Binding of dye to albumin, as studied by ultrafiltration

We describe a procedure for measuring the binding of small molecules to proteins by means of a dynamic ultrafiltration system. A continuous binding curve is obtained by continuously monitoring the absorbance of ultrafiltrate as a function of ultrafiltrate volume, and corrections are made for the retention of the small molecule by the membrane and the leakage of the protein through the membrane. The method is applied to the binding of bromcresol green to human albumin. Some major sources of uncertainties in the method are discussed.