Pulsed ultrafiltration characterization of binding.
We describe a new method for measuring binding constants, pulsed ultrafiltration. In this technique, a single injection or "pulse" of ligand is passed through a cell containing macromolecules confined by a conventional ultrafiltration membrane. Any binding of the ligand to the macromolecule alters the elution profile of the ligand. We describe this interaction by a set of coupled differential equations whose solution allows us to extract the binding density as a function of free ligand concentration eluting from the cell. A method of comparing elution profile areas which leads to values for both binding affinity and stoichiometry is also presented. We show that the pulsed ultrafiltration method can generate an extensive binding isotherm with a dense set of data 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 stoichiometry, the accuracy and precision of the method, and temperature dependence of binding. In general, our results agree with those from the literature, and they show that the approach is a fast and flexible method for characterizing ligand-macromolecule binding.
Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois, 60612, USA.
C J
CJ
Chen
S
S
Chen
C P
CP
Woodbury
D L
DL
Venton
D002851:Chromatography, High Pressure Liquid; D008956:Models, Chemical; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet; D014462:Ultrafiltration / Q000379:methods*