Binding of 4-Methylumberlliferyl beta-d-galactosyl-(13)-N-acetyl-beta-d-galactosaminide to Peanut Agglutinin. Characterisation and Application in Substitution Titrations

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Abstract
Binding of 4-methylumbelliferyl-2-acetamido-2-deoxy-3-O-(β-d-galactopyranosyl)-β-d-galactopyranoside [MeUmbβGal(β1→3)GalNAc] to peanut agglutinin was characterized by equilibrium dialysis and by measurement of the increase in ultraviolet absorption or fluorescence of the chromophoric glycoside upon continuous titration with excess of the lectin. All data in the 4–30°C range correspond to ΔG°=−(26.5 ± 0.1) kJ mol−1, ΔH°=−(58.4 ± 2) kJ mol−1 and ΔS°=−(107 ± 8) J mol−1 K−1. Values of the association constants are e. g. K= 2.5 × 105 M−1 at 4°C and K= 4.5 × 104 M−1 at 25°C. MeUmbβGal(β1→3)GalNAc was used as an indicator ligand to determine K values for nonchromophoric carbohydrates by continuous displacement titrations, measuring either fluorescence or difference in absorption of the indicator. The data were analyzed in terms of the general expression for a non-ideal indicator system (as detailed in the appendix). Thus, the values of K are not underestimated. They are K= 4.8 × 103 M−1 for methyl α-d-galactopyranoside [MeαGal], 2.0 × 103 M−1 for methyl β-d-galactopyranoside [MeβGal] and 4.7 × 103 M−1 for lactose [Gal(β1→4)Glc], all at 14.5°C. The MeUmb difference absorption spectra resulting from binding of the lectin with MeUmbβGal(β1→3)GalNAc and MeUmbβGal(β1→4)Glc are larger than for MeUmbβGal and MeUmbαGal. These observations are consistent with the extended nature of the combining site of peanut agglutinin.