41] Separation of peptides by countercurrent distribution

Abstract

Countercurrent distribution offers several advantages over other separation methods for the fractionation of peptide mixtures. This chapter describes the method of separation of peptides by countercurrent distribution. The countercurrent distribution method is useful as a preliminary step in separating the complex mixture of peptides usually obtained from chymotryptic and peptic digests of proteins. Countercurrent distribution also serves as a criterion of purity, since the presence of contaminants, even though they do not appear as discrete bands, can be detected as deviations of the experimental distribution pattern from the theoretical curve. A large number of solvent systems are used for the countercurrent distribution of peptides. Most of these include 1-butanol or 2- butanol as the organic component equilibrated with aqueous solutions of acetic acid, di- or trichloroacetic acid, and pyridine. The methods of dissolving the sample and carrying out the distribution are also discussed. The concentration of solute in each tube of the distribution train or each effluent fraction after a given number of transfers is calculated. The calculation of recovery and effluent series is estimated. The arbitrary cuts from the countercurrent distribution are subjected to ion-exchange chromatography to isolate homogeneous peptides. The methods of analysis used for the countercurrent distribution of proteins can also be used for determining the location of peptides that are subjected to countercurrent distribution.