An affinity-ligand gradient technique for purification of enzymes by counter-current distribution

Abstract

Enzymes related to the glycolysis, present in an extract of baker's yeast, have been fractionated by counter-current distribution using an aqueous dextran—polyethyleneglycol two-phase system containing affinity ligands. To minimize the peak width a gradient of a general affinity ligand (triazine dye) was included in the stationary (liquid) phase. A centrifugal counter-current distribution machine was used to speed up the settling of the aqueous phases after equilibration. With 55 transfer cycles several of the measured enzymes increased 4–15 times in purity. Furthermore, a number of the enzymes showed clear heterogeneity indicating two or more isoenzymic forms. The use of this multistep liquid—liquid extraction in large scale for specific isolation of intracellular enzymes is discussed.