Conventional isoelectric focusing (IEF) in soluble, amphoteric buffers known as carrier ampholytes (CAs) was reported by Svensson-Rilbe in 1961 (1-3). In this technique, the macromolecules of interest are subjected to electrophoresis in a continuous pH gradient. They migrate electrophoretically toward the pH of their isoelectric point (pI), where they have zero mobility. Therefore, the system comes to equilibrium, with each species of macromolecule tightly focused at its pI; any diffusion away is reversed by the electrophoretic migration. At about the same time, Meselson et al. (4) described isopycnic centrifugation, a related high-resolution technique that is another member of a family called isoperichoric focusing by Kolin (5). Unlike conventional chromatographic and electrophoretic techniques, where peaks of material are constantly dissipated by diffusion, IEF and isopycnic centrifugation have built-in mechanisms opposing this. As the analyte reaches an environment (the perichoron in Greek) in which its physicochemical parameters are equal (iso) to those of its surroundings, it focuses, or condenses, in an ultrathin zone, kept stable and sharp in time by two opposing force fields: diffusion (tending to dissipate the zone) and external fields (voltage gradients in IEF or centrifugal fields in isopycnic centrifugation) forcing the "escaping" analyte back into its "focusing" zone.
Both conventional IEF using carrier ampholytes and the new version using immobilized pH gradients (IPG) (6) rely on the equation for resolving power (expressed as DpI, i.e., the difference in isoelectric points between a protein and a just resolved, nearest contaminant) (7):
where Dt is the protein translational diffusion coefficient, E is the voltage gradient (V/cm) applied, d (pH)/d x is the slope of the pH gradient along the separation axis, and du/d(pH) is the titration curve of the protein in terms of its mobility as a function of pH. Experimental conditions minimizing DpI offer the greatest resolution, which are shallow pH gradients and high voltage gradients. The best DpI attainable is of the order of 0.01 pH unit in CA-IEF but 0.001 in IPG, where more shallow pH gradients and higher voltages are possible. Consequently, complex mixtures of macromolecules can be resolved into very many species, and what appears to be a single species by many separation techniques is frequently resolved into several by IPG (Fig. 1). A brief description of both methods will be given.
1. Conventional Isoelectric Focusing in Soluble, Amphoteric Buffers
Figure 1. An example of the separations possible with isoelectric focusing. This soluble form of the receptor for epidermal growth factor (EGF) gave a single band on SDS-PAGE, but it was resolved by IPG into three major isoforms and several minor bands. The pH gradient was from pH 5 to pH 8, in a 10-cm-long 5% T, 4% C polyacrylamide gel equilibrated with 30% ethylene glycol. A total of 50 ^g of protein was loaded at the anode, and focusing was carried out with the cathode uppermost. The resulting gels were stained with Coomassie Brilliant Blue R-250 (in ethanol, acetic acid, and copper sulfate) on the left and by Western blotting on the right. The pI values of the major components are indicated.
Carrier ampholytes set up the pH gradient for IEF by distributing themselves electrophoretically at their isoelectric points. For a range of pH values, an appropriate mixture of ampholytes is necessary. They must not only be amphoteric, but must also have suitable buffering power and conductivity at their pI (7). The hallmark of a carrier ampholyte is the absolute value of the difference between its pI and the p Ka of its buffering groups; the smaller this value, the greater its conductivity and buffering capacity at its pI (8). Very few natural compounds have this property.
A breakthrough came in 1964 with Versterberg’s synthesis of Ampholines (trade name of the commercial product from LKB). They are "oligoprotic amino carboxylic acids, each containing at least four weak protolytic groups, at least one being a carboxyl group and at least one a basic nitrogen atom, but no peptide bonds" (9). Figure 2 reports a hypothetical general structural formula of these ampholytes. Very narrow pH ranges of ampholytes are produced, except for the very extreme pH 2.5 to 4 and pH 9 to 11 ranges, by subfractionating the "wide pH-range" mixture in multicompartment electrolyzers. The synthesis of Ampholines is a genuine "chaotic" process, in that very heterogeneous mixtures of different oligoamines (typically pentaethylene hexamine, tetraethylene pentamine, and triethylene tetramine, including their branched isomers) were reacted with an a-b unsaturated acid (typically acrylic acid) at an appropriate ratio (usually 2 N atoms/1 carboxyl group); b-propionic acid residues result. By this synthetic approach, >600 ampholyte species could be generated in the pH 3 to 10 interval. Subsequently, Pharmalyte species containing >5000 chemically distinct amphoteres in the pH 2.5 to 11 interval were produced. Almgren (10) had predicted theoretically (assuming equimolar distribution of the CA species, even distribution of their DpI along the pH scale, and the same DpKa) that only 30 amphoteres/pH unit had to be present to generate a stepless pH course. Because the oligoamines prepared had pKa values well distributed along the pH scale, they could provide the buffering power and conductivity required for IEF. Additionally, the extra p Ka value of the "titrant" acrylic acid grafted onto the oligoamino backbone endowed acidic carrier ampholytes with extra buffering power. This is one of the reasons why focusing with CA buffers is usually more successful in acidic pH ranges than alkaline. The other reasons are that this synthesis generates fewer alkaline ampholytes and that open-face IEF gel slabs at pH >8 absorb atmospheric CO2, unless submerged under a thin layer of light paraffin oil.
Figure 2. Composition of ampholines. (a) Representative chemical formula (aliphatic oligo-amino, oligo-carboxylic acid ( b) Portions of hypothetical titration curves of Ampholines. (c) Different pH cuts for wide- and narrow-range carrier ampholytes.
