Discontinuous Electrophoresis

In 1959 Raymond and Weintraub described the use of polyacrylamide gels (PAG) in ZE, which offered UV and visible transparency (starch gels are opalescent) and the ability to sieve macromolecules over a wide range of sizes. Figure 2 gives a scheme of reaction for producing polyacrylamide gels from the standard mixture of monomers, acrylamide and the cross-linker Bis. It should be noted that although this matrix should be neutral (except where accidental hydrolysis of acrylamide to acrylic acid occurs), in reality it is not completely devoid of charges; at the

Figure 2 The polymerization reaction of acrylamide. The chemical formula of acrylamide, A/,W-methylenebisacrylamide (Bis) and of the initiators (peroxysulfate and A/,A/,W,W-tetramethylethylendiamine, TEMED) are shown. On the right-hand side, growing polyacrylamide chains, in equilibrium with free monomers, are illustrated. In this particular case, it is assumed that the chain termini are TEMED molecules, although peroxysulfate could be just as well incorporated.

Figure 2 The polymerization reaction of acrylamide. The chemical formula of acrylamide, A/,W-methylenebisacrylamide (Bis) and of the initiators (peroxysulfate and A/,A/,W,W-tetramethylethylendiamine, TEMED) are shown. On the right-hand side, growing polyacrylamide chains, in equilibrium with free monomers, are illustrated. In this particular case, it is assumed that the chain termini are TEMED molecules, although peroxysulfate could be just as well incorporated.

chain termini either initiator, N,N,N',N'-tetra-methylethylenediamine (TEMED), or sulfate, could be incorporated which would impart positive or negative charges, respectively. The fact that polyac-rylamides always exhibit a residual electroosmotic flow towards the cathode suggest that an excess of negative charges is incorporated over positive ones (TEMED).

In 1964, Ornstein and Davis created discontinuous (disc) electrophoresis by applying to PAG a series of discontinuities (of leading and terminating ions, pH, conductivity, and porosity), thus further increasing the resolving power of the technique. In discontinuous disc electrophoresis (the principle of which is outlined in Figure 3), the proteins are separated on the basis of two parameters: surface charge and molecular mass. The matrix is divided into three sections (from bottom up): a 'separation', or 'running' gel, a 'spacer' or 'stacking' gel, and a sample gel. A sharp discontinuity exists at the running/stacking interface: the bottom gel is a tightly knit sieve (with small pores), while the second and third layers are minimally sieving, open-pore structures. At the same interface, a second discontinuity exists in pH. In fact, the running gel is titrated at pH 8.9, whereas spacer and sample gels are buffered at pH 6.7. This gel region at pH 6.7 is also a low conductivity region (third discontinuity), which means that a voltage gradient will be generated in this zone when an electric current is passed through it. Below and above it (in the cathodic chamber) high-conductivity regions are found. A fourth discontinuity exists at the interface between the upper gel end and the liquid in the cathodic compartment: below it only Cl" (leading, L) ions are present, while above it only glycinate (trailing or terminating, T) ions are found.

Why is there the need for such a complicated system? This intricate set up must satisfy the Kohlrausch regulating function, which is at the heart of ITP (in fact, movement of ions in the first two gel segments will be according to ITP rules). If all the ions in the system are arranged in such a way that ¿uL > ¿uP > ¿uT (where ¿u is the mobility of leading, protein and terminating ions, respectively), then, upon playing a voltage gradient, they will migrate down the gel cylinder with equal velocities and the boundary between each adjacent species will be maintained. As soon as the electric circuit is closed, Cl~ (fastest moving) ions are swept down the column towards the anode. Just behind this boundary, all protein ions will start arranging themselves in order of their mobilities, with the lowest pi component next to the Cl~ boundary and

Figure 3 Principle of discontinuous disc electrophoresis. (A) Sample in sample gel; (B) sample concentration in stacking gel; (C) sample separation in running gel. From top to bottom, the following phases are encountered: glycine buffer at pH 8.3 in the cathodic reservoir; sample gel and spacer gel, both titrated to pH 6.7; small-pore running gel, titrated to pH 8.9; and glycinate buffer again in the anodic reservoir at the bottom. In part (C) it is seen that, as the glycinate boundary sweeps down the gel past the protein zones, the pH increases from 8.9 (in A and B) up to 9.5. (Reproduced with permission from Ornstein, 1964.)

