Chen Jianzhong, Hage David S
Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588-0304, USA.
Anal Chem. 2006 Apr 15;78(8):2672-83. doi: 10.1021/ac052017b.
A new chromatographic method was developed for characterizing allosteric interactions between an immobilized binding agent and low-solubility compounds. This approach was illustrated by using it to characterize the interactions between tamoxifen and warfarin during their binding to the protein human serum albumin (HSA), with beta-cyclodextrin being employed as a solubilizing agent for these drugs. It was confirmed in this work through several experiments that warfarin had a single binding site on HSA with an association equilibrium constant of (2-5) x 10(5) M(-1) (average, 3.9 x 10(5) M(-1)) at 37 degrees C, in agreement with previous reports. It was also found that tamoxifen had a single major binding site on HSA, with an association equilibrium constant of (3-4) x 10(7) M(-1) (average, 3.5 x 10(7) M(-1)) at 37 degrees C. When warfarin was used as a mobile-phase additive in competition studies with tamoxifen, this had a positive allosteric effect on tamoxifen/HSA binding, giving a coupling constant of 2.3 (+/-0.3). Competitive studies using tamoxifen as a mobile-phase additive indicated that tamoxifen had a negative allosteric effect on warfarin/HSA binding, providing a coupling constant of 0.79 (+/-0.03). A unique feature of the technique described in this report was its ability to independently examine both directions of the warfarin/tamoxifen allosteric interaction. This approach is not limited to warfarin, tamoxifen, and HSA but can also be used to study other solutes and binding agents.
开发了一种新的色谱方法，用于表征固定化结合剂与低溶解度化合物之间的变构相互作用。通过用该方法表征他莫昔芬和华法林在与蛋白质人血清白蛋白（HSA）结合过程中的相互作用来说明这种方法，其中β-环糊精用作这些药物的增溶剂。在这项工作中，通过几个实验证实，华法林在HSA上有一个单一结合位点，在37℃时缔合平衡常数为(2 - 5)×10⁵ M⁻¹（平均为3.9×10⁵ M⁻¹），与先前报道一致。还发现他莫昔芬在HSA上有一个单一的主要结合位点，在37℃时缔合平衡常数为(3 - 4)×10⁷ M⁻¹（平均为3.5×10⁷ M⁻¹）。当华法林在与他莫昔芬的竞争研究中用作流动相添加剂时，这对华法林/ HSA结合具有正变构效应，耦合常数为2.3（±0.3）。使用他莫昔芬作为流动相添加剂的竞争研究表明，他莫昔芬对华法林/ HSA结合具有负变构效应，耦合常数为0.79（±0.03）。本报告中描述的技术的一个独特之处在于其能够独立研究华法林/他莫昔芬变构相互作用的两个方向。这种方法不仅限于华法林、他莫昔芬和HSA，还可用于研究其他溶质和结合剂。
