LIGAND-BINDING TO A HUMAN SERUM-ALBUMIN STATIONARY-PHASE - USE OF SAME-DRUG COMPETITION TO DISCRIMINATE PHARMACOLOGICALLY RELEVANT INTERACTIONS

A technique based on a human serum albumin (HSA) stationary phase high -pressure liquid chromatography (HPLC) has been successfully used for the past few years to characterize the interactions between HSA and ne w substrates. Immobilized HSA conserves the binding properties of the protein in solution, allowing fast and reliable analyses of binding in teractions. Nevertheless, clear evidence that all binding mechanisms o f HSA-HPLC are pharmacologically relevant is so far lacking. In partic ular, nonstoichiometric interactions of injected ligands with stationa ry phase components such as silica and the amino acid medium (other th an protein binding areas) might interfere with the correlation between chromatographic retention and HSA binding. Here we present a quantita tive method to distinguish between the molecular interactions of a lig and with binding areas of potential pharmacological interest and other , non-saturable binding mechanisms. Such a method, based on HPLC same ligand displacement, is simple and reliable, as confirmed by in situ p rotein denaturation. Consequently, we were able to distinguish between different types of competitions detected in the co-binding of two dru gs to HSA. (C) 1998 John Wiley & Sons, Ltd.