Determination of the carbohydrate-binding properties of peanut agglutinin by ultraviolet difference spectroscopy

The anti-T lectin from peanuts was purified on a new affinity matrix, and the number of carbohydrate binding sites was determined by equilibrium dialysis with [14C]lactose to be four per tetramer. Methyl-alpha- and methyl-beta-D-galactopyranoside and lactose were found to perturb the UV spectrum of the lectin in the aromatic region and their association constants were determined by UV difference spectroscopy to be 1.8, 1.0, and 1.3 X 10(3) M-1, respectively, at 25 degrees C. Thermodynamic parameters were also obtained for the two galactosides from measurements at several temperatures. For the alpha anomer, delta H degrees = -42 kJ mol-1 and delta S degrees = -78 J K-1 mol-1; for the beta anomer, delta H degrees = -43 kJ mol-1, and delta S degrees = -86 J K-1 mol-1. Analysis of the lectin for metal atoms disclosed 0.98 mol of Ca2+ and 0.78 mol of Mg2+ subunit, while manganese was present in trace amounts only. The results of the present study indicate that recent improvements in instrumentation should make UV difference spectroscopy more widely applicable to studies of protein-ligand interactions.