Studies on the specific interaction of concanavalin A and saccharides by affinity chromatography. Application of...

Quantitative affinity chromatography of concanavalin A (Con A) was studied in order to clarify its mechanism of saccharide binding. Another aim of this work was to confirm the validity of frontal affinity chromatography, which we have developed for the study of specific interactions, for multivalent systems. Since Cona A exists as either a dimer or a tetramer, it is a suitable multivalent test system. Immobilized p-aminophenyl-beta-D-glucopyranoside (AP beta-Glc Sepharose) was prepared and the interactions of homogeneous preparations of Con A with this affinity adsorbent were analyzed. Experiments were carried out under conditions where Con a exists as the dimer (5 degrees C, pH 7.9, I=0.19). The intrinsic dissociation constant of Con A for the immobilized ligand (Kd) could be determined from the extent of retardation. alpha-Con A (intact Con A) had a stronger affinity than beta-Con A. Changes in pH and ionic strength had different effects on the affinity of alpha- and beta-Con A. The dissociation constants of soluble saccharides and their derivatives (counter ligands) for Con A (K1) could be determined from their ability to diminish the elution volume of Con A. K1 values of various monosaccharides for beta-Con A including those having very weak affinity, were determined. Further, the K1 values of a series of glucobioses were determined and compared. It was found that the binding mode of the reducing terminal glucose residue must be taken into account. The results are discussed in relation to their configurations. Frontal affinity chromatography proved to be very useful as a tool to analyze specific interactions in multivalent systems.