New approaches in biomembrane chromatomatography and microchromatographic techniques

Abstract: Development of methods for rendering chromatography more applicable to studies in lifesciences remains an area of active research. In the present work, new methods inbiomembrane chromatography and microchromatographic techniques have been developedand applied successfully to quantitative analysis of biological interactions and separations ofproteins on a micro-scale.Biomembranes, specifically: human red cells/ghosts, membrane vesicles and proteoliposomes, were immobilized in gel beds for chromatographic analyses of solutes specificallybound to the glucose transporter Glut1 and the nucleoside transporter of the red cellmembrane. The resultant binding parameters revealed that this system can be a practical toolfor quantitative analysis of biological interactions involving integral membrane proteins andfor monitoring procedures for purification and reconstitution of membrane proteins. Analternative method based on Hummel and Dreyer analysis was introduced and proved usefulin analyses of cytochalasin-B binding to Glut1 in membrane vesicles and proteoliposomes.Chromatographic analysis of non-specific partitioning of a drug into lipid bilayers wasperformed on a microcolumn containing liposomes immobilized in the gel bed. The resultsshowed that this method can be utilized for predicting absorption of a drug in vivo.Hydrophobic interaction microchromatography of proteins and a blotting membranetechnique for detection of minute amounts of proteins have been developed on the basis ofcontinuous beds which can be synthesized directly in the microcolumn tubes. Thesedevelopments extend the uses of continuous beds to a broader field in life science.