Hage D S, Sengupta A
Chemistry Department, University of Nebraska, Lincoln 68588-0304, USA.
Anal Chem. 1998 Nov 1;70(21):4602-9. doi: 10.1021/ac980734i.
High-performance affinity chromatography and zonal elution studies were used to examine the binding that takes place between the drug clomiphene and the protein human serum albumin (HSA). Equations were derived to describe the behavior of zonal elution experiments in which a solubilizing agent is present in the mobile phase to aid in the dissolution of a competing agent or injected analyte. These equations were then used to determine the association equilibrium constants for the clomiphene/HSA system, with beta-cyclodextrin being used as a complexation agent to improve the water solubility of cis- and trans-clomiphene without affecting the nature of their binding to HSA. It was found in these studies that both cis- and trans-clomiphene have 1:1 interactions at a common binding region on HSA (association constants at pH 7.4 and 37 degrees C: cis, 7.5 x 10(6) M-1; trans, 1.3 x 10(6) M-1). Further competition experiments between cis- or trans-clomiphene and various site-selective probes indicated that the clomiphene-binding region is the same as the proposed tamoxifen site of HSA. The approach and equations used within this report are general ones that can be applied to zonal elution studies of other solute-ligand systems in which one or more of the test components have limited solubility in the desired mobile phase.
采用高效亲和色谱法和区域洗脱研究来检测药物克罗米芬与蛋白质人血清白蛋白（HSA）之间发生的结合。推导了一些方程来描述区域洗脱实验的行为，在这些实验中，流动相中存在一种增溶剂以帮助竞争性试剂或注入的分析物溶解。然后使用这些方程来确定克罗米芬/HSA系统的缔合平衡常数，β-环糊精用作络合剂以提高顺式和反式克罗米芬的水溶性，而不影响它们与HSA结合的性质。在这些研究中发现，顺式和反式克罗米芬在HSA上的一个共同结合区域都有1:1的相互作用（在pH 7.4和37℃时离解常数：顺式，7.5×10⁶ M⁻¹；反式，1.3×10⁶ M⁻¹）。顺式或反式克罗米芬与各种位点选择性探针之间的进一步竞争实验表明，克罗米芬结合区域与HSA上推测的他莫昔芬位点相同。本报告中使用的方法和方程是通用的，可应用于其他溶质-配体系统的区域洗脱研究，其中一种或多种测试组分在所需流动相中的溶解度有限。
