PULSED ULTRAFILTRATION CHARACTERIZATION OF BINDING - I - A NEW METHOD

We describe a new method for measuring binding constants, pulsed ultra filtration. in this technique, a single injection or ''pulse'' of liga nd is passed through a cell containing macromolecules confined by a co nventional ultrafiltration membrane. Any binding of the ligand to the macromolecule alters the elution profile of the ligand. We describe th is interaction by a set of coupled differential equations whose soluti on allows us to extract the binding density as a function of free liga nd concentration eluting from the cell. A method of comparing elution profile areas which leads to values for both binding affinity and stoi chiometry is also presented. me show that the pulsed ultrafiltration m ethod can generate an extensive binding isotherm with a dense set of d ata points over a wide range of binding densities. We apply the method to several model Ligand-macromolecule binding systems to demonstrate the measurement of equilibrium association constants and binding stoic hiometry, the accuracy and precision of the method, and temperature de pendence of binding. In general, our results agree with those from the literature, and they show that the approach is a fast and flexible me thod for characterizing Ligand-macromolecule binding. (C) 1998 Academi c Press.