Protocols for use of ultrafiltration in determination of free ligand concentration and of complexity of ligand/protein interactions.

Analysis of a simple ligand/binding-protein association model predicts a decrease in the percentage of free hormone during ultrafiltration. Re-analysis of the original data validating ultrafiltration in free-ligand determinations (Sophianopoulos et al., Arch Biochem Biophys 1978;187:132-7) confirms this prediction. Binding data indicating a constant percentage of free hormone (Hammond et al., J Biol Chem 1980;255;5023-6) require a more complex ligand-protein interaction model. Application of these procedures to any new ligand system requires measurement of the percentage free, or of free-ligand concentration, as a function of filtration time or volume. The protocol becomes important if values for percentage free are to be accurate.