Affinity partitioning of enzymes using dextran-bound procion yellow HE-3G. Influence of dye-ligand density

The dye Procion Yellow HE-3G was bound to dextran of molecular weight 70,000 and the partitioning of this dye-polymer within an aqueous two-phase system containing dextran and poly(ethylene glycol) was studied as function of ligand density, polymer concentration, type of salt, concentration of salt and concentration of dye-dextran. Even moderate dye:dextran molar ratios (5-8) make the partitioning strongly salt-dependent. The dye-dextran can be directed to either the upper or the lower phase with partition coefficients from 0.02 to 28 by using salts. The dye-dextran in the two-phase system affects the partitioning of dye-binding enzymes (lactate dehydrogenase, glucose-6-phosphate dehydrogenase, 3-phosphoglycerate kinase) towards the dye-containing phase. Measurements of competition with nucleotide binding show an increased affinity of the dye for the enzyme with increasing ligand:dextran ratio. Theoretical considerations indicate that 1-2 dextran molecules are attached per enzyme molecule when affinity partitioning is fully developed.