Parallel isoelectric focusing chip

Fast isoelectric focusing (IEF) is becoming a key method in modern protein analysis. We report here the theory and experimental results of new parallel isoelectric devices (PID) for fast IEF. The main separation tool of any PID is a dielectric membrane with conducting channels filled by immobiline gels of varying pH. The pH value of the surrounding aqueous solution is not equal to the pH of any of the channels. The membrane is held perpendicular to the applied electric field. Proteins are collected (trapped) in the channels whose pH values are equal to the pI of the proteins. The fast particle transport between different channels takes place due to convection in the aqueous solution. We developed a mathematical model for PID. Experiment duration is shown to be proportional to the number of different bands N (the peak capacity in standard IEF) in contrast with N(2) for usual IEF devices. This model was validated with experimental results. Parallel IEF accelerates the fractionation of proteins by their pI values (down to several minutes) allowing a more desirable collection efficiency to be achieved. The main theoretical limitation of PID resolution is the sensitivity of proteins to pH change due to the Coulomb blockade effect. The existence of a minimal pH change deltapH(min) for each type of protein is shown: deltapH(min) approximately r(-1) for globular molecules with radius r.     

pH gradients in immobilized amidases and their influence on rates and yields of beta-lactam hydrolysis

The pH gradients developing within immobilized biocatalysts during hydrolysis of penicillin G and glutaryl-7-aminocephalosporanic acid have been estimated both theoretically and experimentally. For the latter a fluorimetric method for the direct measurement of the average pH value within the carrier during reaction has been developed using the pH-dependent fluorescence intensity of an enzyme-bound fluorophore determined with a fiber bundle. The theoretical calculations were based on a model for the hydrolysis with immobilized enzymes using a kinetic expression with five pH-dependent, measurable kinetic and equilibrium constants. The transport reaction differential equation which considers the laminar boundary layer has been solved numerically for the key component. The calculated values agreed well with the experimental data. Under the typical reaction conditions of penicillin G hydrolysis the average pH value in the carrier was 1 and 2.5 pH units below the bulk pH (=8) with and without buffer, respectively. The corresponding changes for the hydrolysis of glutaryl-7-aminocephalosporanic acid at bulk pH 8 in the presence of buffer was 0.5. This demonstrates the existence of considerable pH gradients in carriers during hydrolytic reactions, even in buffered systems with negligible mass transfer resistance. The low pH value causes suboptimal reaction rates, reduced equilibrium conversion, and reduced enzyme stability. These pH gradients can be minimised by using buffers with pK values approximately equal to the bulk pH used for the hydrolysis. The prediction quality of the model has been tested applying it to fixed bed reactor design. The reduction in rate and yield due to concentration and pH gradients can be overcome with simple measures such as high initial pH value and pH adjustments in segmented or recycling fixed bed reactors. Thus, enzymatic conversions with high yield and high operational effectiveness are achieved.     

Comparison of isoelectric focusing in Sephadex vs immobiline flatbeds for the recovery of follicle regulatory protein from porcine follicular fluid

We describe and compare the use of isoelectric focusing (IEF) in a granulated Sephadex matrix and in polyacrylamide immobilized pH gradients to separate an aromatase inhibitor (follicle regulatory protein: FRP) in preparative amounts from porcine follicular fluid (PFF). The starting material for IEF was derived from pFF after passage through agarose immobilized textile dye Orange A (0.5 KC1 eluent). Before IEF, some Orange A bound (OAB) material was further purified on a FPLC employing a Mono-Q anion exchange column. Previous use of chromatofocusing indicated that aromatase inhibitory activity is largely concentrated in OAB fractions with a pI in the ranges of pH approximately 4.5 and approximately 6.5. The current study revises these findings to provide a more precise measure of the isoelectric points in question to pH 4.73 +/- 0.05 and pH 6.41 +/- 0.06. The use of Sephadex was limited by gradient instability and the selection of pH ranges available. IEF using immobilized pH gradients had several advantages over Sephadex: 1) broader selection of gradients from 0.1 to 7.0 pH units; greater resolving power, and enhanced stability. The principal disadvantage of the immobiline system was the recovery of focused material from the gel matrix. The use of isoelectric focusing with immobilized pH gradients on a preparative scale to purify FRP from OAB resulted in a greater than 50% recovery with a substantial increase in specific activity (from ID50 approximately 300 micrograms/ml to 20 ng/ml).     

