Dynamic Dialysis as a Method for Studying Protein Binding I: Factors Affecting the Kinetics of Dialysis through a Cellophane Membrane

Abstract

A potentially useful method for determining the extent to which binding occurs in a protein-small molecule system consists of studying the kinetics of dialysis of the small molecule in the absence and presence of protein. The kinetics of dialysis of a number of compounds, in the absence of protein, was studied in some detail in order to characterize the nature of the dialytic process and its dependency on experimental variables. It was shown that the rate of escape of the small molecule from a dialysis cell was a first-order process provided that sink conditions were maintained. The influence of such variables as cell size, solution volume, stirring rates, temperature, pH, ionic strength, and viscosity on the rate process was considered and investigated. It was found that stirring rate (above a minimum), ionic strength, and viscosity did not markedly affect the kinetic picture. Temperature, pH (for one compound), the size of the cell, and the volume of solution contained by the cell did affect the rate of escape. Binding by the cellophane membrane was not encountered as a problem with the compounds studied. Experimental variables could be readily controlled to yield reproducible and dependable results.