Studies of protein binding to nonpolar solutes by using zonal elution and high-performance affinity chromatography:: Interactions of cis- and trans-clomiphene with human serum albumin in the presence of β-cyclodextrin

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.