Immobilized pH gradients: New pK values of acrylamido buffers in poly(N‐acryloylaminoethoxyethanol) matrices

Abstract

A novel matrix consisting of N-acryloylaminoethoxyethanol, a hydrophilic monomer extremely resistant to hydrolysis, was recently reported by Chiari et al. (Electrophoresis, 1994, 15, 177-186). When using it as a matrix for grafting immobilized pH gradients for isoelectric focusing, a shift in protein spot position was noticed. This was attributed to a shift in pK values of the Immobiline buffers when changing from the standard poly(acrylamide) to a poly(N-acryloylaminoethoxyethanol) matrix. A series of 1 pH unit gradients was constructed, where a single buffering Immobiline was used and titrated with a counterion having a pK removed by at least 3 pH units from the nearest extreme of the generated pH interval. It was noted that all compounds became weaker acids and bases, with a delta pK ranging from -0.02 to -0.06 for the acids (pK 3.6, 4.4, and 4.6) and a delta pK ranging from -0.12 tp -0.20 for the bases. The new pK values for the seven commercially available buffers are thus pK 3.6-->pK 3.58, pK 4.4-->pK 4.36, pK 4.51-->4.45, pK 6.21-->6.09, pK 7.06-->6.94, pK 8.50-->pK 8.37, and pK 9.59-->pK 9.39. These values refer to 10 degrees C in the gel phase, the first value in poly(acrylamide) and the second in poly(N-acryloylaminoethoxyethanol).

Focusing of alkaline proteases (subtilisins) in pH 10–12 immobilized gradients

“…Analogous results were obtained by using a poly(AAEE) matrix (Fig. 2B), although the train of bands was focused more closely to the cathode, due to a matrix effect which weakens all Immobiline buffers [12].…”

Combined cup loading, bis(2‐hydroxyethyl) disulfide, and protein precipitation protocols to improve the alkaline proteome of Lactobacillus hilgardii

“…A prefractionation approach, employing either 2-D LC [21,22] or miniaturized liquid IEF [23][24][25] prior to 2-DE, has been applied to enrich the selected proteins. Other attempts to enhance the resolution at alkaline pI during IEF include: (i) addition of isopropanol [19] or sorbitol [26] to the gel matrix to counteract ionized water migration toward the anode; (ii) replacement of DTT with nonionic reducing agents such as tributyl phosphine [27]; (iii) the use of N-substituted acrylamide derivatives (such as N,N-dimethylacrylamide or piperazine diacrylamide) that sterically prevent amido-group hydrolysis [19]. Good improvements in the basic IEF, for example, have led to the setting up of the alkaline proteomes of three sequenced organisms, Caulobacter crescentus [2], H. pylori [17], and L. lactis [3].…”

Production of D-phenylglycine from racemic (D,L)-phenylglycine via isoelectrically-trapped penicillin G acylase

“…The pro-cessing rate will then be proportional to the available membrane area; all other experimental conditions being kept constant (voltage gradient, substrate concentration in the feed, etc.). The cost of the membranes would not be prohibitive too, because we have described novel membranes, made of extremely resistant and highly hydrophilic Nsubstituted acrylamide monomers, capable of operating in an electric field for long periods of time , with minimal variations of the Immobiline pK values (Bossi et al, 1994). Additionally, our isoelectric membranes are known not to be fouled by adsorption of proteins and enzymes, thus they can be reused for several MIER operations (Righetti et al, 1992).…”