxide to produce hydrolytically and mechanically stable hydrogels, supported on a PVA substrate, for use as buffering membranes in isoelectric trapping (IET) separations. The pH values of the DACAPVA membranes were determined with the help of small-molecule pI markers and proteins and were found to be in the 6 < pH < 8.5 range. The membranes were successfully used to isoelectrically trap small ampholytes, desalt ampholyte solutions in IET mode, and effect the binary separation of chicken egg white proteins.