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.
D001665:Binding Sites; D003208:Concanavalin A / Q000031:analogs & derivatives / Q000378:metabolism*; D003956:Dialysis; D005456:Fluorescent Dyes; D006003:Glycogen / Q000378:metabolism; D006027:Glycosides / Q000378:metabolism*; D006923:Hymecromone / Q000031:analogs & derivatives* / Q000378:metabolism; D008362:Mannosides / Q000378:metabolism*; D009391:Nephelometry and Turbidimetry; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014468:Umbelliferones / Q000031:analogs & derivatives*
D005456:Fluorescent Dyes; D006027:Glycosides; D008362:Mannosides; D014468:Umbelliferones; D003208:Concanavalin A; D006923:Hymecromone; D006003:Glycogen