Advantages of quantitative affinity chromatography for the analysis of solute interaction with membrane proteins.
The use of membrane proteins as chromatographic stationary phases for the quantitation of biospecific interaction between the proteins and solutes is reviewed. This method is one among the few where a membrane protein is immobilized for repeated analyses of solute binding. To our knowledge, five transmembrane proteins have been immobilized in chromatographic matrices: the glucose and nucleoside transporters from human red blood cells, the photosynthetic reaction center from Rhodobacter sphaeroides, the nicotinic acetylcholine receptor from rat brain and a recombinant P-glycoprotein. Proteoliposomes and membrane vesicles have thereby been entrapped in size-exclusion beads, such as Superdex 200, and membrane proteins have been adsorbed on 'immobilized artificial membrane' monolayers of lipid analogs grafted to silica beads. Encouragingly, immobilized glucose transporter and P-glycoprotein showed constant interactant affinities for months. Analysis is done in the frontal mode at equilibrium because there is no separation between bound and free ligand. Both the affinity constant, which generally coincides with the corresponding constant determined by use of nonchromatographic methods, and the amount of active binding sites are obtained. The method has been successfully applied to functional analysis of membrane proteins in cells or reconstituted in lipid mono- or bilayers, screening of low-molecular interactants, investigation of protein-protein interaction and studies of effects of physico-chemical parameters on solute-protein interaction. The analyses require sensitive detection of the analyte and matching between amount of binding sites and affinity.
Lundqvist
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Molecular Biology, AstraZeneca R&D Mölndal, SE-431 83 Mölndal, Sweden.
Lundahl
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D000818:Animals; D001921:Brain / Q000502:physiology; D002352:Carrier Proteins / Q000032:analysis / Q000097:blood; D002846:Chromatography, Affinity / Q000379:methods*; D004912:Erythrocytes / Q000737:chemistry; D051272:Glucose Transporter Type 1; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis* / Q000097:blood; D009004:Monosaccharide Transport Proteins / Q000097:blood; D045322:Photosynthetic Reaction Center Complex Proteins / Q000032:analysis; D051381:Rats; D011978:Receptors, Nicotinic / Q000032:analysis; D011994:Recombinant Proteins / Q000032:analysis; D012996:Solutions
D002352:Carrier Proteins; D051272:Glucose Transporter Type 1; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D045322:Photosynthetic Reaction Center Complex Proteins; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins; C495677:SLC2A1 protein, human; D012996:Solutions