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.     

Preparative free-flow isoelectric focusing: modeling and experiments

Free-flow isoelectric focusing was adapted to preparative scale separations and chemical engineering methods were used to describe the main mechanisms operating in the apparatus. A mixture of human serum albumin (pI 4.6) and beta-lactoglobulin (pI 5.22) was separated in pH gradients, generated with carrier ampholytes of different origin and covering the pH ranges 4-6.5, 3.5-5, 4-5.5 and 4.5-5.0. Best results were obtained in the pH 4-5.5 range. The experimental results have validated the results obtained with a numerical model.     

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.     

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.     

Protein tryptic digests analyzed by carrier ampholyte-based capillary electrophoresis coupled to ESI-MS

In this study, narrow pH cuts of carrier ampholytes have been used as buffers in CE for the analysis of protein tryptic digests. Their low conductivity allows very efficient separations under high electric field strength without inducing any significant Joule heating. In this study, the capabilities of narrow pH cuts of carrier ampholytes for the separation of protein tryptic digests have been assessed. Three proteins of different molecular masses have been studied: cytochrome C (horse heart), beta-lactoglobulin B (bovine) and human transferrin. Efficient, rapid and repeatable separations of the peptides resulting from the tryptic digestion have been achieved in this buffer. Moreover, the feasibility of the coupling of carrier ampholyte-based capillary electrophoresis with ESI-MS has been demonstrated through the study of the cytochrome C tryptic digest.     

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.     

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.     

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.     

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.     

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.     

Polarography of lead itaconate complexes

The complexation of lead ions with itaconic acid (H(2) A) has been studied polarographically at 30 degrees . At pH < pK(1), the complex species Pb(HA)(+) was identified. At pK(1) < pH < pK(2) and pH > pK(2) the formation of Pb(A) and Pb(A)(2)(2-) respectively, was confirmed. The dissociation constant of Pb(A)(2)(2-) was found to be 8.3 x 10(-5). The electrode reactions were established.     

Human alpha-1-antitrypsin subtyping by hybrid isoelectric focusing in miniaturized polyacrylamide gel

The polymorphism of alpha-1-antitrypsin (PI) has been studied by hybrid isoelectric focusing in miniaturized immobilized pH gradient gels, with an interelectrode distance of 55 mm, in two narrow ranges of pH 4.35-4.75 and 4.35-4.55), following rehydration with pH 4.2-4.9 carrier ampholytes. The use of the separator N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) in combination with carrier ampholytes for gel rehydration has been shown to enhance PI band sharpness. The influence of different additives (sucrose, sorbitol and glycerol) on the PI band pattern has also been evaluated. Glycerol has been shown to be responsible for the change in the relative mobility of the M3 band. The analysis of the minor M-7 isoprotein zone by hybrid isoelectric focusing followed by silver staining has permitted a more reliable classification of PIM subtypes. A population study carried out with 164 unrelated individuals living in Spain is also presented.     

Direct detection of carrier ampholytes in immobilized pH gradients using picric acid precipitation.

A protocol is described for monitoring the heterogeneity of end products of organic syntheses yielding amphoteric molecules containing two or more amino groups. This protocol was found to be a valuable aid in synthesis of carrier ampholytes for specific isoelectric focusing applications. This method does not depend on the ampholytes themselves to dictate the conditions under which they are analyzed. Carrier ampholytes have been found previously to be insoluble in picric acid and the insolubility property was not dependent upon the pI of individual ampholyte species. This insolubility property was exploited in the protocol. Immobilized pH gradients were used to focus the carrier ampholytes. Ampholytes were then visualized in situ by picric acid precipitation. The data shows that the protocol is useful for analyzing the results of chemical manipulations for enhancing the resolution of carrier ampholytes. A direct relationship was shown between carrier ampholyte heterogeneity as demonstrated by this protocol and the resolution of complex protein mixtures in isoelectric focusing gels. Picric acid formed visible precipitates with a variety of organic compounds which contained more than one amino group.     

