Characterization of synthetic carrier ampholytes for isoelectric focusing.

The synthesis of carrier ampholytes suitable for isoelectric focusing is described. The mixture of hexamethylenetetramine (HMTA), triethylenetetramine (TETA), tetraethylenepentamine (TEPA) and pentaethylenehexamine (PEHA) ampholytes closely resembles commercial Ampholine, and covers the pH range 3-9.5. We have been able to detect focused ampholytes in a gel slab, taking advantage of their different refractive indices, and to assess their relative amounts along the pH gradient. PEHA ampholytes contain up to 20% of chromophoric structures, with two UV peaks at 368 and 315 nm, in a pH-dependent equilibrium, associated with a very weak nitrogen function having a pK of 1.1. This could be the pK6 of the last amino group in PEHA. However, NMR spectra failed to reveal any nitrogen heterocyclic structure formed during the synthesis. This mixture of ampholytes exhibits good conductivity, produces smooth pH gradients and allows sharp protein separations in the pH range 3-9.5. Their synthesis is very easy and their cost is extremely low. Their availability sould make feasible large-scale preparative isoelectric focusing, and attract more interest to continuous-flow techniques, where large amounts of ampholytes are required.     

Itaconic acid carrier ampholytes for isoelectric focusing.

Commercial carrier ampholytes, obtained by coupling polyethylene polyamines to acrylic acid, exhibit a conductivity minimum in the pH range 5.5-6.5 owing to the lack of appropriate pK values of the polyamine in this pH region. By replacing acrylic with itaconic acid, it has been possible to effect substantial improvements in the pH range 5.5-6.5 as itaconic acid has a pK2 value of 5.45. Upon coupling, the pK of the gramma-carboxyl group remains virtually unaltered. With itoconic acid carrier ampholytes it has been possible to improve the conductivity in the pH range 5.5-6.5 by as much as 400% compared with conventional carrier ampholytes. It is suggected that the commercial products should be supplemented with itaconic acid carrier ampholytes in order to obtain a more uniform conductivity and buffering capacity in the pH range 3-10.     

Advanced online mass spectrometry detection of proteins separated by capillary isoelectric focusing after sequential injection

Capillary isoelectric focusing hyphenated with mass spectrometry detection, following the sequential injection of the carrier ampholytes and the sample zone, is highly efficient for the characterization of proteins. The main advantage of the sequential injection protocol is that ampholytes, with pH ranges, which are not supposed to cover the isoelectric points of the sample components, can be used for separation. The method then allows online mass spectrometry detection of separated analytes either in the absence (substances that have left the pH gradient) or in the presence of low‐level ampholytes (substances that are migrating within the pH gradient). The appearance of the substances within, or outside the pH gradient depends on, e.g., the composition of the ampholytes (broad or narrow pH range) or on the composition of electrolyte solutions. The experiments performed in coated capillaries (with polyvinyl alcohol or with polyacrylamide) show that the amount and the injection length of the ampholytes influence the length of the pH gradient formed in the capillary.     

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.     

Isoelectric buffers, part 3: determination of pKa and pI values of diamino sulfate carrier ampholytes by indirect UV-detection capillary electrophoresis

Ampholytes with close pK(a) values (i.e., good carrier ampholytes (CAs)) are needed as buffers in pH-biased isoelectric trapping (IET) separations. The syntheses of two families of such good CAs were reported recently. Members of the family of diamino sulfate ampholytes (first series) had pI values in the 5.7 < pI < 9.0 range. Members of the family of quaternary ammonium dicarboxylic acid ampholytes (second series) had pI values in the pI < 4.3 range. To further characterize the diamino sulfate ampholytes, their effective mobilities were measured by indirect UV-absorbance detection capillary electrophoresis in a series of background electrolytes (BGEs) with different pH values. The pK(a) and limiting ionic mobility values of the CAs were obtained by fitting these mobility values, as a function of the pH and the ionic strength of the BGEs, to the theoretical mobility expression. These diamino sulfates complete the list of CAs suitable for IET separations.     

