Ligand binding to macromolecules or micelles: Use of centrifugal ultrafiltration to measure low-affinity binding

We describe a method for estimating ligand binding to a macromolecular samp le under conditions where this binding is of low affinity and must be measu red under equilibrium conditions, without removal of the unbound Ligand. Th e method is based on centrifugal ultrafiltration through a membrane with a molecular mass cut-off intermediate between that of the ligand and that of the target, and the amount of bound ligand is calculated from the differenc e between the (total) ligand in the concentrated sample and the (free) liga nd in the ultrafiltrate. Centrifugal ultrafiltration makes it possible to s eparate free ligand from bound ligand (without changing its concentration) and to simultaneously concentrate the target (such that the proportion of b ound ligand becomes significant, even under low-affinity binding conditions ). We applied this technique, using Centricon 10 (Amicon) devices, to sever al cases (soluble proteins, intact membranes, detergent-solubilized protein s, and pure detergent micelles) and assessed its value with respect to the common artifacts that occur in other protocols involving protein retention on nitrocellulose biters (nonspecific ligand adsorption and protein denatur ation). (C) 1998 Academic Press.