Characterization of minor site probes for human serum albumin by high-performance affinity chromatography.

This study used high-performance affinity chromatography (HPAC) and immobilized human serum albumin (HSA) columns to examine the specificity and cross-reactivity of various compounds that have been proposed as markers for the minor binding sites of HSA. These agents included acetyldigitoxin and digitoxin as probes for the digitoxin site, phenol red as a probe for the bilirubin site, and cisor trans-clomiphene as markers for the tamoxifen site. None of these probes showed any significant binding at HSA's indole-benzodiazepine site. However, phenol red did bind at the warfarin-azapropazone site of HSA, and cis/trans-clomiphene gave positive allosteric effects caused by the binding of warfarin to HSA. Digitoxin and acetyldigitoxin were found to bind to a common, unique region on HSA; cis- and trans-clomiphene also appeared to interact at a unique site, although trans-clomiphene displayed additional direct competition with phenol red. From these results it was possible to develop a model that described the general relationship between these binding regions on HSA. This information should be useful in future studies that employ HPAC for characterizing the binding of HSA to other drugs or clinical agents.