Tweed S A, Loun B, Hage D S
Department of Chemistry, University of Nebraska, Lincoln 68588-0304, USA.
Anal Chem. 1997 Dec 1;69(23):4790-8. doi: 10.1021/ac970565m.
This study examined how the binding capacities and equilibrium constants measured by frontal analysis are affected by ligand heterogeneity in affinity columns. Equations derived for n- and two-site systems gave good agreement with results obtained for the binding of L-thyroxine to a column containing human serum albumin (HSA) and for the binding of (R)-warfarin to coupled columns containing HSA or pigeon serum albumin. The same equations were used to examine how different degrees of ligand heterogeneity affected the apparent binding capacities or equilibrium constants measured using the linear range of double-reciprocal frontal analysis plots. A large proportion of two-site systems gave good estimates (i.e., less than 10-20% error) for the true total column capacity and for the association constant of the highest affinity ligand in the column. A smaller, but still appreciable, fraction of all three- and four-site cases also produced good estimates of these values. The results of this work are not limited to protein-based affinity columns but should be applicable to any type of stationary phase that has well-defined binding regions and relatively fast, reversible interactions with solutes.
本研究考察了亲和柱中配体异质性如何影响通过前沿分析法测得的结合容量和平衡常数。针对多位点和双位点体系推导的方程，与L-甲状腺素与人血清白蛋白（HSA）柱的结合以及（R）-华法林与含HSA或鸽血清白蛋白的偶联柱的结合所获得的结果吻合良好。相同的方程用于考察不同程度的配体异质性如何影响使用双倒数前沿分析图的线性范围测得的表观结合容量或平衡常数。很大比例的双位点体系对柱的真实总容量和柱中最高亲和力配体的缔合常数给出了良好估计（即误差小于10 - 20%）。所有三位点和四位点情况中较小但仍可观的一部分也对这些值给出了良好估计。这项工作的结果不仅限于基于蛋白质的亲和柱，还应适用于任何具有明确结合区域且与溶质有相对快速、可逆相互作用的固定相类型。
