Study of high-affinity interactions by quantitative affinity chromatography. Analytical expressions in terms of total...

Analytical expressions are derived for the description of ligand-facilitated and ligand-retarded desorption of partitioning solute in terms of total ligand concentration in quantitative affinity chromatography. Their application is then illustrated by consideration of results from recycling partition equilibrium studies of the heparin-facilitated desorption of thrombin from heparin-Sepharose, and of the competition between methyl-alpha-D-mannoside and p-nitrophenyl-alpha-D- mannoside for concanavalin A immobilized on CPG-170. Finally, published frontal affinity chromatographic data for the NADH-dependent elution of rabbit muscle lactate dehydrogenase from oxamate-Sepharose are reanalysed using these equations to demonstrate the characterization of a system reflecting the binding of a solute-ligand complex to an affinity matrix. This investigation extends the scope of quantitative affinity chromatography to include not only the study of solute-ligand interactions governed by larger binding constants but also the characterization of interactions in which the partitioning solute and ligand are both macromolecular, and eliminates the need for prior dialysis to establish the free ligand concentration required for application of earlier analytical expressions.