Paper
Binding of 4‐Methylumbelliferyl α‐Manno‐oligosaccharides to Concanavalin A: Equilibrium Study
Published Feb 1, 1978 · A. Landschoot, F. G. Loontiens, C. Bruyne
FEBS Journal
25
Citations
0
Influential Citations
Abstract
Previous binding studies with concanavalin A and 4-methylumbelliferyl α-D-mannopyranoside (I) [Loontiens, F. G., Clegg, R. M. & Jovin, T. M. (1977) Biochemistry, 16, 159–166] were extended to 4-methylumbelliferyl α-mannobioside (II) and 4-methylumbelliferyl α-mannotrioside (III) using difference absorption spectrometry and titration of ligand fluorescence quenching. Compounds II and III were prepared through their acetochloro derivatives, obtained from the separated acetolysis products of the cell-wall mannan from Saccharomyces cerevisiae. Their fluorescence decreases as a function of solvent polarity. Upon binding of compounds I, II and III to concanavalin A, the changes in optical properties of the 4-methylumbelliferyl group are specific for the binding of carbohydrates and depend upon the length of the carbohydrate moiety. With I the absorption spectrum shows a pronounced blue shift, with II and III there is a red shift; the changes in Δɛ at 334 nm are -2210 M−1 cm−1 for I, + 1040 M−1 cm−1 for II and + 435 M−1 cm−1 for III. Upon binding to concanavalin A the fluorescence of I is totally quenched, whereas the quenching is 65% for II and 60% for III. It is suggested that with II and III the 4-methylumbelliferyl group is in less close contact with the protein than with I. As determined by continuous titration of ligand fluorescence quenching, there is no pronounced or systematic increase of the association constants in the series I, II, III: at 25°C the values are (4.26 ± 0.07) × 104 M−1, (1.39 ± 0.05) M−1 and (8.2 ± 0.3) × 104 M−1. In addition, the binding enthalpies are -8.5 ± 0.2, -8.6 ± 0.2 and -8.8 ± 0.2 kcal mol−1; (-35.6, -36.0 and -36.8 kJ mol−1) and the binding entropies -7.4 ± 0.6, -5.3 ± 0.8 and -7.0 ± 0.9 cal mol−1 K−1; (-31, -22 and -29 J mol−1 K−1) are almost constant. The data are consistent with specific binding to a single α-D-mannopyranosyl residue; if any additional binding subsites for one or two mannopyranosyl residues exist, they should have contributions in the binding enthalpies that are less than 2.6 kcal (10.9 kJ) mol−1 for one or 2.8 kcal (11.7 kJ) mol−1 for two α-D-mannopyranosyl residues.
4Methylumbelliferyl -mannooligosaccharides specifically bind to a single -D-mannopyranosyl residue in concanavalin A, with binding enthalpies and entropies being almost constant.
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