A NEW APPROACH TO EXAMINE CONFORMATIONAL-CHANGES OCCURRING UPON BINDING OF LIGAND BY BIOMOLECULES

Liquid-liquid partition chromatography in an aqueous poly(ethylene gly col)/dextran two-phase system (LLPC) is shown to be a quick and sensit ive method for detecting conformational changes occurring upon binding of ligands by biospecific molecules. Two groups of well-characterized proteins, enzymes and monoclonal antibodies, were employed. As an exa mple, LLPC demonstrated that isoforms of lactate dehydrogenase as well as of hexokinase existed in a ligand-dependent equilibrium between tw o forms and that conformational changes occurred when monoclonal antib odies bound haptens. We also demonstrate that the method could be used to detect and separate subfractions in preparations of unliganded pro teins that appeared to be homogeneous when analysed by other technique s.