Chemistry Department, University of Nebraska, Lincoln, Lincoln, NE 68588-0304, USA.
J Chromatogr A. 2011 Sep 28;1218(39):6892-7. doi: 10.1016/j.chroma.2011.08.026. Epub 2011 Aug 17.
Peak profiling and high-performance columns containing immobilized human serum albumin (HSA) were used to study the interaction kinetics of chiral solutes with this protein. This approach was tested using the phenytoin metabolites 5-(3-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) as model analytes. HSA columns provided some resolution of the enantiomers for each phenytoin metabolite, which made it possible to simultaneously conduct kinetic studies on each chiral form. The dissociation rate constants for these interactions were determined by using both the single flow rate and multiple flow rate peak profiling methods. Corrections for non-specific interactions with the support were also considered. The final estimates obtained at pH 7.4 and 37°C for the dissociation rate constants of these interactions were 8.2-9.6 s(-1) for the two enantiomers of m-HPPH and 3.2-4.1 s(-1) for the enantiomers of p-HPPH. These rate constants agreed with previous values that have been reported for other drugs and solutes that have similar affinities and binding regions on HSA. The approach used in this report was not limited to phenytoin metabolites or HSA but could be applied to a variety of other chiral solutes and proteins. This method could also be adopted for use in the rapid screening of drug-protein interactions.
采用峰形分析和键合固定化人血清白蛋白（HSA）的高效柱，研究手性溶质与该蛋白的相互作用动力学。以苯妥英代谢物 5-(3-羟苯基)-5-苯基海因（m-HPPH）和 5-(4-羟苯基)-5-苯基海因（p-HPPH）为模型分析物，对该方法进行了测试。HSA 柱对每种苯妥英代谢物的对映体提供了一定程度的分辨率，从而可以同时对每种手性形式进行动力学研究。使用单流速和多流速峰形分析方法确定了这些相互作用的离解速率常数。还考虑了与载体的非特异性相互作用的校正。在 pH 7.4 和 37°C 下，获得了这些相互作用的离解速率常数的最终估计值，对于 m-HPPH 的两种对映体为 8.2-9.6 s(-1)，对于 p-HPPH 的对映体为 3.2-4.1 s(-1)。这些速率常数与先前报道的其他具有相似亲和力和结合区域的药物和溶质在 HSA 上的速率常数一致。本报告中使用的方法不仅限于苯妥英代谢物或 HSA，也可应用于多种其他手性溶质和蛋白质。该方法还可用于快速筛选药物-蛋白质相互作用。
