| If a continuous ultrafiltration process could be operated near equilibrium, the change of the total ligand concentration in a solution containing a fixed quantity of macromolecule has the potential to generate a series of bound versus free ligand data points needed for measuring binding parameters. It is this concept, i.e. a dynamic measurement taken at slow enough ultrafiltration rates to accurately approximate an equilibrium process with two sub-processes, binding and elution, which lies at the center of the pulsed ultrafiltration method discussed in this thesis.;Such a system has been used to determine the equilibrium and kinetic binding constants associated with small ligand/macromolecule interactions. Accuracy of this method was assessed by comparing the apparent equilibrium dissociation constants for L-Trp/BSA, L-Trp/HSA, warfarin/HSA and N-Acetyl-L-Trp/BSA with those determined by other methods.;An experimental approach, called pulsed ultrafiltration, has been developed to test this concept by introducing a pulse of free ligand into a rapidly stirred solution of macromolecule retained in a continuous ultrafiltration cell, to the extent that the ligand and the macromolecule interact, the appearance of the ligand in the effluent is initially retarded relative to the situation without macromolecule. Given sufficient time, however, all of the ligand elutes from the cell. This ligand retardation is related to the binding interaction, and a series of bound versus free ligand data can be obtained by graphically analyzing the elution profiles. |
