Focusing of pepsin in strongly acidic immunobilized pH gradients

Abstract

A new acrylamido buffer has been synthesized, for use in isoelectric focusing in immobilized pH gradients. This compound (2-acrylamido glycolic acid) has a p K = 3.1 (at 25°C, 20 mM concentration during titration) and is use, by titration with the p K 9.3 Immobiline, to produce a linear pH gradient in the pH 2.5–3.5 interval. Pepsin (from pig stomach) focused in this acidic pH gradient is resolved into four components, two major (with p I values 2.76 and 2.78) and two minor (having p I values 2.89 and 2.90). This is the first time that such strongly acidic proteins could be focused in an immobilized pH gradient. AEven in conventional isoelectric focusing in amphoretic buffers it has been impossible to focus reproducibly very-low-p I macromolecules.