An automated continuous-flow dynamic dialysis technique for investigating protein-ligand binding

An automated, continuous-flow dynamic dialysis technique has been developed to investigate protein-ligand binding. The method depends on a comparison of the diffusion of the low molecular mass ligand, in the presence and absence of protein, through a semipermeable membrane. The ligand passes from the sample compartment of a dialysis cell into the sink compartment through which a constant flow of eluting buffer is maintained. Digitized spectrophotometric determinations of the ligand concentration in the eluting buffer at successive, equally spaced time intervals, punched onto paper tape, provide the primary data (normally about 1000 data points). A mathematical treatment of the data based on a model of the diffusion system, whereby the protein-ligand binding isotherm may be evaluated, is discussed. The validity of the method is demonstrated from studies of the binding of phenol red to bovine serum albumin (BSA) at 15, 20, and 25°C. The method yields a large number of points on the binding isotherm (usually several hundred) which, in terms of a Scatchard model, provide values for the number of binding sites on the BSA molecule and binding constants for the phenol red-BSA interaction. The results obtained are consistent with values reported in the chemical literature but which are based on much scantier data.