Determination of the affinity constants of concanavalin A for monosaccharides by fluorescence affinity probe capillary electrophoresis.
Dissociation constants between a legume lectin, concanavalin A (Con A), and unlabeled neutral monosaccharides were determined by affinity probe capillary electrophoresis (APCE). An affinity probe was synthesized by coupling Lissamine rhodamine B and p-amino-phenyl-alpha-D-mannopyranoside, with glutathione as a negatively charged linker. Electrophoresis of the affinity probe was carried out at neutral pH in a fused silica capillary coated with succinylpolylysine on its inner surface, using laser-induced fluorescence detection. In the presence of Con A, the mobility of the affinity probe decreased and this decrement was analyzed as a function of the concentration of Con A. In the presence of a mixture of Con A and an unlabeled monosaccharide, APCE can thus measure the concentration of Con A unoccupied by the unlabeled monosaccharide, and this allows the determination of the dissociation constant of the Con A-monosaccharide complex. The method should be useful for the analysis of a wide range of molecular interactions on a microscale.
Shimura
K
K
Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa, Japan.
Kasai
K
K
D003208:Concanavalin A / Q000378:metabolism*; D004586:Electrophoresis / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D005978:Glutathione / Q000031:analogs & derivatives / Q000138:chemical synthesis; D006601:Hexoses / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008766:Methylmannosides / Q000378:metabolism*; D008956:Models, Chemical; D011485:Protein Binding; D012235:Rhodamines / Q000138:chemical synthesis; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D006601:Hexoses; D008024:Ligands; D008766:Methylmannosides; C095538:N-(6-(diethylamino)-9-(4-((2,5-diaza-1,9-dicarboxy-4-(((((mannopyranos-1-yloxy)phenylamino)carbonyl)methyl)thiomethyl)-3,6-dioxononyl)aminosulfonyl)-2-sulfophenyl)-3H-xanthene-3-ylidene)-N-ethylethanaminium; D012235:Rhodamines; D003208:Concanavalin A; C008466:methylmannoside; D005978:Glutathione