Abstract

Studies of the binding of low molecular mass analogues of polyphenolic vegetable tannins to soluble hide collagen have prompted the development of a novel method for investigating protein-ligand interactions. This method, referred to as continuous-flow dynamic dialysis (CFDD), differs from conventional equilibrium dialysis procedures for measurement of protein-ligand binding in that the ligand concentration in the diffusate is monitored automatically at successive closely spaced time intervals, while being continuously eluted from a dialysis cell. Details of the method and its validation using a bovine serum albumin-phenol red binding system are reported. The method has been applied to investigate the binding to collagen of various phenolic tannin subunits which can be considered as monomeric precursor analogues of the polymeric vegetable tannins. Binding of these ligands to collagen is shown to be characterized by high capacity, low affinity binding in which ligand uptake increases linearly with concentration. No indication of site saturation was observed over the experimentally accessible ligand concentration range.