Quantitative assay of deoxyribonuclease activity after isoelectric focusing in polyacrylamide gels: pH control and effects of enzyme diffusion

A method for the quantitative assay of nuclease activity in crude cell lysates after isoelectric focusing (IEF) in polyacrylamide slab gels is described. After IEF, an agarose overlay gel containing DNA is placed on the IEF gel and the nuclease activity quantified by the loss of ethidium bromide fluorescence of the DNA. With this method a linear response was obtained for 1 to 10 ng of DNase I. Various methods of pH equilibration after IEF were also evaluated. The use of a high buffer concentration in the overlay gel is recommended to control the pH during the enzyme reaction. An analytical solution for the diffusion of enzymes from the IEF gel to the overlay gel is also presented and an equation that may be used to choose optimum times for transfer of the enzyme from the IEF gel to the overlay gel is given.     

Systematic interpolation method predicts protein chromatographic elution with salt gradients,pH gradients and combined salt/pH gradients

A methodology is presented to predict protein elution behavior from an ion exchange column using both individual or combined pH and salt gradients based on high‐throughput batch isotherm data. The buffer compositions are first optimized to generate linear pH gradients from pH 5.5 to 7 with defined concentrations of sodium chloride. Next, high‐throughput batch isotherm data are collected for a monoclonal antibody on the cation exchange resin POROS XS over a range of protein concentrations, salt concentrations, and solution pH. Finally, a previously developed empirical interpolation (EI) method is extended to describe protein binding as a function of the protein and salt concentration and solution pH without using an explicit isotherm model. The interpolated isotherm data are then used with a lumped kinetic model to predict the protein elution behavior. Experimental results obtained for laboratory scale columns show excellent agreement with the predicted elution curves for both individual or combined pH and salt gradients at protein loads up to 45 mg/mL of column. Numerical studies show that the model predictions are robust as long as the isotherm data cover the range of mobile phase compositions where the protein actually elutes from the column.     

Electrical behavior of polymer hydrogel composed of poly(vinyl alcohol)-hyaluronic acid in solution

Interpenetrating polymer networks (IPN), as polymer hydrogels composed of poly(vinyl alcohol) (PVA) and hyaluronic acid (HA), which exhibited electrical sensitive behavior were prepared. The swelling behavior of the IPN/HA IPN was studied by immersing the gel in various concentrations of aqueous NaCl solutions and various pH buffer solutions. The response of the PVA/HA IPN to electric fields stimuli was also investigated. When swollen IPN was placed between a pair of electrodes, and an electric field applied, it exhibited bending behavior. The PVA/HA IPN also displayed stepwise bending behavior, depending on the magnitude of the electric stimulus. Also, for use in biosensors application, their bending behavior was studied in Hank's solution at pH 7.4.     

Isoelectric focusing of proteins using a pH gradient created by a concentration gradient of nonelectrolytes in solution.

A new method of producing a stable pH gradient in buffer solutions is suggested, obtained by the concentration gradient of a nonelectrolyte in buffer solutions as a result of the gradual change in the dielectric properties of the solution. The maximal concentrations of nonelectrolyte which do not influence the protein configuration allow a pH gradient with a range of two pH units to be produced. It is suggested that the properties of some polyols (i.e. glycerol or mannitol) be used to change the pH of the borate buffer for the production of greater pH gradient with a range of up to 4-5 pH units. Creating the gradient of concentration of polyols, one can obtain a pH gradient in borate buffer solutions. Though the polylydroxyl compound-borate complexes posses mobility in an electric field, a stable pH gradient can be achieved during 12 days of electrophoresis. The isoelectric focusing of haemoglobin, human serum albumin and immunoglobulins was carried out in both systems suggested. These findings were compared with isoelectric focusing in Ampholines. There was a good agreement between the methods compared. The possible differences are discussed.     

Separation of bacterial cells by isoelectric focusing, a new method for analysis of complex microbial communities.

A simple isoelectric focusing (IEF) method for whole bacterial cells was developed. In a pH gradient of 2 to 10 and an electric field of 11.5 V cm-1, mixtures of cells from the three different bacterial strains Chlorobium limicola 6230, Pseudomonas stutzeri DSM 50227, and Micrococcus luteus DSM 20030 could be separated. A density gradient of Ficoll prevented convective currents in the system. The method was tested with a concentrated mixture of bacteria from a shallow eutrophic lake and yielded up to 10 different bands. Species composition in each IEF band was analyzed by PCR plus denaturing gradient gel electrophoresis (DGGE). Each IEF band exhibited a different species composition. After the separation of cells by IEF three times more 16S ribosomal DNA signals could be detected by DGGE than in the unfractionated natural bacterial community. It is concluded that the resolution of these molecular biological methods is significantly enhanced if cells are first separated by IEF. At the same time, the IEF fractions are enriched for certain species, which can be used in subsequent cultivation experiments.     

