Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588-0304, USA.
J Chromatogr A. 2011 Apr 15;1218(15):2072-8. doi: 10.1016/j.chroma.2010.09.070. Epub 2010 Oct 16.
This report examined the use of silica monoliths in affinity microcolumns containing human serum albumin (HSA) to measure the dissociation rates for various drugs from this protein. Immobilized HSA and control monolith columns with dimensions of 1 mm × 4.6 mm i.d. were prepared for this work and used with a noncompetitive peak decay method. Several drugs known to bind HSA were examined, such as warfarin, diazepam, imipramine, acetohexamide, and tolbutamide. Items that were studied and optimized in this method included the sample volume, sample concentration, and elution flow rate. It was found that flow rates up to 10 mL/min could be used in this approach. Work with HSA silica monoliths at these high flow rates made it possible to provide dissociation rate constants for drugs such as warfarin in less than 40s. The dissociation rate constants that were measured gave good agreement with values reported in the literature or that had been obtained with other solutes that had similar binding affinities for HSA. This approach is a general one that should be useful in examining the dissociation of other drugs from HSA and in providing a high-throughput method for screening drug-protein interactions.
本报告研究了在含有人血清白蛋白 (HSA) 的亲和微柱中使用硅胶整体柱来测量各种药物与该蛋白的解离速率。为这项工作制备了尺寸为 1mm×4.6mm id 的固定化 HSA 和对照整体柱,并使用非竞争性峰衰减法进行了研究。研究了几种已知与 HSA 结合的药物,如华法林、地西泮、丙咪嗪、醋己脲和甲苯磺丁脲。在这种方法中研究和优化的项目包括样品体积、样品浓度和洗脱流速。结果发现,在这种方法中可以使用高达 10mL/min 的流速。在这些高流速下使用 HSA 硅胶整体柱,使得能够在不到 40 秒的时间内提供华法林等药物的解离速率常数。测量得到的解离速率常数与文献中报道的值或与具有相似 HSA 结合亲和力的其他溶质获得的值吻合良好。这种方法是一种通用方法,应该有助于研究其他药物与 HSA 的解离,并提供用于筛选药物-蛋白质相互作用的高通量方法。