Experimental and theoretical studies of rate constant evaluation by affinity chromatography Determination of rate constants for the interaction of saccharides with concanavalin A

Abstract

Experimental studies have been combined with numerical simulations of frontal affinity chromatography to illustrate the potentials of this technique for obtaining rate constants for the interaction of the solute with affinity matrix and for a reaction in competition with the solute—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 dissociation of the solute-immobilized lectin complex on the basis of the flow-rate dependence of boundary spreading in the elution profile: this constant ( k −1) has been obtained by extrapolating the apparent dissociation rate constant ( k obs −1) to zero solute concentration to eliminate the consequences of non-linear (Langmuir) kinetics. Numerical simulations of the affinity chromatographic behaviour of p-nitrophenylmannside (A) in the presence of a fixed concentration of a second saccharide (B) that competes for matrix sites have shown that the corresponding extrapolation of k obs −1 to zero solute concentration again yields k −1, but that the presence of competing saccharide is manifested as a change in slope of the essentially linear dependence of k obs −1 upon p-nitrophenylmannoside concentration ([A]). A calibration plot of the variation of d( k obs −1/d[A] with dissociation rate constant for the competing ligand—matrix interaction ( k −2) has been used to obtain a rate constant of 1 s −1 for the dissociation of methylmannoside from the immobilized lectin on the basis frontal affinity chromatography of p-nitrophenylmannoside in the presence of 100μ M competing saccharide. Finally, the potential of affinity chromatography for the evaluating the dissociation rate constant for a competing interaction between ligand and solute has been illustrated by numerical simulation of elution profiles 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 rate constants so obtained to zero solute concentration to take into account the effects of Langmuir kinetics on chromatographic migration.