Electrical behavior of a natural polyelectrolyte hydrogel: chitosan/carboxymethylcellulose hydrogel

An amphoteric hydrogel film was prepared by solution blending of two natural polyelectrolytes, chitosan and carboxymethylcellulose, and cross-linking with glutaraldehyde. The bending of the film in an electric field was studied in different electrolyte solutions. Because of its amphoteric nature, the hydrogel can bend toward either anode or cathode depending on the pH of the solution. Other factors such as ionic strength and electric field strength also influence the electromechanical behavior of the hydrogels. The equilibrium bending angle of the hydrogel was found to reach a maximum at about 90 degrees in pH = 6 Britton-Robinson buffer solution with an ionic strength of 0.2 M. The sensitivity of the films over a wide range of pH and the good reversibility of this natural amphoteric electric-sensitive hydrogel suggest its future use in microsensor and actuator applications, especially in the biomedical field.     

Experimental generation and computational modeling of intracellular pH gradients in cardiac myocytes

It is often assumed that pH(i) is spatially uniform within cells. A double-barreled microperfusion system was used to apply solutions of weak acid (acetic acid, CO(2)) or base (ammonia) to localized regions of an isolated ventricular myocyte (guinea pig). A stable, longitudinal pH(i) gradient (up to 1 pH(i) unit) was observed (using confocal imaging of SNARF-1 fluorescence). Changing the fractional exposure of the cell to weak acid/base altered the gradient, as did changing the concentration and type of weak acid/base applied. A diffusion-reaction computational model accurately simulated this behavior of pH(i). The model assumes that H(i)(+) movement occurs via diffusive shuttling on mobile buffers, with little free H(+) diffusion. The average diffusion constant for mobile buffer was estimated as 33 x 10(-7) cm(2)/s, consistent with an apparent H(i)(+) diffusion coefficient, D(H)(app), of 14.4 x 10(-7) cm(2)/s (at pH(i) 7.07), a value two orders of magnitude lower than for H(+) ions in water but similar to that estimated recently from local acid injection via a cell-attached glass micropipette. We conclude that, because H(i)(+) mobility is so low, an extracellular concentration gradient of permeant weak acid readily induces pH(i) nonuniformity. Similar concentration gradients for weak acid (e.g., CO(2)) occur across border zones during regional myocardial ischemia, raising the possibility of steep pH(i) gradients within the heart under some pathophysiological conditions.     

A systematic study of field inversion gel electrophoresis.

The mobilities of oligomers of phage lambda DNA and of yeast chromosomes in agarose gels during field inversion gel electrophoresis (FIGE) were measured at different pulse times and electric fields. Also the ratios between forward and backward pulse times and/or field gradients were varied. The problem of 'band inversion' during FIGE, leading to an ambiguity in the mobility of large DNA fragments, was solved by using two dimensional gel electrophoresis with different parameters in the first and second dimension. The results are compared with those obtained with other pulsed electrophoresis systems and with a theoretical model.     

Induction of DNA single-strand breaks in barley by sodium azide applied at pH 3.

Sodium azide (1 to 50 mM), adjusted to pH 3 and applied for 2 h to presoaked barley seeds, induced a dose-dependent frequency of single-strand breaks in DNA of non-germinating embryos. This was demonstrated by sedimentation analyses of isolated DNA samples in alkaline sucrose gradients and in neutral sucrose gradients with 80% formamide. The doses applied also inhibited dose dependently the root length, seed germination and partially the seedling height. Only the sub-lethal doses (10 and 12.5 mM) induced a low frequency of chromatid breaks and translocations in the root tip metaphases. The sedimentation rate (in alkaline sucrose gradients) of calf thymus DNA treated with sodium azide at pH 3, was similar to that of the control DNA treated with buffer (pH 3) alone.     

Responses of forest snail assemblages to soil acidity buffer system and liming

Physiological constraints from harsh environmental conditions, such as from calcium limitation on acidic soils, is expected not only to affect species richness, but also species abundance distributions. Also, the effects of amendments by calcium addition (soil liming) on these assemblage characteristics are poorly understood. Because of their sensitivity towards calcium availability, we use snails as model organisms and integrate field surveys and literature data. Temperate forest snail data supported a rule-of-thumb calibration with pH measurements in water being one unit higher than in KCl buffer. The resulting large data set suggests stepwise changes in snail richness that occur at transitions in soil buffer systems, especially at pH 3.2. Species abundance distributions follow the logseries model in most soil buffer systems, except for the iron buffer range (pH ≤3.2) where they swap to the geometric model. Our findings thus suggest several smaller soil pH thresholds for snail assemblages associated with shifts between soil buffer systems, and a tipping point at the threshold to pH ≤3.2. Liming with ground carbonate rocks is a technique to temporarily increase soil pH and calcium availability in forest soils, but its effects on snail assemblages produced inconsistent results that did not meet expectations from the ameliorated soil pH and might warrant a re-evaluation of liming applications.     

