Determination of binding constants by ultrafast affinity extraction: Theoretical and experimental studies of optimum conditions for analysis
Determination of binding constants by ultrafast affinity extraction: Theoretical and experimental studies of optimum conditions for analysis
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DOI：
10.1016/j.chroma.2023.464307
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发表时间：
2023-08-22
影响因子：
4.1
通讯作者：
Hage，David S.
中科院分区：
文献类型：
--
作者：
Iftekhar，Sazia;Rauhauser，Madeleine;Hage，David S.
超快亲和萃取（UAE）是一种微量亲和HPLC，可用于快速测定溶液中溶质-结合剂相互作用的平衡常数。本研究使用色谱和平衡理论与通用图来检查阿联酋所需的一般条件，以获得准确，精确和稳健的测量平衡常数的这种相互作用。将预测结果与UAE在研究各种药物与两种转运蛋白（人血清白蛋白和α1-酸性糖蛋白）结合的研究中获得的结果进行了比较。这些结合研究的最精确和耐用条件发生在溶质平衡游离分数具有中间值的系统（F0 = 0.20-0.80）。这些趋势与使用UAE的先前研究中观察到的趋势显示出良好的一致性。进一步确定了溶质的表观自由分数与该溶质的解离速率、UAE过程中溶质解离所需的时间以及溶质的平衡自由分数之间的关系。这些结果也与华法林和格列齐特与人血清白蛋白结合的实验结果一致。最后一节研究了如何在表观自由分数的变化，所造成的溶质解离，影响的准确性的平衡常数是由阿联酋测量。此外，生成理论图以允许选择UAE的条件，其在平衡常数的测量期间提供给定水平的准确度。为UAE创建的方程和确定的趋势是通用的，可以在未来的工作中扩展到其他溶质和结合剂。
Ultrafast affinity extraction (UAE) is a form of microscale affinity HPLC that can be employed to quickly measure equilibrium constants for solute-binding agent interactions in solution. This study used chromatographic and equilibrium theory with universal plots to examine the general conditions that are needed in UAE to obtain accurate, precise, and robust measurements of equilibrium constants for such interactions. The predicted results were compared to those obtained by UAE in studies that examined the binding of various drugs with two transport proteins: human serum albumin and α1-acid glycoprotein. The most precise and robust conditions for these binding studies occurred for systems with intermediate values for their equilibrium free fraction for the solute (F0≈ 0.20–0.80). These trends showed good agreement with those seen in prior studies using UAE. It was further determined how the apparent free fraction of a solute was related to the dissociation rate of this solute, the time allowed for solute dissociation during UAE, and the equilibrium free fraction for the solute. These results also agreed with experimental results, as obtained for the binding of warfarin and gliclazide with human serum albumin. The final section examined how a change in the apparent free fraction, as caused by solute dissociation, affected the accuracy of an equilibrium constant that was measured by UAE. In addition, theoretical plots were generated to allow the selection of conditions for UAE that provided a given level of accuracy during the measurement of an equilibrium constant. The equations created and trends identified for UAE were general ones that can be extended in future work to other solutes and binding agents.