pH-dependent binding analysis, a new and rapid method for isoelectric point estimation

Abstract

Based upon the pH-dependent binding affinity of amphoteric molecules for an ion exchanger, and by taking advantage of batch procedures, a facile method was developed for estimating isoelectric points of these molecules. The new method allows pI measurements to be accomplished within 1 h. Moreover, any possible protein-ampholyte interaction or artifact formation, as may be introduced from the presence of carrier ampholytes when conventional focusing methods are employed, is eliminated by the method. In addition, because of the short processing time, isoelectric points of proteins can be measured at any desired temperature without much risk of protein denaturation. Seven proteins with well-defined isoelectric points were examined by the method. The measured pI values were within a range of 0.2 pH unit or less of the reported values. The precision of pI measurements by the method can be even further improved with the employment of a narrower pH gradient. Since the isoelectric point is an important parameter which governs much of the art of separating proteins, the advent of a simple and rapid method for its measurement would be of use for selecting the proper strategy for protein isolation and purification.