CA-IEF offers unrivaled resolving power in a vast number of separations and is now a standard technique in molecular biology. In addition to its current uses, at least two particular applications are worth mentioning: (a) its use in two-dimensional (2-D) electrophoresis to generate 2-D maps (11, 12) and (b) for generating titration curves of macromolecules. In the latter case, the CA-IEF slab gel is focused to generate the desired pH gradient. The slab is then turned 90° and the sample is loaded in a long, thin trench spanning from anode to cathode. The sample is then subjected to electrophoresis perpendicular to the stationary pH gradient. As a result, each protein exhibits its own pH/mobility curve in the gel slab (13).
There are, however, a number of problems with the CA-IEF gels: They have uncertain chemical environments of low ionic strength and uneven buffering capacity and conductivity; cathodic drift results in extensive loss of proteins at the gel cathodic extremity upon prolonged runs; and the pH gradients possible are limited by the nature of the carrier ampholytes available. The low ionic strength within the gel often induces near-isoelectric precipitation and smears of proteins, even in analytical runs with small amounts of protein. For all these reasons, the technique of IPG was launched in 1982 (6).
2. Immobilized pH Gradients
Immobilized pH gradients (IPGs) are unique in that they are based on the insolubilization of the entire set of buffers and titrants responsible for generating and maintaining the pH gradient along the separation axis. Contrary to CA-IEF, where a multitude of soluble, amphoteric buffers is expected to create and sustain the pH gradient during the electrophoretic run, IPGs are based on only a few well-defined, nonamphoteric buffers and titrants, able to perform in a highly reproducible manner.
Advantages of the IPG technique are: (a) increased resolution (by at least one order of magnitude); (b) unlimited stability of the pH gradient; (c) flexibility in the choice of pH interval; (d) increased loading ability; (e) high reproducibility; (f) minimal distortion by salts in the sample; (g) full control of pH, buffering capacity and ionic strength; and (h) easy separation of sample from buffering ions in preparative runs.
In IPGs, the pH gradient exists prior to the IEF run and is copolymerized, and thus immobilized, within the polyacrylamide matrix. This is achieved by using, as buffers, a set of six nonamphoteric weak acids and bases, having the general chemical composition CH2CH—CO—NH—R, where R denotes either two different weak carboxyl groups, with pKa values of 3.6 and 4.6, or four tertiary amino groups, with pKa values of 6.2, 7.0, 8.5, and 9.3 (available under the trade name Immobiline from Pharmacia-LKB). A more extensive set, comprising 10 chemicals (with the addition of a pKa 3.1 acidic buffer, a pKa 10.3 alkaline species, and two strong titrants, a pK a 1 acid and a pK a >12 quaternary base), is available as "pI select" from Fluka AG, Buchs, Switzerland. During gel polymerization, these buffering species are efficiently incorporated into the gel (84% to 86% conversion efficiency at 50°C for 1 h). Immobiline-based pH gradients can be cast in the same way as conventional gradient polyacrylamide gels, by using a density gradient to stabilize the Immobiline concentration gradient, with the aid of a standard two-vessel gradient mixer (see Transverse Gradient Gel Electrophoresis (Tgge)). The buffers are not amphoteric, but are bifunctional: At one end of the molecule is located the buffering (or titrating) group, and at the other is the acrylic double bond, which will participate in the polymerization process. Acidic and alkaline Immobilines have different temperature coefficients (dpKJBT), so temperature affects Immobiline pH gradients, as do the ionic strength and additives that change the water structure (chaotropic agents, such as urea) or lower its dielectric constant. The largest changes are due to the presence of urea: Acidic Immobilines increase their pK a values in 8 M urea by as much as 0.9 pH units, and basic Immobilines increase their pKa values by only 0.45 pH unit. Detergents in the gel (up to 2% w/v) do not alter the Immobiline pKa,suggesting that acidic and basic groups attached to the gel are not incorporated into surfactant micelles. For generating extended pH gradients, one should use two additional strong titrants having pKa values well outside the desired pH range: QAE (quaternary amino ethyl)-acrylamide (pKa >12) and AMPS (2-acrylamido-2-methyl propane sulfonic acid, pKa ^1).
With the IPG technology, ultranarrow (0.1), narrow (1.0), and extended (up to 8.0) pH gradients can be engineered with high precision and reproducibility. Recipes have been tabulated listing 58 1-pH-unit-wide gradients, separated by 0.1 pH unit increments, starting with the 3.8 to 4.8 pH interval and ending with the pH 9.5 to 10.5 range (14). If a narrower pH gradient is needed, it can be derived from any of the 58 pH intervals tabulated by a simple linear interpolation of intermediate Immobiline molarities. Recipes for gradients with pH spans from 2 pH units up to 6 pH unit spans are available (15). All the formulations are normalized to give the same average value of buffering power of 3mequiv.L-1pH-1, adequate for producing highly stable pH gradients. For pH intervals covering >4pH units, the best solution is to mix a total of 10 Immobiline species, 8 of them buffering ions and two the strong acidic and basic titrants. Nonlinear (eg, concave and convex exponential, as well as sigmoidal) IPG gradients can be generated and optimized (16).
After casting, IPG gels should be washed extensively, so as to remove nonpolymerized material, salts, and polymerization catalysts. This also produces clean matrices, devoid of toxic materials (eg, unreacted acrylic double bonds) that could modify proteins by reacting with thiol groups and terminal amino groups. As IPG gels are cast onto plastic supports and are rather thin (0.5 mm) and porous, they can be stored dry and then reswollen with any desired additives just prior to use. IPG are very reproducible and effective at very alkaline pH values, up to pH 12, where CA-IEF would simply fail; histones could thus be focused to steady state (17).