Effect of electric field gradients on lipid monolayer membranes.

Externally applied nonuniform electric fields can strongly affect thermodynamic phases in a lipid monolayer when applied under conditions of temperature, pressure, and composition that are near phase boundaries. Under such conditions nonuniform applied fields can produce or suppress phase separations. Field-induced phase-separated domains have sizes that are in good agreement with calculations. Field gradients can also produce large concentration gradients in binary mixtures just above their critical points. The present work elaborates our earlier studies of these field effects using thermodynamic models of the phase behavior of two-component liquid mixtures. The calculations are of interest in connection with biological membranes that, at the growth temperature, are in a liquid state close to a phase boundary.     

Effect of anomalous ion inertia and oblique ion viscosity on the radial electric field in FT-2 tokamak experiments

Results are presented from numerical simulations that show that, in a plasma with well-developed turbulence, the radial electric field can be positive in the region where the gradients of the plasma parameters are steep. In a plasma in which the turbulence is suppressed (as is the case with auxiliary lower hybrid heating), the radial electric field is found to exhibit a nearly neoclassical behavior during the formation of a transport barrier and transition to the H-mode.     

Two-stage methods for alkaline-range analytical isoelectric focusing in thin-layer polyacrylamide gel: Application to cationic proteins in serum

Two-stage methods for alkaline-range analytical isoelectric focusing in thin-layer polyacrylamide gels have been developed for the analysis of the cationic proteins in complex mixtures of protein. A pH gradient covering the “entire” pH range but emphasizing only a narrow portion of this gradient is produced. During the first stage a nonlinear pH gradient is nearly established. The anodic electrode is then moved to a position on the gel such that the applied potential is exclusively across the alkaline portion of the gradient. The second stage results in a significant increase in the electric field strength and, consequently, the resolving power. The two-stage methods, exploiting the advantages of narrow pH gradients and high field strengths, have enabled excellent resolution of the highly heterogeneous cationic proteins in serum.     

Isolation of dipeptidyl peptidase IV (DP 4) isoforms from porcine kidney by preparative isoelectric focusing to improve crystallization

Abstract In the present studies we resolved the post-translational microheterogeneity of purified porcine dipeptidyl peptidase IV (DP 4) from kidney cortex. Applying SDS-homogeneous DP 4 onto an analytical agarose isoelectric focusing (IEF) gel, pH 4-6, activity staining resulted in at least 17 isoforms between pH 4.8-6.0. These could be separated into fractions with only two to six isoforms by means of preparative liquid-phase IEF, using a Rotofor cell. Starting off with three parallel Rotofor runs under the same conditions at pH 5-6, the fractions were pooled according to the specific activity of DP 4, pH and analytical IEF profile, and further refractionated without any additional ampholytes. Since excessive dilution of ampholytes and proteins was kept to the minimum, a second refractionation step could be introduced, resulting in pH gradients between 0.022 and 0.028 pH increments per fraction. By performing two consecutive refractionation steps, the high resolution necessary for the separation of DP 4 isoforms could be achieved. This represents an alternative method if isolation of isoforms with similar pI's results in precipitation and denaturation in presence of a narrow pH range. Furthermore, it demonstrates that preparative IEF is a powerful tool to resolve post-translational microheterogeneity of a purified protein required for crystallization processing.     

Direct detection of beta-1,3-glucanase isozymes on polyacrylamide electrophoresis and isoelectrofocusing gels

A procedure to assay isozymes of beta-1,3-glucanase directly on polyacrylamide gel electrophoresis (PAGE) and isoelectrofocusing (IEF) gels by using 2,3,5-triphenyltetrazolium chloride is described. The reagent reacts with reducing sugars released by beta-1,3-glucanases from the substrate laminarin. Acidic and neutral isozymes of beta-1,3-glucanase were detected and quantified on 17.5% native PAGE gels run with an anodic buffer system. A significant linear relationship (alpha = less than 0.01, R = 0.991) was observed between amounts of beta-1,3-glucanase loaded and intensity of bands stained with the reagent on native PAGE gels. A full isozyme pattern was obtained on 7.5% IEF gels with a pH range of 3.5-9.5. The IEF gels were heated in a microwave oven during the staining process to minimize diffusion.     

Electric field effects on immobilized urease activity.

The behavior of an enzyme/membrane system containing urease is studied when an external electric field is applied. The device using a potential difference across the enzyme/membrane system is first described. Optimal operating conditions with respect to substrate concentration, ionic strength and pH are studied. Possible mechanisms of the change in membrane activity by electric field are discussed.