INTERACTION OF CIBACRON-BLUE WITH POLYMERS - IMPLICATIONS FOR POLYMER-SHIELDED DYE-AFFINITY CHROMATOGRAPHY OF PHOSPHOFRUCTOKINASE FROM BAKERS-YEAST

Interactions between Cibacron Blue F3GA and water-soluble non-ionic polymers were investigated by monitoring the spectral shift that accompanies the binding phenomena. Polyvinylpyrrolidone (PVP) and poly(vinyl alcohol) were the only polymers among those tested found to interact effectively with the dye. The difference spectra for the PVP-dye complex was typical of ''electrostatic interaction spectra'' at low ionic strength and typical of ''hydrophobic interaction spectra'' in the presence of 1.5 M KCl. The binding constant and the number of binding sites per polymer molecule were calculated using the simplest model of independent binding sites. One dye molecule was bound by a PVP segment with a molecular mass of 1000-1300. Regardless of the size of the polymer molecules, the binding constants were in the micromolar range. Poly(vinyl alcohol) bound less efficiently to Cibacron Blue than PVP. One dye molecule was bound by a polymer segment with a molecular mass of about 10 000. The data on PVP complexing with Cibacron Blue were used to develop the concept of polymer-shielded dye-affinity chromatography. This concept was successfully applied to the chromatography of phosphofructokinase (EC 2.7.1.11) from baker's yeast. Specific elution of the bound enzyme from PVP-shielded column resulted in an efficient process with 27-fold purification.