Isoelectric focusing by free solution capillary electrophoresis.

A reproducible, quantitative isoelectric focusing method using capillary electrophoresis that exhibits high resolution and linearity over a wide pH gradient was developed. RNase T1 and RNase ba are two proteins that have isoelectric points (pI's) at the two extremes of a pH 3-10 gradient. Site-directed mutants of the former were separated from the wild-type form and pI's determined in the same experiment. The pI's of RNase T1 wild-type, its three mutants, and RNase ba were determined for the first time as 2.9, 3.1, 3.1, 3.3, and 9.0, respectively. The paper describes the protocol for isoelectric focusing by capillary electrophoresis, as well as presenting data describing the linearity, resolution, limits of mass loading, and reproducibility of the method.     

Isoelectric focusing using non-amphoteric buffers in free solution: I. Determination of stable concentration profiles

For large-scale separations of proteins, the use of simple non-amphoteric buffers in free solution and in multicompartment electrolyzers seems promising for industrial applications. The stabilization of a pH profile with this type of buffer requires the strict observation of two conditions: choice of an adequate buffer; stationary profiles of concentrations. During electrolysis in free solution, the ions of the buffer are displaced across the compartments by migration and by diffusion. To keep a stationary composition, the inflow and outflow of all individual ionic species through each compartment must be identical. At high current, diffusion may be neglected against migration and the ionic flows will be identical if the transport number of each ion is constant at each location within the cell. In these conditions, stationary compositions will be independent of the electric current. This condition of constant transport numbers implies the use of profiles of buffer concentrations different from those published up to now. The new equations for these profiles of concentrations are given in the present paper. The constant migration of the ions must be compensated in the end compartments of the isoelectric focusing cell to provide a stable steady state. Two methods are proposed in the literature: the buffer renewal method and the external recycling method (rheoelectrolysis). Here modified buffer renewal method is proposed. Using stationary mass balances, analytical equations are given to calculate the flows and the composition of the solutions to be recycled or added. Using these equations and the profiles of concentrations to keep constant transport numbers, it is demonstrated that only a renewal of the buffers in the end compartments may lead to stable pH profiles and thus to valid conditions of separation.     

Isoelectric focusing of mycoplasma proteins.

Polyacrylamide gel isoelectric focusing (PAGIF) in thin layer was used to resolve proteins of Mycoplasma spp., Acholeplasma spp., and eight strains of Ureaplasma urealyticum (T-strain). A mixture of urea, Triton X-100, and dithioerythritol was used to solubilize sonically disrupted cells. PAGIF was performed in the range of pH 3 to 10. Protein patterns were carefully compared, demonstrating resolved and distinguishable species-specific protein bands. The eight serotypes of U. urealyticum (T-strain) gave identical protein patterns in the pH 3 to 10 range. The characteristic "fingerprints" of a species appeared to correlate with the biochemical nature and not the habitat in each case. Arginine-hydrolyzing species seemed to show more diverse focusing than those that ferment glucose, or prefer an acid environment. Characterization and identification of highly resolved species-specific proteins, ease of performance, and reproducibility of this method suggest that PAGIF might be employed as a taxonomic aid.     

Isoelectric focusing using non-amphoteric buffers in free solution: II. Apparatus and measures of pH stability

Isoelectric focusing of amino acids or proteins requires a time-invariant pH gradient. The maintenance, in an electric field, of such profiles with simple non-amphoteric buffers is possible in multicompartment cells using well determined concentration profiles. The method for determining these concentration profiles has been proposed in a preceding paper. A multicompartment cell has been built for the purpose of verifying the assumptions made. The technical characteristics of this cell will be described here. The time-stability of pH profiles obtained with sodium acetate/acetic acid buffers has been measured for concentration profiles determined so as to keep constant the transport number of each ion throughout the cell. Measured over a time span of 10 h and with a current density of 96 mA cm-2, the pH shift is, in the worst case, 0.009 pH unit/h. This corresponds to a much better stability than the one obtained with a constant concentration of sodium acetate in all compartments, and justifies the chosen concentration profiles based on the condition of constant transport numbers. The above mentioned method for the computation of the buffer concentration assumed a constant relative mobility of the ions. The experiments have shown the limits of this assumption, i.e. the variation of ionic mobilities with the ionic strength and the temperature diminishes the stability of the pH gradient. However, slight adjustments of the concentration in the end compartments allow some improvement of the stability. If the temperature could be precisely controlled in each compartment, a better pH stability than what has been achieved in these experiments should be reached. Two methods of compensation of the migration of ions in the end compartments (buffer renewal method and external recycling method) have been tested and will be discussed.     

Isoelectric focusing of myelin membrane proteins

Human myelin was isolated from the white matter of autopsy brains. Myelin proteins were characterized by isoelectric focusing in ultrathin slab gels in a pH range from 3.5 to 10 after solubilization with urea and Nonidet P 40. The protein profile in the acidic region (pH below 6.2) revealed at least twelve faint bands which comprised only a few percent of the total myelin proteins. Most of the myelin proteins were focused in the neutral range (pH 6.2–7.8) which showed two sharper and three broader major bands, the total number of bands in this region being about twenty. The basic pH range (pH above 7.8) contained about 30% of the proteins, and revealed a very intense band near the cathode with seven to nine weaker bands below pH 9.0. When the myelin was partially delipidated prior to solubilization, an additional broad band was observed at the area pH 8.0–8.5.     

