Proton nuclear magnetic resonance studies of soybean lectin-monosaccharide interactions: computer analysis of complex binding behavior

Abstract 1 H NMR was used to quantify soybean lectin binding to monosaccharides, using presaturation of HOD plus a spin-echo sequence to observe sugar -NHCOCH 3 and -OCH 3 to below 0.01 m m . Binding is in the very-slow-exchange limit; there is no broadening or shifting and only unbound sugar is observed for pH 5 to 8 and 25 to 75 °C. Preliminary results were consistent with those previously reported for methyl 2-acetamido-2-deoxy-α- d -galactopyranoside (Me α- d -GalNAc p ) ( K = 3 × 10 4 liters mol −1 with four sites per tetramer). More detailed studies, however, gave concave Scatchard plots for methyl 2-acetamido-2-deoxy-α- and β- d -galactopyranoside, (Me α- and Me β- d -GalNAc p ), best fitted using K 1 values of (6–12) × 10 4 liters mol −1 , K 2 values ⩽ 0.5 × 10 4 liters mol −1 , and four sites of each type in D 2 O or 80% H 2 O at 25 °C and pH 7.2. Data for methyl α- d -galactopyranoside were fitted with K = 0.5 × 10 4 liters mol −1 and eight sites of the same K . Monosaccharides may be binding in the recently reported “hydrophobic sites” of soybean lectin. Both methyl 2-acetamido-2-deoxy-β- d -galactofuranoside (Me β- d -GalNAcƒ) and methyl 2-acetamido-2-deoxy-α- d -glucopyranoside (Me β- d -GlcNAcƒ) showed some binding by 1 H NMR; K 's were similar to those for the high-affinity sugars, but the occupancy was much lower. The soybean lectin in this study was saturated in Ca 2+ (⩾4 mol/tetramer), but low in Mn 2+ , with Mn 2+ plus Mg 2+ less than 4. We report new melting points for d - N -acetylgalactosamine, Me α- d -GlcNAc p , Me β- d -GalNAc p , and Me β- d -GalNAcƒ, and a fully listed program for fitting curved Scatchard plots using Apple IIc and IIe computers.