EXPERIMENTAL AND THEORETICAL-STUDIES OF RATE-CONSTANT EVALUATION BY AFFINITY-CHROMATOGRAPHY - DETERMINATION OF RATE CONSTANTS FOR THE INTERACTION OF SACCHARIDES WITH CONCANAVALIN-A
Pd. Munro et al., EXPERIMENTAL AND THEORETICAL-STUDIES OF RATE-CONSTANT EVALUATION BY AFFINITY-CHROMATOGRAPHY - DETERMINATION OF RATE CONSTANTS FOR THE INTERACTION OF SACCHARIDES WITH CONCANAVALIN-A, Journal of chromatography, 646(1), 1993, pp. 3-15
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.