Isoelectric focusing of sparingly soluble proteins in immobilized pH gradients, exemplified by microvillar membrane hydrolases

A novel fractionation technique is described for analysis of membrane-bound enzymes and sparingly soluble proteins: isoelectric focusing in a mixed-type matrix, containing a primary, immobilized pH gradient with a superimposed, secondary carrier ampholyte pH gradient. Three microvilli hydrolases: dipeptidyl peptidase IV, gamma-glutamyl transferase and alkaline phosphatase exhibit an array of sharply focused, enzyme active bands in the pH 4-6.5 range. The separation pattern obtained is by far superior to any separation achieved by either technique separately.     

Isoelectric focusing of cells using zwitterionic buffers.

A simple method of isoelectric focusing of cells is described. The pH gradient, superimposed on a density gradient, is developed by generating opposing concentration gradients of two zwitterionic buffers. The method can be used as a cell separation technique or as a means of characterizing the cell type on the basis of the focusing pH. Focusing is rapid and thus the method is of special advantage in its application to cells.     

Isoelectric focusing using non-amphoteric buffers in free solution: III. Separation of amino acids

Separation of proteins or amino acids by isoelectric focusing in multicompartment devices has been proposed for large-scale purifications of biological mixtures. In the perspective of industrial applications, the present authors built a multicompartment apparatus and studied the pH profiles stabilized by simple non-amphoteric buffers (acetic acid and sodium acetate). Mixtures of two amino acids were separated to test this device. A theoretical model comprising one dimensionless separation parameter is proposed to characterize these separations. This model allows one to calculate the purity of the recovered amino acids, the yield of a separation at steady-state or the time necessary to obtain a given concentration of an amino acid in one of the compartments of the isoelectric focusing cell. The separation parameter contains the physical parameters which intervene in the electric migration and in the diffusion. Values of this separation parameter have been experimentally determined for three amino acids under various experimental conditions. The results confirm the usefulness of this model in designing a multicompartment isoelectric focusing apparatus.     

Isoelectric focusing of basic proteases in immobilized pH gradients

By exploiting a new, alkaline immobilized pH gradient spanning the pH 10-11 interval, it has been possible to focus and to detect, by in situ zymogramming with cellulose acetate foils impregnated with fluorogenic substrates, 2 alkaline proteases, namely elastase and trypsin. Elastase gave a sharp array of 3 bands, with the following pIs (at 10 degrees C): 10.60 (major component), 10.53 (intermediate species) and 10.45 (minor isoform). Trypsin was resolved into 2, about equally abundant, species having pIs of 10.70 and 10.53. However, the latter enzyme gave smears in between these 2 forms and also anodic to the lower pI species. As hydrophobic interaction with the Immobiline matrix was excluded, it is suggested that these smears represent product of auto-digestion due to the very alkaline pH during the focusing process.     

Isoelectric focusing of human head hair proteins. Preliminary research

The examination of human hair keratin to obtain genetic information, which may be useful also in forensic sciences, has been carried out with the use of isoelectrophoretic procedure obtaining considerable evidence for the existence of specific-species patterns. In this paper the keratins extracted from hairs of 280 subjects belonging to Sardinian people (113 males, 167 females, aged from 1 to 89, belonging to 52 families) were analyzed using IEF in thin-layer polyacrylamide gel (0.5 mm) in the pH range 2.5–7.0, followed by the silver staining method. Number, position and colour of the bands were the same in all the analyzed samples but a large individual variability was revealed for the relative intensity of some bands. Differences for a long time storage were not revealed as well hair's sample as protein extract: Neither were differences in the number and position of the bands analyzing samples of hair from several sites of the head of the same individual revealed. The results obtained are a useful indication to continue this research considering the numerous fields of application of this analysis system.     

Isoelectric focusing in immobilized pH gradients: Generation of extended pH intervals

A new technique for generatiing extended pH gradients (3–4 pH units) in Immobiline gels for isoelectric separations is described. A five-chamber gradient mixer has been built, based on the ‘Varigard’-type mixers of Peterson and Sober (Anal. Chem. 31, 1959, 857–862). Each chamber contains one of the following Immobilines, in this order: pK values 4.4, 4.6, 6.2, 7.0 and 8.5, titrated in the pH 4–8 interval with non-buffering Immobilines pK 9.3 (in the case of the two acidic Immobilines) and pK 3.6 (in the case of the three basic Immobilines). In this way it is possible to cast, in a highly reproducible way, an immobilized pH gradient in thepH range 4.0 to 7.5, which should be ideal for isoelectric separations in the first dimension of two-dimensional techniques. A computer program is also described which, given the molarities and pK values of the different Immobilines in the chambers of the Varigrad mixer, can generate the theoretical pH profile, together with the buffering capacity (β) and ionic strength (I) courses.     

Isoelectric focusing in immobilized pH gradients of a snake venom fibrinolytic enzyme

A fibrinolytic enzyme with a molecular weight between 23,000 and 25,000 Da has been purified from southern copperhead snake venom. Immobilized pH gradient isoelectric focusing with an ultranarrow pH interval (pH 6.65-6.95) resolved two isoforms of the fibrinolytic enzyme that were not resolved by standard isoelectric focusing. Attempts at purification of the individual isoenzymes by semi-preparative scale IPG and elution of enzyme by macerating the gel yielded only 20-40% recovery of activity. In attempts to improve recovery, a semi-preparative IPG canal-isoelectric focusing technique has been utilized.