High-resolution computer simulation of the dynamics of isoelectric focusing: in quest of more realistic input parameters for carrier ampholytes

The impact of the systematic variation of either DeltapK(a) or mobility of 140 biprotic carrier ampholytes on the conductivity profile of a pH 3-10 gradient was studied by dynamic computer simulation. A configuration with the greatest DeltapK(a) in the pH 6-7 range and uniform mobilities produced a conductivity profile consistent with that which is experimentally observed. A similar result was observed when the neutral (pI = 7) ampholyte is assigned the lowest mobility and mobilities of the other carriers are systematically increased as their pI's recede from 7. When equal DeltapK(a) values and mobilities are assigned to all ampholytes a conductivity plateau in the pH 5-9 region is produced which does not reflect what is seen experimentally. The variation in DeltapK(a) values is considered to most accurately reflect the electrochemical parameters of commercially available mixtures of carrier ampholytes. Simulations with unequal mobilities of the cationic and anionic species of the carrier ampholytes show either cathodic (greater mobility of the cationic species) or anodic (greater mobility of the anionic species) drifts of the pH gradient. The simulated cationic drifts compare well to those observed experimentally in a capillary in which the focusing of three dyes was followed by whole column optical imaging. The cathodic drift flattens the acidic portion of the gradient and steepens the basic part. This phenomenon is an additional argument against the notion that focused zones of carrier ampholytes have no electrophoretic flux.     

Colored pI standards and gel isoelectric focusing in strongly acidic pH

Colored, low molecular weight pI markers have been developed for isoelectric focusing (IEF) in acidic pH range. Their isoelectric points (pIs) were determined by direct measurement of the pH of the focused bands after completion of IEF on polyacrylamide gels. The practicable suitability of the proposed pI markers as pI standards for IEF was tested by applying gel IEF. The acidic pH gradient was created either by commercial synthetic carrier ampholytes or by mixture of simple buffers consisting of acids (non-ampholytes) and ampholytic buffers. By applying simple acids, it was possible to extend the acidic pH range beyond those achievable with commercial synthetic carrier ampholytes. By using an experimental arrangement without electrode electrolyte reservoirs with electrodes creating the fixed end of the gel, the strongly acidic pH gradient was stable even for prolonged focusing time.     

The adsorption of large proteins in electrofocusing on immobilized pH gradients: I. Protein specificity and dependence on Immobiline and carrier ampholyte concentrations

Phycoerythrin, ferritin, urease, beta-galactosidase and thyroglobulin, with molecular masses in excess of 200 kDa, adsorb and consequently fail to migrate to, and focus at, their pI positions in electrofocusing in immobilized pH gradients at a total Immobiline concentration of 20 mM while they do focus normally in pH gradients formed by carrier ampholytes. The addition of carrier ampholytes (pH range 3.5-9.5) at concentrations of 0.1 to 5% to the Immobiline-containing gels reduces adsorption (desorbs) some but not all of the 5 proteins at specific Immobiline concentrations. The adsorption is not due to water redistribution and consequent reduction in gel porosity; nor is it due to conductivity minima across the pH gradient. The hypothesis that the presence of oligomeric Immobiline contributed to the protein adsorption is the subject of the accompanying report.     

Effects of ampholyte concentration on protein behavior in on-chip isoelectric focusing

The effects of mobility corrections on carrier ampholytes are studied at various ampholyte concentrations to understand protein behavior during IEF. IEF simulations are conducted in the presence of 25 biprotic carrier ampholytes within a pH range of 6-9 after applying the Onsager-Debye-Hückel correction to the carrier ampholytes. Two model proteins with ten charge states but without ionic strength corrections are allowed to focus under an electric field of 300 V/cm in a 1 cm long channel. The IEF simulation results show that higher ionic strengths (50 - 100 mM) cause significant changes in the transient movement as well as the final focused profiles of both ampholytes and proteins. The time required for a single, well-defined peak to form increases with ionic strength when Onsager corrections are applied to the carrier ampholytes. For a particular ampholyte concentration, the space-averaged conductivity does not change during the final focusing stage, but the magnitude of space averaged conductivity is different for different ampholyte concentration. The simulation results also reveal that at steady-state ionic strength profiles remain flat throughout the channel except at the locations of proteins where a significant change in ampholyte concentration is obtained.     

Titration curves of polypeptide chains by combined isoelectric focusing-electrophoresis in 8 M urea

Titration curves of reduced and alkylated polypeptide chains can be successfully performed in 8 M urea-polyacrylamide gel plates by electrophoresis perpendicular to a stationary stack of focused carrier ampholytes. All buffers and thiol reagents with pK values in the range pH 3--10 should be removed, since their pH-dependent ionization affects the migration and apparent pI values of the protein chains. No blurring of the patterns below pH 4.5 is observed, as usually found in titration curves in the absence of urea, thus allowing the direct titration of Glu and Asp residues. It is not possible by the present technique to titrate any group below pH ca. 3 and above pH ca. 10, due to the lack of suitable carrier ampholytes and to a "flooding" phenomenon, with concomitant identical electrophoretic mobility for all protein species, irrespective of their relative pI values and amino acid composition. The "electrophoretic titration curves" thus obtained were well correlated with the overall amino acid composition of the polypeptide chains analyzed.     

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.     

