Temperature-induced ultraviolet difference absorption spectrometry for determination of enthalpy changes Binding of 4-methylumbelliferyl glycosides to four lectins

AbstractRaising the temperature in a single mixture of a lectin and a chromophoric glycoside allows determination of the binding enthalpy. This is made possible by continuously monitoring the displacement of the complex from its equilibrium concentration with a sensitive difference absorption spectrophotometer. The method is illustrated with the following lectins: concanavalin A, soybean agglutinin, peanut agglutinin and Erythrina cristagalli agglutinin. The ligands are 4-methylumbelliferyl glycosides. The binding enthalpies found range from −60 kJ · mol−1 for the Galß1 → 3GalNAc-ßglycoside and peanut agglutinin to −30 kJ · mol−1 for a monosaccharide glycoside and the other lectins

Binding of 4-methylumbelliferyl α-manno-oligosaccharides to Concanavalin A: equilibrium study

Previous binding studies with concanavalin A and 4-methylumbelliferyl alpha-D-mannopyranoside (I) [Loontiens, F. G., Clegg, R. M. & Jovin, T. M. (1977) Biochemistry, 16, 159–166] were extended to 4-methylumbelliferyl alpha-mannobioside (II) and 4-methylumbelliferyl alpha-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 alpha-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 alpha-D-mannopyranosyl residues

Binding kinetics of methyl α-D-mannopyranoside to Concanavalin A: temperature-jump relaxation study with 4-methylumbelliferyl α-D-mannopyranoside as a fluorescence indicator ligand

The binding of methyl alpha-D-mannopyranoside and methyl alpha-D-glucopyranoside to concanavalin A has been investigated by the temperature-jump relaxation kinetic technique using the competitive inhibitor 4-methylumbelliferyl alpha-D-mannopyranoside as an indicator of the binding reaction. The analysis shows that these saccharides bind to concanavalin A in a single bimolecular step. The binding parameters are compared to those of derivatized carbohydrates which have previously been used to study the binding of saccharides to concanavalin A. The similarity of the association rate constants indicates that a common process is involved in the binding of all carbohydrates to concanavalin A. The different affinities of saccharides for the lectin are primarily due to the different dissociation rate constants. A discussion of the proposed mechanism is given under the Appendix to clarify the fact that one of the observed relaxation times is faster than is possible with only the kinetic indicator reaction

Binding of 4-methylumbelliferyl α-d-mannopyranoside to tetrameric Concanavalin A: fluorescence temperature-jump relaxation study

The kinetics of saccharide binding to the tetramer form of concanavalin A have been studied at pH 7.2 with the temperature-jump method. 4-Methylumbelliferyl alpha-D-mannopyranoside was used as a ligand; its fluorescence is totally quenched upon binding. A single relaxation of ligand fluorescence (τ= 20–400 ms) was observed and was investigated at three different temperatures, using kinetic titration and dilution types of experiments. The concentration dependence of the relaxation time and amplitude were consistent with a single-step bimolecular association and independent binding sites. In the temperature range 13–24 °C the association and dissociation rate parameters are in the range (6–10) × 104 M−1 s−1 and (1.4–3.2) s−1 respectively, corresponding to activation energies for the forward and reverse reactions equal to approx. 13 and 8 kcal/mol (54 and 33 kJ/mol) respectively. Two additional relaxations of protein fluorescence (3 ms and larger than 1 s at 25 °C) were unaffected by carbohydrate binding. Tetrameric concanavalin A shows carbohydrate binding parameters that are almost identical to those of native or derivatized dimeric concanavalin A

Binding kinetics of 4-methylumbelliferyl α-mannobioside to Concanavalin A by fluorescence stopped-flow measurements

The stopped-flow binding kinetics of concanavalin A with fluorescently labelled (alpha 1 → 2)-linked mannooligosaccharides are characterized by signal changes which are monophasic for 4-methyl-umbelliferyl alpha-D-mannopyranoside (I), biphasic for 4-methylumbelliferyl alpha-mannobioside(II) and triphasic for 4-methylumbelliferyl alpha-mannotrioside (III). The results indicate that the carbohydrate-binding site interacts specifically with a single mannopyranosyl group, resulting in as many possible complexes as there are mannopyranosyl groups in the oligosaccharide chain. Pseudo-first-order kinetics with II and concanavalin A in the range 6–334 μM yield a faster time constant (110–37 ms) with a saturable amplitude and a slower one (3.9–0.29s) with an amplitude which reaches a peak at 30 μM. The concentration dependencies of the two apparent rate constants and the corresponding amplitudes are compatible with two equivalent mechanisms for which the reaction-rate parameters are calculated. Each involves two complexes with spectroscopic parameters which are dependent upon the mechanism. The first mechanism consists of binding followed by an isomerisation, interpreted as sliding of the internal mannopyranosyl residue to bind preferentially via the terminal residue. In the second mechanism, these two binding modes would alternate; here, the binding via the terminal residue is preferred at equilibrium by a factor of 68; both association-rate parameters are comparable and similar to those for I or simple glycosides. Consistent results were obtained by temperature-jump relaxation kinetics