Affinity partitioning of phosphofructokinase from baker's yeast using polymer-bound Cibacron blue F3G-A.

1. Phosphofructokinase from baker's yeast is partitioned between the phases of an aqueous two-phase system, containing dextran (Mr = 500000) and poly(ethyleneglycol) (Mr = 6000), in favour of the dextran-rich phase. By covalent binding of the dye Cibacron blue F3G-A to poly(ethyleneglycol) the enzyme can be extracted to the phase rich in this polymer, i.e. affinity partitioning. 2. The affinity partitioning effect, measured as the logarithmic increase of the partition coefficient by introducing polymer-bound Cibacron blue, depends on several factors. The influence of dye-polymer concentration, polymer concentration, polymer molecular weight, kind of salt and salt concentration, pH and temperature has been studied. 3. The effect of ATP, ADP, AMP, ITP, fructose 1,6-bis-phosphate and fructose 6-phosphate show large differences in the binding strength of these substances to the Cibacron blue binding sites. AMP cannot compete with Cibacron blue while ATP is strongly competing. 4. The use of affinity partitioning for enzyme isolation and determination of ligand binding is discussed, as well as possible mechanisms concerning this type of liquid/liquid extraction.