Affinity chromatography without immobilized ligands; A new method for studying macromolecular interactions using high-performance liquid chromatography

Abstract A new method for studying macromolecular interactions was devised. The principle is based on affinity chromatography with a mobile zone of affinity ligand instead of a column with immobilized ligand. In this method, the difference in migration velocities between the moving zone of affinity ligand and a sample in a conventional gel-permeation column is utilized. The fast migrating zone ( A zone), which is later injected into the column, and the slow migrating zone ( B zone), which is injected beforehand, interfere with each other at the passing point in the column if A and B interact such as A + B ⇆ AB . The zone interference deforms each elution profile of A and B , because the complex AB has a migration velocity different from the others. The elution profiles in zone-interference chromatography are calculated by computer simulation in the framework of the plate theory. The binding constant is calculated from the peak shift of elution volume of B in the zone-interference chromatogram. The interaction between single-strand DNA ( A zone) and ribonuclease A ( B zone) was studied.