Gong Maojun, Wehmeyer Kenneth R, Limbach Patrick A, Heineman William R
Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221-0172, USA.
Electrophoresis. 2007 Mar;28(5):837-42. doi: 10.1002/elps.200600398.
Equilibrium constants, such as the dissociation constant (K(d)), are a key measurement of noncovalent interactions that are of importance for the proper functioning of molecules in living systems. Frontal analysis (FA) is a simple and accurate CE method for the determination of K(d). Microchip CE coupled with LIF detection was used to determine K(d) of protein-DNA interactions using the FA method. A model system of IgE and the IgE-binding aptamer was selected to demonstrate the capability of FA in microchip CE. Because the fluorescence emission was dependent on the dye migration velocity, the velocity of the free aptamer was adjusted to be the same as that of the aptamer-IgE complex by setting up individual separation voltage configurations for the free and bound aptamers. The ratio of the free and bound aptamers in the equilibrium mixture was directly measured from the heights of their plateaus detected at 1.0 cm from the intersection of the microchip, and no internal standard was needed. The K(d) of the IgE-aptamer pair was determined as 6 +/- 2 nM which is consistent with the reported results (8 nM).
平衡常数，如解离常数（K(d)），是非共价相互作用的关键测量指标，对于生物系统中分子的正常功能至关重要。前沿分析（FA）是一种用于测定K(d)的简单且准确的毛细管电泳方法。采用微芯片毛细管电泳结合激光诱导荧光检测，利用FA方法测定蛋白质 - DNA相互作用的K(d)。选择IgE与IgE结合适体的模型系统来证明微芯片毛细管电泳中FA的能力。由于荧光发射取决于染料迁移速度，通过为游离和结合适体设置单独的分离电压配置，将游离适体的速度调整为与适体 - IgE复合物的速度相同。平衡混合物中游离和结合适体的比例直接从微芯片交叉点1.0 cm处检测到的平台高度测量，无需内标。IgE - 适体对的K(d)测定为6 +/- 2 nM，与报道结果（8 nM）一致。
