Analysis of Hormone-Protein Binding in Solution by Ultrafast Affinity Extraction: Interactions of Testosterone with Human Serum Albumin and Sex Hormone Binding Globulin

Ultrafast affinity extraction was used to study hormone-protein interactions in solution, using testosterone and its transport proteins human serum albumin (HSA) and sex hormone binding globulin (SHBG) as models. Both single column and two-dimensional systems based on HSA microcolumns were utilized to measure the free fraction of testosterone in hormone/protein mixtures at equilibrium or that were allowed to dissociate for various lengths of time. These data were used to determine the association equilibrium constants (K_a) or global affinities (nK_a′) and dissociation rate constants (k_d) for testosterone with soluble HSA and SHBG. This method was also used to measure simultaneously the free fraction of testosterone and its equilibrium constants with both these proteins in physiological mixtures of these agents. The k_d and K_a values obtained for HSA were 2.1-2.2 s^-1 and 3.2-3.5 × 10⁴ M^-1 at pH 7.4 and 37°C. The corresponding constants for SHBG were 0.053-0.058 s^-1 and 0.7-1.2 × 10⁹ M^-1. All of these results gave good agreement with literature values, indicating that this approach could provide information on a wide range of rate constants and binding strengths for hormone-protein interactions in solution and at clinically relevant concentrations. The same method could be extended to alternative hormone-protein systems or other solutes and binding agents.