USE OF QUANTITATIVE AFFINITY-CHROMATOGRAPHY FOR CHARACTERIZING HIGH-AFFINITY INTERACTIONS - BINDING OF HEPARIN TO ANTITHROMBIN-III

The versatility of quantitative affinity chromatography (QAC) for evaluating the binding of macromolecular ligands to macromolecular acceptors has been increased substantially as a result of the derivation of the equations which describe the partitioning of acceptor between matrix-bound and soluble forms in terms of total, rather than free, ligand concentrations. In addition to simplifying the performance of the binding experiments, this development makes possible the application of the technique to systems characterized by affinities higher than those previously amenable to investigation by QAC. Addition of an on-line data acquisition system to monitor the concentration of partitioning solute in the liquid phase as a function of time has permitted the adoption of an empirical approach for determining the liquid-phase concentration of acceptor in the system at partition equilibrium, a development which decreases significantly the time required to obtain a complete binding curve by QAC. The application of these new QAC developments is illustrated by the determination of binding constants for the interactions of high-affinity heparin (Mr 20,300) with antithrombin III at three temperatures. Association constants of 8.0 ± 2.2 × 107, 3.4 ± 0.3 × 107, and 1.0 ± 0.2 × 107 m-1 were observed at 15, 25, and 35°C, respectively. The standard enthalpy change of -4.2 ± 0.6 kcal/mol that is calculated from these data is in good agreement with a reported value obtained from fluorescence quenching measurements. © 1991.