EXPERIMENTAL AND THEORETICAL-STUDIES OF RATE-CONSTANT EVALUATION BY AFFINITY-CHROMATOGRAPHY - DETERMINATION OF RATE CONSTANTS FOR THE INTERACTION OF SACCHARIDES WITH CONCANAVALIN-A

Experimental studies have been combined with numerical simulations of frontal affinity chromatography to illustrate the potential of this te chnique for obtaining rate constants for the interaction of the solute with affinity matrix and for a reaction in competition with the solut e-matrix interaction. Frontal chromatography of p-nitrophenylmannoside on a standard HPLC column of concanavalin A-CPG 3000 at flow-rates of 2-10 ml/min has yielded a rate constant of 0.42 s-1 for the dissociat ion of the solute-immobilized lectin complex on the basis of the flow- rate dependence of boundary spreading in the elution profile: this con stant (k-1) has been obtained by extrapolating the apparent dissociati on rate constant (k-1obs) to zero solute concentration to eliminate th e consequences of non-linear (Langmuir) kinetics. Numerical simulation s of the affinity chromatographic behaviour of p-nitrophenylmannoside (A) in the presence of a fixed concentration of a second saccharide (B ) that competes for matrix sites have shown that the corresponding ext rapolation of k-1obs, to zero solute concentration again yields k-1, b ut that the presence of competing saccharide is manifested as a change in slope of the essentially linear dependence of k-1obs upon p-nitrop henylmannoside concentration ([A]). A calibration plot of the variatio n of d(k-1obs)/d[A] with dissociation rate constant for the competing ligand-matrix interaction (k-2) has then been used to obtain a rate co nstant of 1 s-1 for the dissociation of methylmannoside from the immob ilized lectin on the basis of frontal affinity chromatography of p-nit rophenylmannoside in the presence of 100 muM competing saccharide. Fin ally, the potential of affinity chromatography for evaluating the diss ociation rate constant for a competing interaction between ligand and solute has been illustrated by numerical simulation of elution profile s and their interpretation in terms of a published expression for the flow-rate dependence of boundary spreading under conditions of linear kinetics; and by extrapolation of the apparent rate constants so obtai ned to zero solute concentration to take into account the effects of L angmuir kinetics on chromatographic migration.