Figure 3 Principle of discontinuous disc electrophoresis. (A) Sample in sample gel; (B) sample concentration in stacking gel; (C) sample separation in running gel. From top to bottom, the following phases are encountered: glycine buffer at pH 8.3 in the cathodic reservoir; sample gel and spacer gel, both titrated to pH 6.7; small-pore running gel, titrated to pH 8.9; and glycinate buffer again in the anodic reservoir at the bottom. In part (C) it is seen that, as the glycinate boundary sweeps down the gel past the protein zones, the pH increases from 8.9 (in A and B) up to 9.5. (Reproduced with permission from Ornstein, 1964.)

the highest pi species closing the procession. The last 'wagon of the train' is the glycinate (terminating) ion; and explains why the sample and spacer gels are titrated at pH 6.7. Gly has a theoretical pi of 6.1 but, as shown by its titration curve, it is almost isoelectric, even at pH 6.7; its anionic mobility, therefore, is extremely small, in any event smaller than the slowest protein ion.

Thus, in the sample and spacer gels, two basic phenomena occur: (1) all protein ions are sorted out and physically separated according to their pis, and (2) each protein ion is strongly concentrated in extremely thin starting zones (the disc barely a few micrometres thick and a concentration process of up to 1000- to 10 000-fold). This isotachophoretic 'train', however, does not have a long life; as it enters the running gel, the train 'runs off the tracks'. Only the 'locomotive' (Cl~) of the train is unaffected; the various protein wagons now overrun each other, since they experience a strong frictional force, due to the highly sieving matrix, so that now their velocity is a function of their charge/size ratio. In addition, as the almost isoelectric Gly enters the pH 8.9 zone, its negative charge density strongly increases so that it jumps ahead and closely follows the Cl" ion. As Gly sweeps down the running gel, the pH increases from pH 8.9 to 9.5 (approaching the pK value of the Gly amino group) so that now the net charge on Gly is — 0.5. As a consequence of this further jump in pH, all proteins experience an additional mobility increment. One might wonder why, after taking on such an experimental burden in forming the ITP train, one should then destroy it and continue the run in the plain zone electrophoretic mode. There are reasons for this. First, the ITP train, while maintaining high resolution due to lack of degradation of zone boundaries, has the main defect that the zones are contiguous and continuous, i.e. they are not separated by blank zones of plain buffer. As a result, when staining the gel, one would only see a single, continuous zone of protein ions, with no visible separation between zones. Second, whereas the sharp protein discs formed during the stacking (ITP) process are separated solely by surface charge, during migration in the running gel, separations continue on the basis of an additional parameter i.e. the mass. The small loss of resolution due to diffusion of the protein discs in the running gel is more than compensated for by the resolution increments due to size (coupled to charge) fractionation in this gel zone. Although disc electrophoresis is no longer in vogue, it was an extremely useful analysis technique for at least 20 years after its inception. Moreover, the general principle has not been abandoned and it is used today as a stacking technique in both SDS and capillary elec-trophoresis.

Disc electrophoresis could also be used for deriving physico-chemical parameters of the proteins under analysis. In 1964, Ferguson showed that one can derive parameters which are proportional to both the surface charge and the mass of the macromolecule. This can be accomplished by plotting the results of a series of experiments with polyacrylamide gels of varying porosity. For each protein under analysis, the slope of the curve log mT (electrophoretic mobility) vs gel density (%T) is proportional to molecular mass, while the y-intercept (Y0) is a measure of surface charge. Examples of these plots are shown in Figure 4. In Figure 4A the two parallel lines indicate charge isomers; in Figure 4B, the fanning out lines indicate a family of constant charge and different mass; in Figure 4C, the two crossing lines indicate proteins differing in both charge and mass. Recently, non-linear Ferguson plots have been reported (Chrambach, 1988), related to the reptation mode of DNA in sieving media.

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