Binding Of 4-methylumbelliferyl Research Articles

Binding of 4-methylumbelliferyl beta-D-galactopyranoside to Momordica charantia lectin: fluorescence-quenching studies.

The binding of 4-methylumbelliferyl beta-D-galactopyranoside (MeUmb-Galp), to Mormordica charantia lectin was studied by equilibrium dialysis and quenching of ligand fluorescence. The fluorescence of MeUmb-Galp decreases as a function of solvent polarity. On binding to M. charantia lectin, its fluorescence was nearly 100% quenched, showing that the binding of the glycoside takes place in hydrophobic environment. The binding of the fluorescent sugar was saccharide-specific as evidenced by reversal of MeUmb-Galp fluorescence quenching by lactose. The association constant is independent of the experimental method used and at 25 degrees C the value is (1.96 +/- 0.05) X 10(4) M-1. The number of binding sites as determined by equilibrium dialysis and fluorescence quenching agree very well with each other; n being equal to 1.98 +/- 0.02. The Ka value for the glycoside was also determined by competition studies employing reversal of fluorescence quenching of MeUmb-Galp by lactose. The value of ka obtained for lactose is 1.21 X 10(4) M-1 at 30 degrees C. The internal consistency of the association constant and number of binding site values at low and high saturation indicates the absence of additional subsite on M. charantia lectin. The thermodynamic parameters do not differ greatly with change in temperature; the values of - delta H degrees and - delta S degrees are equal to 30 +/- 9.63 kJ mol-1 and 21 +/- 0.3 J mol-1 K-1 respectively in the range of 15--35 degrees C indicting that the binding of M. charantia lectin to saccharide is exothermic in nature.

Fluorescence-polarization studies on binding of 4-methylumbelliferyl beta-D-galactopyranoside to Ricinus communis (castor-bean) agglutinin.

The binding of Ricinus communis (castor-bean) agglutinin 1 to saccharides was studied by equilibrium dialysis and fluorescence polarization by using the fluorescently labelled sugar 4-methylumbelliferyl beta-D-galactopyranoside. No appreciable change in ligand fluorescence of 4-methylumbelliferyl beta-D-galactopyranoside was considerably polarized on its binding to the lectin. The association constants obtained by Scatchard analysis of equilibrium-dialysis and fluorescence-polarization data do not differ much from each other, and at 25 degrees C, Ka = 2.4 (+/- 0.2) X 10(4)M-1. These values agree reasonably well with that reported in the literature for Ricinus agglutinin 1. The number of binding sites obtained by the different experimental procedures is 1.94 +/- 0.1 per molecule of 120 000 daltons and is equal to the reported value of 2. The consistency in the values of Ka and number of binding sites indicate the absence of additional subsites on Ricinus agglutinin 1 for its specific sugars. In addition, the excellent agreement between the binding parameters obtained by equilibrium dialysis and fluorescence polarization indicate the potential of ligand-fluorescence-polarization measurements in the investigation of lectin-sugar interactions.

Binding of 4-methylumbelliferyl β- d-ribopyranoside to β- d-xylosidase from Bacillus pumilus

The determination of the binding parameters of 4-methylumbelliferyl β- d-ribopyranoside, a fluorescent ligand of β- d-xylosidase (exo-1,4-β-xylosidase, EC 3.2.1.37) from Bacillus pumilus, is described. A single binding site per polypeptide chain (60 000 daltons) was found and the homogeneity of the binding sites in the dimeric or tetrameric forms of the enzyme were shown. The association constants, as a function of temperature and ionic strength, were obtained from equilibrium binding experiments and compared to the kinetically determined inhibition constants. The apparent discrepancies are attributed to a temperature, ionic strength and concentration dependent shift in the dimer-tetramer equilibrium of the enzyme and different affinities of the ligand for both oligomeric forms. Sedimentation velocity experiments seem to corroborate this hypothesis.

Binding of 4-methylumbelliferyl n-acetyl-chitooligosaccharides to wheat-germ agglutinin. A reinvestigation of equilibrium studies.

