Determination of reserve albumin-equivalent for ligand binding, probing two distinct binding functions of the protein

A method is reported for determination of albumin binding capacity for various ligands in 50-μl sample volumes. A small amount of a radioactively labeled test ligand is added to the undiluted sample and the rate of dialysis of the free ligand into an identical sample without added ligand is measured. The reserve albumin-equivalent concentration is defined as the concentration of a standard albumin preparation which in buffered solution gives the same rate of dialysis and hence the same ratio of free/bound concentrations of the added ligand. It is shown that the reserve albumin-equivalent concentration, thus defined, is identical with the sum of concentrations of carrier species, each multiplied by the first stoichiometric binding constant of the test ligand to the carrier and divided by its first stoichiometric binding constant to the standard albumin. Determinations of this parameter are suitable for studies of the chemical potential and transfer affinities of individual ligands and for determination of interaction among several binding substances. Two test ligands have been used, monoacetyldiaminodiphenyl sulfone and diazepam. The former is bound competitively with bilirubin while diazepam engages another, independent binding function. The method can thus be used for separate determinations of the degree of saturation of two distinct binding functions of albumin. Complex mixtures of several carrier proteins with interacting ligands can be studied.