Binding isotherms by continuous-flow dynamic dialysis

Abstract

The classical dynamic dialysis technique for the determination of a protein—ligand binding isotherm has been modified by the introduction of a flow cell in which the dialysate on the sink side of the membrane is continuously eluted with a constant flow of eluting buffer and its ligand concentration measured. This new experimental method is termed continuous-flow dynamic dialysis (CFDD). A transfer function procedure for extracting the binding isotherm from the dialysis data is described. This is a more general technique (requiring only a verifiable assumption of linearity) than that previously used, in which the system was modelled using Fick's first law and which relied on the establishment of quasi-steady state conditions across the membrane. The present analysis uses the Laplace transform to effect deconvolution of the impulse response function of the cell from the dialysis data and, using a Fourier series approach, directly yields numerical data representing the free ligand concentration in equilibrium with the protein—ligand complex. The protein—ligand binding isotherm is obtained in parametric form, with time as the parameter.