Thermodynamic and kinetic analysis of carbohydrate binding to the basic lectin from winged bean (Psophocarpus tetragonolobus).
A basic lectin (pI approximately 10.0) was purified to homogeneity from the seeds of winged bean (Psophocarpus tetragonolobus) by affinity chromatography on Sepharose 6-aminocaproyl-D-galactosamine. The lectin agglutinated trypsinized rabbit erythrocytes and had a relative molecular mass of 58,000 consisting of two subunits of Mr 29,000. The lectin binds to N-dansylgalactosamine, leading to a 15-fold increase in dansyl fluorescence with a concomitant 25-nm blue shift in the emission maximum. The lectin has two binding sites/dimer for this sugar and an association constant of 4.17 X 10(5) M-1 at 25 degrees C. The strong binding to N-dansylgalactosamine is due to a relatively positive entropic contribution as revealed by the thermodynamic parameters: delta H = -33.62 kJ mol-1 and delta S0 = -5.24 J mol-1 K-1. Binding of this sugar to the lectin shows that it can accommodate a large hydrophobic substituent on the C-2 carbon of D-galactose. Studies with other sugars indicate that a hydrophobic substituent in alpha-conformation at the anomeric position increases the affinity of binding. The C-4 and C-6 hydroxyl groups are critical for sugar binding to this lectin. Lectin difference absorption spectra in the presence of N-acetylgalactosamine indicate perturbation of tryptophan residues on sugar binding. The results of stopped flow kinetics with N-dansylgalactosamine and the lectin are consistent with a simple one-step mechanism for which k+1 = 1.33 X 10(4) M-1 s-1 and k-1 = 3.2 X 10(-2) s-1 at 25 degrees C. This k-1 is slower than any reported for a lectin-monosaccharide complex so far. The activation parameters indicate an enthalpically controlled association process.
D000818:Animals; D001667:Binding, Competitive; D002236:Carbohydrate Conformation; D050260:Carbohydrate Metabolism; D002846:Chromatography, Affinity; D003619:Dansyl Compounds / Q000378:metabolism; D007887:Fabaceae / Q000378:metabolism*; D005688:Galactosamine / Q000031:analogs & derivatives / Q000378:metabolism; D006384:Hemagglutination; D007700:Kinetics; D037102:Lectins / Q000378:metabolism*; D008433:Mathematics; D008970:Molecular Weight; D037121:Plant Lectins; D010946:Plants, Medicinal; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D003619:Dansyl Compounds; D037102:Lectins; D037121:Plant Lectins; D005688:Galactosamine; C041456:dansylgalactosamine