Abstract
Titration calorimetry measurements of the binding of methyl alpha-D-mannopyranoside (Me alpha Man), D-mannopyranoside (Man), methyl alpha-D-glucopyranoside (Me alpha Glu), and D-glucopyranoside (Glu) to concanavalin A (Con A), pea lectin, and lentil lectin were performed at 281 and 292 K in 0.01 M dimethylglutaric acid-NaOH buffer (pH 6.9) containing 0.15 M NaCl and Mn+2 and Ca+2 ions. The site binding enthalpies, delta H, are the same at both temperatures and range from -28.4 +/- 0.9 (Me alpha Man) to -16.6 +/- 0.5 kJ mol-1 (Glu) for Con A, from -26.2 +/- 1.1 (Me alpha Man) to -12.8 +/- 0.4 kJ mol-1 (Me alpha Glu) for pea lectin, and from -16.6 +/- 0.7 (Me alpha Man) to -8.0 +/- 0.2 kJ mol-1 (Me alpha Glu) for lentil lectin. The site binding constants range from 17 +/- 1 x 10(3) M-1 (Me alpha Man to Con A at 281.2 K) to 230 +/- 20 M-1 (Glu to lentil lectin at 292.6 K) and exhibit high specificity for Con A where they are in the Me alpha Man:Man:Me alpha Glu:Glu ratio of 21:4:5:1, while the corresponding ratio is 5:2:1.5:1 for pea lectin and 4:2:2:1 for lentil lectin. The higher specificity for Con A indicates more interactions between the amino acid residues at the binding site and the carbohydrate ligand than for the pea and lentil lectin-carbohydrate complexes. The carbohydrate-lectin binding results exhibit enthalpy-entropy compensation in that delta Hb (kJ mol-1) = -1.67 +/- 0.06 x 10(4) + (1.30 +/- 0.12)T(K) delta Sb (J mol-1K-1). Differential scanning calorimetry measurements on the thermal denaturation of the lectins and their carbohydrate complexes show that the Con A tetramer dissociates into monomers, while the pea and lentil lectin dimers dissociate into two submonomer fragments. At the denaturation temperature, one carbohydrate binds to each monomer of Con A and the pea and lentil lectins. Complexation with the carbohydrate increases the denaturation temperature of the lectin and the magnitude of the increases yield binding constants in agreement with the determinations from titration calorimetry.
Highlights
Titration calorimetry measurements of the binding Lectins are proteins which bind carbohydrates with a high of methyl a-D-mannopyranoside (MeaMan),D-mannO- degree of specificity
MeaMan:Man:MeaGlu:Glu which is close to the ordeor f the differential scanning calorimetry (DSC) Measurements-TypicalDSC scans of concanavalin A (Con A), pea binding affinities of 16:4:4:1 from inhibition studies (Debray lectin,andlentillectinin 0.01 M dimethyl glutaric acid (DMG) buffer containing et al, 1981).In Table11,the corresponding ordeforr pea lectin Mn2+, Ca2+, and0.150 M sodium chloride at pH 6.90 f 0.05 is 4.8 f 0.5:2.2 f 0.1:1.5 f 0.1:1 a t room temperature which are shown in Fig. 3.The concentrationsof the Mn2+ and Ca2+
Earlier interpretations (Goldstein et al, 1965; Poretz and Goldstein, 1970; Bhattacharyyaand Brewer, 1988) of the binding mechanism of carbohydrates to the legume lectins have relied on comparisons of the binding constants
Summary
Materials-Con A, lentil lectin, 3,3'-dimethyl glutaric acid (DMG), Tris, and the monosaccharides were obtained from Sigma' and used without any furtherpurification. A Pharmacia Phast native electrophoresis gel with 0.1 M Tris-HC1 buffer (pH 6.9) containing 0.2% bromphenol blue and 20% glycerolexhibited one major band for pea lectin and lentil lectin and one major band and a weak band for ConA. 4-10-pl aliquots of the 530 mM ligand solution were added via a rotatingstirrer-syringe to the 0.1-1 mM protein solution contained in a 1.38-ml cell.The additions were 3 min apart to allow the exothermic heat peak accompanying each addition to return to the base line prior to the addition. This was continued until the last titration peak was less than 25% of the initial peak. The transition temperature, T,, is the temperature at half the peak area and thecalorimetric enthalpy is the peak area per number of moles of lectin monomer in the DSC cell
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