Electrophoretic phenotyping of erythrocyte enzymes.

Erythrocyte acid phosphatase (EAP), esterase D (ESD) and phosphoglucomutase (PGM) phenotypes among the erythrocyte enzyme types of blood groups are surveyed and a modified cellulose acetate membrane isoelectric focusing (CAM-IEF) method for their exploration is described. The phenotyping procedures are usually classified as either equilibrium or non-equilibrium IEF. Equilibrium IEF, which is based on differences in pI values, includes three methods: (i) a narrow pH range of carrier ampholytes, (ii) a relatively narrow pH range of carrier ampholytes containing chemical separators and (iii) immobilized pH gradient gels. Among the three methods, immobilized pH gradients provides a better resolution of isozymes. Conversely, the disadvantages of immobilized pH gradients include longer focusing times and complex gel preparations. Moreover, immobilized pH gradients are unsuitable for stain analysis because of the insensitivity of PGM1 detection. A hybrid IEF system and a commercial immobilized pH gradient dry plate have overcome these problems. However, EAP typing is extremely expensive and ESD typing is not well distinguished by hybrid IEF. As each method has both merits and demerits, the most suitable technique should be selected based on the kind of erythrocyte enzyme types and sample conditions. On the other hand, non-equilibrium IEF is a rapid method because isozymes are detected on the basis of their charge differences under non-equilibrium conditions. Moreover, the appropriate addition separators increases the charge difference and provides a good resolution within a shorter time. Addition of more separators produces a narrow pH range in the gel and takes a substantially longer time to reach the optimum pH range for charge difference.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sampling strategies for capillary isoelectric focusing with electroosmotic zone mobilization assessed by high-resolution dynamic computer simulation

The impact of initial sample distribution on separation and focusing of analytes in a pH 3–11 gradient formed by 101 biprotic carrier ampholytes under concomitant electroosmotic displacement was studied by dynamic high-resolution computer simulation. Data obtained with application of the analytes mixed with the carrier ampholytes (as is customarily done), as a short zone within the initial carrier ampholyte zone, sandwiched between zones of carrier ampholytes, or introduced before or after the initial carrier ampholyte zone were compared. With sampling as a short zone within or adjacent to the carrier ampholytes, separation and focusing of analytes is shown to proceed as a cationic, anionic, or mixed process and separation of the analytes is predicted to be much faster than the separation of the carrier components. Thus, after the initial separation, analytes continue to separate and eventually reach their focusing locations. This is different to the double-peak approach to equilibrium that takes place when analytes and carrier ampholytes are applied as a homogenous mixture. Simulation data reveal that sample application between two zones of carrier ampholytes results in the formation of a pH gradient disturbance as the concentration of the carrier ampholytes within the fluid element initially occupied by the sample will be lower compared to the other parts of the gradient. As a consequence thereof, the properties of this region are sample matrix dependent, the pH gradient is flatter, and the region is likely to represent a conductance gap (hot spot). Simulation data suggest that sample placed at the anodic side or at the anodic end of the initial carrier ampholyte zone are the favorable configurations for capillary isoelectric focusing with electroosmotic zone mobilization.     

Finite element simulation of Off-Gel trade mark buffering

The protonation of an aqueous solution of two ampholytes AH and BH next to a gel buffered by immobilized acid moieties IH has been studied by finite element simulation in an iterative scheme. A ten species model has been formulated, taking into account transient diffusion and equilibrium kinetics of the two amphoteric species AH and BH, of water and of the immobilized species IH. This model has been developed to illustrate the pH evolution between an ampholyte solution and an Immobiline gel, and to study the influence of the Immobiline concentration on protons and ampholyte distributions. It has been demonstrated that a minimum initial Immobiline concentration of 10(-2) M is necessary to maintain the pH in the gel in contact with a closed chamber, when the difference between the isoelectric points of AH and BH is 4 and when the initial concentration of the ampholytes in solution is in the micromolar range. This approach provides a first theoretical framework for the recently developed Off-Gel trade mark electrophoresis.     

Synthesis and characterization of quaternary ammonium dicarboxylic acid isoelectric buffers and their use in pH-biased isoelectric trapping separations

Two approaches are described in this paper for the synthesis of isoelectric buffers that have pI values in the 1.5 < pI < 4.3 range. The first synthesis relies on the alkylation of existing aminodicarboxylic acids and recovery of the ampholyte as an inner salt. The second synthesis method forms low-pI ampholytes by reacting a secondary amine with two equivalents of an alkylester of a haloalkanecarboxylic acid, followed by hydrolysis of the intermediate in an alkaline solution and recovery of the ampholyte as an inner salt. The new ampholytes have been analytically characterized by capillary electrophoresis, high-resolution electrospray ionization-mass spectrometry, one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy, and X-ray crystallography. The isoionic solutions of the new ampholytes have high buffering capacity and conductivity, making them good pH biasers in the receiving stream in preparative-scale pH-biased isoelectric trapping separations.     

