Chromatographic Studies of Solute Interactions with Immobilized Red Blood Cells and Biomembranes

Abstract: Specific and non-specific interactions of solutes with immobilized biomembranes were studied using chromatographic methods. Liposomes, proteoliposomes and red blood cell (RBC) membrane vesicles were immobilized by a freeze-thawing procedure, whereas whole RBCs were adsorbed in the gel beds using electrostatic interaction, binding to wheat germ agglutinin (WGA) or the streptavidin-biotin interaction.Superporous agarose gel with coupled WGA was the most promising matrix for RBC adsorption and allowed frontal chromatographic analyses of the cells for about one week. Dissociation constants for the binding of cytochalasin B and glucose to the glucose transporter GLUT1 were determined under equilibrium conditions. The number of cytochalasin B-binding sites per GLUT1 monomer was calculated and compared to corresponding results measured on free and immobilized membrane vesicles and GLUT1 proteoliposomes. This allowed conclusions about the protein´s binding state in vitro and in vivo.Partitioning of drugs into biomembranes was quantified and the system was suggested as a screening method to test for possible intestinal absorption of drug candidates. We also studied how membrane partitioning of drugs is affected by the presence of integral membrane proteins or of charged phospholipids.An attempt to combine the theory for specific binding and membrane partitioning of solutes in a single equation is briefly presented.