Immobilized pH Gradients IPG

IPGs are based on the principle that the pH gradient, which exists prior to the IEF run itself, is copolymerized, and thus immobilized within the polyacrylamide matrix. This is achieved by using as buffers a set of up to 10 non-amphoteric, weak acids and bases, called Immobilines, having the following general chemical composition: CH2=CH-CO-NH-R, where R denotes either three different weak car-boxyls, with pKs of 3.1, 3.6, and 4.6 (Table 1), or five tertiary amino groups, with pKs of 6.2, 7.0, 8.5, 9.3

Table 1 Acidic acrylamido buffers pK Formula

and 10.3 (Table 2). This set of eight weak buffers is complemented by a strong acid (pK of approximately 1, 2-acrylamido-2-methyl propanesulfonic acid) and a strong base (pK > 12, quaternary aminoethyl-acrylamide), used only as titrants. During gel polymerization, these buffering species are incorporated into the gel (84-86% conversion efficiency at 50°C for 1 h), by the same free radical reaction used to activate the acrylamide double bond. Figure 4 shows a segment of a hypothetical structure of an Immobiline matrix and the process of focusing two proteins in it. It is seen that only the proteins migrate to their steady-state position, whereas the Immobilines remain fixed at their original grafting position in the gel, where a fixed ratio of buffering/titrant ions defines the pH locally. This means that the pH gradient is stable indefinitely (but it has to pre-exist before the onset of polymerization) and can only be destroyed if and when the polyacrylamide gel is hydrolyzed. Given the sparse distribution of Immobilines in the gel they behave as isolated charges, able to effectively contribute to the ionic strength of the medium. In conventional IEF, on the contrary, at steady-state the ionic strength is exceedingly low (less than 1 mequivL-1) since the focused carrier ampholytes form an inner salt, and this often results in protein precipitation and smears both at the pi and in its proximity. In IPGs the high ionic strength existing in the matrix (typically 10 mequivL ! 1) induces protein solubilization at the pi value (thus CA-IEF is similar to a 'salting-out' milieu and IPGs to a 'salting-in' environment).

Immobiline-based pH gradients can be cast in the same way as conventional polyacrylamide gradient gels by using a density gradient to stabilize the Immobiline concentration gradient, with the aid of a standard, two-vessel gradient mixer. As shown earlier, these buffers are no longer amphoteric as in

Name Mr

2-Acrylamido-2-methylpropanesulfonic acid 207

2-Acrylamidoglycolic acid

N-Acryloylglycine

4-Acrylamidobutyric acid

Table 2 Basic acrylamido buffers pK Formula Name Mr

6.2 / \ 2-Morpholinoethylacrylamide 184

7.0 / \ 3-Morpholinopropylacrylamide 198

8.5 CH2=CH —CO —NH —(CH2)2 —N(CH3)2 /V,/V-Dimethylaminoethylacrylamide 142

9.3 CH2=CH—CO — NH — (CH2)3 — N(CH3)2 /V,/V-Dimethylaminopropylacrylamide 156 10.3 CH2=CH— CO — NH — (CH2)3 — N(CH2H5)2 /V,/V-Diethylaminopropylacrylamide 184

>12 CH2=CH-CO-NH-(CH2)2-N<CH2Hs)3 NN-N-Triethylaminoethylacrylamide 198

conventional IEF, but are bifunctional: the buffering group is located at one end of the molecule and at the other end there is the acrylic double bond which will disappear during the grafting process. The three car-boxyl immobilines have rather small temperature coefficients of ionization (dpK/dT) in the 10-25°C range due to their small standard heats of ionization (approximately 1 kcal mol"1) and thus exhibit negligible pK variations over this temperature range. On the other hand, the four basic immobilines exhibit rather large ApKs in the same temperature range (as much as ApK = 0.44 for the pK 8.5 species) due to their larger heats of ionization (6-12 kcal mol"1). Therefore, for reproducible runs and pH gradient calculations, all the experimental parameters have been fixed at 10°C. Temperature is not the only variable that will affect immobiline pKs (and therefore the actual pH gradient generated): additives in the gel that will change the water structure (chao-tropic agents such as urea) or lower its dielectric constant, and the ionic strength itself of the solution, will alter pK values.

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