Peak identification in capillary isoelectric focusing using the concept of relative peak position as determined by two isoelectric point markers

In CIEF analysis, sample peaks can be identified by their relative peak positions (RPP) that are determined using only two internal pI markers. The two internal pI marker peaks should bracket, as close as possible, the sample peaks. The RPP values of the sample peaks are then calculated using the pI values, peak positions of the two pI markers, and peak position of the sample. Use of this method can effectively compensate for pH gradient distortions that often occur as a result of salts. Also, as shown by the results of this paper, regardless of the linearity of the pH gradient established by the given carrier ampholytes, sample peaks can be identified within an SD of 0.1 pH unit in RPP (<2% RSD) as long as the sample is run using the same carrier ampholytes and maintaining salt concentrations in the range of 0-15 mM.     

Use of quasi-isoelectric buffers to limit protein adsorption in capillary zone electrophoresis

The use of quasi-isoelectric buffers consisting of narrow pH cuts of carrier ampholytes (NC) has been investigated to limit protein adsorption on capillary walls during capillary zone electrophoresis experiments. To quantify protein adsorption on the silica surface, a method derived from that of Towns and Regnier has been developed. alpha-Lactalbumin (14 kDa, pI 4.8) and alpha-chymotrypsinogen A (25 kDa, pI 9.2) have been used as model proteins. Acidic narrow pH cuts of carrier ampholytes (NC, pH 3.0) obtained from fractionation of Serva 4-9 carrier ampholytes were used as BGE in bare-silica capillaries, and allowed to decrease significantly protein adsorption, as compared to experiments performed with classical formate buffer. The use of NC as BGE appeared to be as efficient as the use of polydimethylacrylamide coating to prevent protein adsorption. This increase of protein recovery when using NC was attributed to the interaction of carrier ampholytes with the silica surface, leading to a shielding of the capillary wall.     

Ampholytes as background electrolytes in capillary zone electrophoresis: sense or nonsense? Histidine as a model ampholyte

A lot of phenomena, occuring in capillary zone electrophoresis (CZE), are linked with the ionic concentration of the background electrolyte (BGE). If weak bases and acids are used as BGEs in CZE, at a pH where they are scarcely ionized, the ionic concentration of the BGE is very low and this brings a strong peak broadening, limited sample stacking and low sample load. Because the electromigration dispersion increases extremely, moreover, the existence of low-conductivity BGEs in CZE is a contradiction in terms. The behavior of ampholytes as BGE in CZE is examined, by means of histidine as a model ampholyte. For BGEs consisting of histidine, important parameters, including the ionic concentrations, buffer capacity, transfer ratio, and the indicator for electromigration dispersion E(1)m(1)/E(2)m(2), are calculated at various pH. Although the transfer ratio is fairly constant over the whole pH traject, the ionic concentration and buffer capacity decrease whereas the electromigration dispersion strongly increases near the pI of histidine. I.e., that ampholytes can be applied as BGEs in CZE, however, just not at pH near their pI value, except as the difference between the pK values of the basic and acidic group, the deltapK value, is very small. For ampholytes with a low deltapK value or at high concentrations, all the before-mentioned effects are less fatal, but in that case we can not speak of a real low-conductivity BGE. If ampholytes are used at pH near their pK values, the use of ampholytes as BGE is not advantageously compared with simple weak bases and acids. This has been confirmed by calculations and experiments.     

Flattening and/or expanding of pH gradients in isoelectric focusing gels exemplified with PhastSystem

A simple method of flattening and/or expanding of pH gradients in isoelectric focusing is described for any pH interval desired: to modify pH gradients near one electrode a paper strip soaked with carrier ampholytes is applied onto the gel close to the opposite electrode. In order to flatten central parts of pH intervals paper strips are applied onto the gel at both electrodes. Conditions and criteria (e.g. amount and pH intervals of carrier ampholytes, width and localization of the paper strip, separation period) for optimization are presented with PhastSystem using ready-made gels with three different pH intervals and pI marker proteins (Pharmacia). Examples utilizing erythrocyte lysates are presented.     

Determination of the operational pH value of a buffering membrane by an isoelectric trapping separation of a carrier ampholyte mixture

The operational pH value of a buffering membrane used in an isoelectric trapping separation is determined by installing the membrane as the separation membrane into a multicompartmental electrolyzer operated in the two-separation compartment configuration. A 3相似文献