Reduced-scale large-zone analytical gel filtration chromatography for measurement of protein association equilibria

The method of large-zone analytical gel filtration chromatography (AGFC) for determination of the energetics and stoichiometries of protein association processes was adapted for use with a semicompressible hydrophilic sieving resin and high-performance liquid chromatography (HPLC) instrumentation. Chromatography was performed using a 2.3-ml glass column, providing a 10-fold reduction in column volume over the conventional AGFC low-pressure chromatography format. Equipment consisted of a Teflon injection valve, HPLC pump, and uv-vis spectrophotometric detector. Method validation included preparation of calibration curves using data obtained from small- and large-zone chromatography of monomeric protein standards. Following confirmation of the agreement of small- and large-zona data obtained for monomeric proteins, the method was further tested by measurement of the dimer-tetramer equilibrium constant for the well-characterized oxygenated human hemoglobin system. Excellent agreement between the experimentally determined equilibrium association constant and the literature value was obtained, as well as a dramatic savings in material and time required to perform the experiment.