Thermodynamic and kinetic analysis of carbohydrate binding to the basic lectin from winged bean (Psophocarpus tetragonolobus)

Abstract

A basic lectin (pI~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 M_r 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 4.17×10⁵ M⁻¹ at 25°C. The strong binding to N-dansylgalactosamine is due to a relatively positive entropic contribution as revealed by the thermodynamic parameters: ΔH=−33.62 kJ mol⁻¹ and ΔS^o=−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 α-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=1.33×10⁴ M⁻¹ s⁻¹ and k₋₁=3.2×10⁻² s⁻¹ at 25°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.