Abstract
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.
Highlights
Dansylgalactosamineis due to a relatively positive entropic contribution as revealed by the thermodynamic parameters: AH = -33.62 k J mol” and AS@= -5.24 J mol“ K-l
Lectin difference absorption spectra in the presence of N acety~g~lactosaminiendicate perturbation of tryptoand 0)types,D-Galactose wasreported to be a better inhibitor than N-ac~tyl-D-gaIacto~aminfeor this lectin
Two groupsof lectins with different isoelectric points (PI5.5 and B9.5) and erythrocyte specificities werereported to occur in thewinged bean [7,8].N-Acetyl-D-galactosaminewas a better inhibitor than D-galactosefor both groups of lectins, the specificity of the basic lectins seems to be directed toward CYgalactosides and that of the acidic lectins toward p-galactosides
Summary
WBA I (4.2 pb! in dimer) was titrated with 1-43 PM DnsGalN. The c a fluorescence of the ligand-protein mixture was compared with the ab) fluorescence of equal concentration of the ligand in the absence of protein. The data obtained by titration of a fixed concentration of WBA I with varying concentrations of DnsGalN at 22.5 "C on analysis accordingto the method of Scatchard [17] gave a straight line (Fig. 3) with an association constant K ,of4.54. A representative plot for DnsGalN bindingto WBA I at 25 "Cis shown in Fig. 4 (inset).The value of the association constant, K,, for DnsGalN determined by this method is 4.17 X lo5M-' at 25 "C,assuming a 1:l stoichiometry withWBA I of M, 29,000. The fluorescence data were analyzed using the followingreaction scheme
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