REDUCED-SCALE LARGE-ZONE ANALYTICAL GEL-FILTRATION CHROMATOGRAPHY FORMEASUREMENT OF PROTEIN ASSOCIATION EQUILIBRIA

The method of large-zone analytical gel filtration chromatography (AGF C) for determination of the energetics and stoichiometries of protein association processes was adapted for use with a semicompressible hydr ophilic sieving resin and high-performance liquid chromatography (HPLC ) instrumentation. Chromatography was performed using a 2.3-ml glass c olumn, providing a 10-fold reduction in column volume over the convent ional AGFC low-pressure chromatography format. Equipment consisted of a Teflon injection valve, HPLC pump, and uv-vis spectrophotometric det ector. Method validation included preparation of calibration curves us ing data obtained from small- and large-zone chromatography of monomer ic protein standards. Following confirmation of the agreement of small - and large-zone data obtained for monomeric proteins, the method was further tested by measurement of the dimer-tetramer equilibrium consta nt for the well-characterized oxygenated human hemoglobin system. Exce llent agreement between the experimentally determined equilibrium asso ciation constant and the literature value was obtained, as well as a d ramatic savings in material and time required to perform the experimen t. (C) 1994 Academic Press, Inc.