Counter-current distribution of yeast enzymes with polymer-bound triazine dye affinity ligands

Abstract

A protein extract from bakers' yeast has been resolved into several of its enzyme components by counter-current distribution using aqueous polymeric two-phase systems comprising water, dextran and polyethylene glycol. The distribution of dehydrogenases and kinases has been effectively changed by binding triazine dyes (Cibacron Blue F3G-A, Procion Yellow HE-3G and Procion Olive MX-3G) to the polyethylene glycol, confined to the upper phase. The effect of the dye ligands on the enzymes decreased roughly in the order: phosphofructokinase, (EC 2.7.1.11), glucose 6-phosphate dehydrogenase, (EC 1.1.1.49), glyceraldehydephosphate dehydrogenase, (EC 1.2.1.12), alcohol dehydrogenase (EC 1.1.1.1), 3-phosphoglycerate kinase (EC 2.7.2.3), phosphoglycerate mutase (EC 2.7.5.3.), hexokinase (EC 2.7.1.1) and enolase (EC 4.2.1.11). However, the distribution of specific enzymes was strongly dependent also on the dye concentration. By appropriate selection of conditions, purifications of up to 10-fold could be achieved for phosphofructokinase and 100-fold for one of the isoenzymes of hexokinase. Furthermore, the two isoenzymes of hexokinase could be almost completely separated from each other. The counter-current distributions were performed in a newly developed apparatus where the separation of phases was enhanced by centrifugation. This allowed 55 transfers to be completed within about 8 h.