Modeling and simulation of IEF in 2-D microgeometries

A 2-D finite-volume model is developed to simulate nonlinear IEF in complex microgeometries. This mathematical model is formulated based on the mass conservation and ionic dissociation relations of amphoteric macromolecules, charge conservation, and the electroneutrality condition. Based on the 2-D model, three different separation cases are studied: an IPG in a planar channel, an ampholyte-based pH gradient in a planar channel, and an ampholyte-based pH gradient in a contraction-expansion channel. In the IPG case, cacodylic acid (pK(1) = 6.21) and Tris (pK(1) = 8.3) are used as the acid and base, respectively, to validate the 2-D IEF model. In the ampholyte-based pH gradient cases, IEF is performed in the pH range, 6.21-8.3 using 10 ampholytes in the planar channel and 20 ampholytes in the contraction-expansion channel. The numerical results reveal different focusing efficiencies and resolution in the narrow and wide sections of the contraction-expansion channel. To explain this, the expressions for separation resolution and peak concentrations of separands in the contraction-expansion channel are presented in terms of the channel shape factor. In a 2-D planar channel, a focused band remains straight all the time. However, in a contraction-expansion channel, initially straight bands take on a crescent profile as they pass through the trapezoidal sections joining the contraction and expansion sections.     

Fractionation of carrier ampholytes for isoelectric focusing.

A simple method for fractionating synthetic carrier ampholytes is reported, based on the principle of continuous-flow isoelectric focusing in gel-stabilized layers. An 8% ampholyte solution, encompassing the pH range 3-9.5, is separated into 12 fractions in a chamber filled with Sephadex G-100 by a continuous-flow technique. We are thus able to obtain ampholytes of narrow pH range, encompassing approximately 2 pH units, whose resolving power is comparable with that obtained with commercial Ampholine covering similar pH ranges.     

Haptoglobin subtyping by isoelectric focusing in miniaturized polyacrylamide gels rehydrated in presence of 2-mercaptoethanol

A fast isoelectric focusing method for routine haptoglobin (Hp) subtyping is presented. This method is based on isoelectric focusing, under reducting conditions, of neuraminidase-treated plasma samples by using dry miniaturized (interelectrode distance: 55 mm) polyacrylamide gel, rehydrated in presence of 2-mercaptoethanol and a mixture of pharmalyte carrier ampholytes (pH 4-6.5 and pH 6-8) followed by immunoblotting. The presence of 2-mercaptoethanol in the gel prevented refolding of the Hp alpha and Hp beta chains during focusing, making it possible to obtain a sharp Hp band pattern with a clear separation of the different Hp alpha allelic products (1S, 1F, 2FS, 2SS and 2FF). A population study carried out with 250 unrelated individuals living in Central Spain is also presented.     

Evaluation of isoelectric focusing runs as specific resistance/volthour plots.

Isoelectric focusing (IEF) runs, e.g. on ultrathin gels, are characterized by an extensive change of gel electric parameters, caused by the rearrangement of carrier ampholyte components from a uniform distribution to a highly ordered pH gradient. A particularly important parameter is the specific resistance rho [Ohm*cm] which has been determined in polymerization mixtures (with and without carrier ampholytes) and in 125 x 0.15 mm ultrathin gels, pH 3-10 with 5% T, 3% C, 5% Servalyt carrier ampholytes, pH 3-10. The starting specific resistance rho of ultra-thin IEF gels, calculated from the geometric gel dimensions and electric current values (V, mA), is in agreement with the data determined directly in 30 mL samples of polymerization mixtures by using a conductivity meter. Electric specific conductivity/Volthour (Vh) plots proved to be a valuable tool for the evaluation of gel systems with and without protein samples during IEP runs. These plots are usually S-shaped, indicating that the key part of pH gradient formation takes places in a relatively short time. A "good" ultrahin gel, after a short lag phase, shows a rapid increase in specific resistance due to a rapid pH gradient formation and a slope of about 18 Ohm*cm/Vh. IEF is finished in about 3000 Vh. After prolonged gel storage, and especially in partially dried gels, the electrical parameters approach equilibrium only slowly, as indicated by the relatively shallow slope (8.9 Ohm*cm/Vh). Accordingly, separations need more than 4000 Vh.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Synthesis of UV-absorbing and fluorescent carrier ampholyte mixtures and their application for the determination of the operational pH values of buffering membranes used in isoelectric trapping separations

Success in isoelectric trapping separations critically depends on the knowledge of the accurate operational pH value of the buffering membranes used. Currently, due to a lack of easy, rapid, accurate methods that can be used for the post-synthesis determination of the operational pH value of a buffering membrane, only nominal pH values calculated from the amounts of the reagents used in the synthesis of the membranes and their acid-base dissociation constants are available. To rectify this problem, UV-absorbing and fluorescent carrier ampholyte mixtures were prepared by alkylating pentaethylenehexamine with a chromophore and a fluorophore, followed by Michael addition of acrylic acid and itaconic acid to the resulting oligoamine. Carrier ampholyte mixtures, with evenly distributed absorbance values across the 3相似文献