Investigation of binding site density: effects on the interaction between cibacron blue-dextran conjugates and lysozyme.

Cibacron-blue-dextran conjugates have been produced with a range of ligand loadings using a dextran preparation of average molecular weight of 2 x 10(6). The equilibrium binding capacity of these ligand conjugates for lysozyme was determined using a gel permeation procedure to separate bound from free protein. The results obtained give clear evidence for at least two types of binding showing a marked difference in affinity. For the higher-affinity interaction the half-saturation constant decreases with increasing ligand loading. The number of dye molecules participating in binding is proportional to loading up to 154 mol dye/mol dextran but is reduced at the highest loading used (315 mol dye/mol dextran). This may be due to steric interference or to dye stacking reducing the number of dye molecules available for binding.