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.
Nenortas
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Department of Chemistry and Biochemistry, University of Maryland Baltimore County 21228.
Beckett
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D
D002138:Calibration; D002850:Chromatography, Gel / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D006441:Hemoglobin A / Q000032:analysis*; D016018:Least-Squares Analysis; D010108:Oxyhemoglobins / Q000032:analysis*; D015203:Reproducibility of Results; D012116:Resins, Plant
D010108:Oxyhemoglobins; D012116:Resins, Plant; D006441:Hemoglobin A; C029774:oxyhemoglobin A