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

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Abstract
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)), measured 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 was 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.degree. C. For unmodified dimeric concanavalin A at 25.0.degree. C, the values are K = (3.36 .+-. 0.04)104 M-1 with .DELTA.H.degree. [enthalpy = -8.3 .+-. O.1 kcal mol-1 and .DELTA.S.degree. [entropy] = -7.2 .+-. 0.3 eu [entropy units]; for tetrameric concanavalin A, the affinity is increased by 25%, and within experimental error the values of .DELTA.H.degree. and .DELTA.S.degree. are identical to those for the dimeric protein. Derivatized concanavalin A shows binding characteristics that are entirely comparable to those of the native protein.

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