Interaction of Cibacron dyes with dehydrogenases and kinases.

In 1969 it was dem onstrated1 that pyruvate kinase binds to Blue Dextran 2000. In a study of the mechanism of this absorption it could be shown that the binding of the enzyme is due to an inter­ action of the enzyme with the blue dye of Blue Dextran 2000 and not with the dextran itself. This property was used for the purification of pyruvate kinase of yeast1 and later on also applied to the ex­ traction of human erythrocyte pyruvate k inase2, phosphofructokinase 3,1 4 and various other enzymes 5. In order to simplify the purification procedure the properties of the dye were examined 5. It was found that the blue dye (Cibacronblue 3 G-A) could be covalently fixed to an insoluble carrier and used suc­ cessfully for affinity chromatography. Later, on the basis of enzymatic and spectrophotometric analysis it was suggested that the blue chromophore specifi­ cally binds to protein ligand sites of NAD-dom a i n s 7. In order to define more specifically the structural requirements for the binding of the dye, we have analysed the binding properties of a number of dyes of the Cibacron-group to a variety of cellular enzymes, and here we report our findings.