The binding of 4‐methylumbelliferyl per‐N‐acetyl‐chitooligosaccharides, MeUmbGlcNAc (I), MeUmb(GlcNAc)2 (II) and MeUmb(GlcNAc)3 (III) to wheat germ agglutinin, was studied by equilibrium dialysis, difference absorption spectrometry and extrinsic fluorescence quenching [Privat, J. P., Delmotte, F. & Monsigny, M. (1974) FEBS Lett. 46, 229–232] titration at a fixed wavelength or above 370 nm. The change in optical properties of the MeUmb group upon binding of the glycosides to the agglutinin depends on the length of the carbohydrate chain. An intense and carbohydrate‐specific difference absorption spectrum was observed with I (‐Δɛ at 316 nm equal to 2770 M−1 cm−1), a weaker one with II (‐Δɛ at 316 nm = 1070 M−1 cm−1), but none was observed with III; the fluorescence quenching is 100% for compounds I and II and 44% for compound III. As determined with I, using equilibrium dialysis and difference absorption spectrometry, there are two identical and independent binding sites per subunit. For I the value of the association constant is independent of the experimental method or the saturation range used. The association constants increase with the chain length of the fluorescent oligosaccharides: at 25°C the values (in M−1) are (1.98 ± 0.12)104 for I, (7.5 ± 0.3)104 for II and (1.17 ± 0.60)105 for III; the thermodynamic parameters do not differ greatly: ΔH0 and ΔS0 are ‐8.2 ± 0.4 kcal (–34.3 kJ) mol−1 and –7.8 ± 1.5 cal (–32.6 J) mol−1 K−1 for I, –10.0 ± 0.3 kcal (–41.8 kJ) mol−1 and –11.2 ± 1.0 cal (–46.9J) mol−1 K−1 for II, –8.4 ± 0.3 kcal (–35.1 kJ) mol−1 and –4.8 ± 1.2 cal (–20.1 J) mol−1 K−1 for III. The results, taken together with data from the literature, are interpreted as binding to a region composed of subsites with a protein – MeUmb interaction for I and II amounting to a contribution of the MeUmb group in the free energy of binding, equal to – 2 kcal (– 8.4 kJ) mol−1 for I.

Binding of 4-methylumbelliferyl alpha-D-mannopyranoside to tetrameric and unmodified or derivatized dimeric concanavalin A: equilibrium studies.

The binding of 4-methylumbelliferyl alpha-D-mannopyranoside (MUM) and concanavalin A, composed of intact polypeptide chains, was studied by equilibrium dialysis, difference spectroscopy, and fluorescence titration (Dean, B.R., and Homer, R.B. (1973), Biochim. Biophys. Acta. 322, 141-144), measured either at a fixed wavelength or above 350 nm. Dimeric and tetrameric concanavalin A samples were used under conditions of apparently full metal saturation. The results are consistent with a single carbohydrate-specific site per protomer, without interaction between sites; no indication for additional unspecific binding could be obtained. The values of the association constant are independent of the method or of the saturation range used and 4-methylumbelliferyl alpha-D-mannopyranoside, bound at a fractional saturation of 0.91 can be totally displaced by methyl alpha-D-mannopyranoside. The thermodynamic binding parameters for acetylated or succinylated concanavalin A, composed of intact polypeptide chains, were obtained by titration of total MUM fluorescence in the temperature range 9-39 degrees C. For unmodified dimeric concanavalin A at 25.0 degrees C, the values are K = (3.36 +/- 0.04) 10(4) M-1 with delta H degrees = -8.3 +/- 0.1 kcal mol-1 and delta S degrees = 7.2 +/- 0.3 eu; for tetrameric concanavalin A, the affinity is increased by 25% and within experimental error the values of delta H degrees and delta S degrees are identical to those for the dimeric protein. Derivatized concanavalin A shows binding characteristics that are entirely comparable to those of the native protein.

Binding of 4-methylumbelliferyl alpha-D-mannopyranoside to dimeric concanavalin A: fluorescence temperature-jump relaxation study.

The kinetics of saccharide binding to the dimer form of concanavalin A (con A) has been studied at pH 5.5 with the fluorescence temperature-jump method. 4-Methylumbelliferyl alpha-D-mannopyranoside, a fluorescent carbohydrate derivative which is quenched upon binding to con A, was used as the ligand. Three relaxation effects were seen. The major relaxation (r = 20-400 ms) was investigated at four different temperatures. The behaviour of this relaxation as a function of reactant concentrations is consistent with a simple one-step bimolecular association reaction. These conclusions result from the analysis of both the relaxation times and amplitudes, and from the comparison of the kinetically determined equilibrium parameters (Kass = 3.5 x 10(4) M-1 at 18.5 degrees C, delta H degrees = -(6-7) kcal/mol) to those obtained from a parallel series of equilibrium experiments (Loontiens, F.G., Clegg R.M., and Jovin, T.M. (1977), Biochemistry 16, preceding paper in this issue). The association and dissociation rate constants are in the range of (6-15) x 10(4) M-1 s-1 and (1.5 - 5.6) s-1, respectively, within a temperature range of 13.5-28.1 degrees C. The activation energies for the forward and reverse reactions are approximately 10 and approximately 15 kcal/mol, respectively. The two additional relaxations which are also present in the absence of saccharides result from changes in the protein fluorescence and are attributed to protein conformational changes which are not affected by the binding of saccharides. These effects were further studied using succinylated, acetylated, and demetallized con A. The faster relaxation (13 ms at 18.5 degrees C) was independent of the concentration of the protein and was not present in the derivatized con A samples. The two derivatized forms of con A show almost identical carbohydrate binding parameters as the underivatized protein. A limited series of stopped-flow experiments yielded results which were fully compatible with those from the relaxation measurements.