Department of Chemistry


Date of this Version



Published in final edited form as: Anal Chem. 2006 April 15; 78(8): 2672–2683. doi:10.1021/ac052017b. Version presented here is from NIH PubMed Central


Copyright Elsevier Inc. Used By Permission.


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 β-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 × 105 M-1 (average, 3.9 × 105 M-1) at 37°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 × 107 M-1 (average, 3.5 × 107 M-1) at 37°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.