title: Binding of simple carbohydrates and some of their chromophoric derivatives to soybean agglutinin as followed by titrimetric procedures and stopped flow kinetics.
abstract: The number of carbohydrate-binding sites of the GalNAc-specific lectin is four per tetramer. The binding parameters of N-acetyl-D-galactosamine and methyl-N-acetyl-alpha-D- galactosaminide , were determined by titrating the perturbation in the absorption spectrum of the protein. For D-galactosides, it was necessary to use p-nitrophenyl-N-acetyl-beta-D- galactosaminide as an indicator in substitution titrations. The association constants K were determined at several temperatures yielding 2.4 X 10(4) M-1 at 25 degrees C with delta H degree' = -45 kJ mol-1 and delta S degree' = -67 J X K-1 mol-1 for methyl-N-acetyl-alpha-D- galactosaminide and 1.0 X 10(3) M-1 at 25 degrees C, delta H degree' = -38 kJ mol-1 and delta S degree' = -69 J X K-1 mol-1 for methyl-alpha-D-galactoside. The increase in K by a factor of 25 caused by the acetamido group is largely enthalpic . Whenever different methods were used to determine the association constant of a given compound, the agreement was excellent. The observed changes in absorption or fluorescence of all chromophoric carbohydrate derivatives used are specific for the binding of carbohydrates. For large aromatic beta- aglycons such as p-nitrophenyl or 4-methylumbelliferyl groups, the increase in K of the N-acetyl-D- galactosaminide moiety is by a factor of 2 or less, but for a large N-5-dimethylaminonaphthalene-1-sulfonyl (dansyl) group this factor is about 20 as compared with the acetyl group. The concomitant 10-fold increase in dansyl fluorescence, also observed with four other GalNAc-binding lectins together with a favorable and large delta S degree' = +60 J X K-1 mol-1 strongly point at the presence of a hydrophobic region in the vicinity of the carbohydrate-binding site. The results of stopped flow kinetics with 4-methylumbelliferyl-N-acetyl-beta-D- galactosaminide and the lectin are consistent with a simple mechanism for which k+ = 1.1 X 10(4) M-1 S-1 and k- = 0.4 S-1 at 25 degrees C. This k- is slower than for any monosaccharide-lectin complex reported so far.
mesh_terms: D000116:Acetylgalactosamine / Q000031:analogs & derivatives* / Q000378:metabolism; D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000378:metabolism; D003619:Dansyl Compounds / Q000378:metabolism; D005688:Galactosamine / Q000031:analogs & derivatives* / Q000378:metabolism; D005697:Galactosides / Q000378:metabolism; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D037102:Lectins / Q000378:metabolism*; D037121:Plant Lectins; D030262:Soybean Proteins; D013053:Spectrophotometry; D013816:Thermodynamics; D010835:Phytohemagglutinins
chemical_list: D000116:Acetylgalactosamine; D000117:Acetylglucosamine; D003619:Dansyl Compounds; D005688:Galactosamine; D005697:Galactosides; D006923:Hymecromone; D037102:Lectins; D037121:Plant Lectins; D030262:Soybean Proteins; C028824:soybean lectin; D010835:Phytohemagglutinins; C014123:4-nitrophenyl-2-acetamido-2-deoxygalactopyranoside; C041455:4-methylumbelliferyl-N-acetylamino-beta-galactoside; C014088:methyl-2-acetamido-2-deoxy-D-glucopyranoside; C041456:dansylgalactosamine