Reduced-scale large-zone analytical gel filtration chromatography for measurement of protein association equilibria.
Reduced-scale large-zone analytical gel filtration chromatography for measurement of protein association equilibria.
复制标题
用于测量蛋白质缔合平衡的缩小规模大区分析凝胶过滤色谱。
DOI：
10.1006/abio.1994.1504
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发表时间：
1994
影响因子：
2.9
通讯作者：
Beckett,D
中科院分区：
文献类型：
--
作者：
Nenortas,E;Beckett,D
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-zone 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.
