Determination of association constants between albumins and steroid hormones by affinity capillary electrophoresis

Albumin, the dominating serum protein, interacts with a wide variety of lipophilic compounds such as drugs, fatty acids and steroid hormones. The affinity for steroids is low, and the dissociation constant, Kd, varies between 10-6 and 10-4 M. However, despite albumin's low affinity for steroids, its high concentration in serum enables it to bind a substantial part of steroids. Generally, in plasma, approximately 96% of steroids are bound to albumin and globulin proteins, while only 4% remains in the free form. Albumin regulates the access of steroids to their receptors, and the association with serum proteins suppresses the biological activity of steroids. Hence the distribution of steroids between plasma components is of great interest. In this study, evaluation of association constants between human and bovine serum albumin (HSA and BSA) and some endogenous steroids is presented. The applied method is affinity capillary electrophoresis (ACE), in which a protein selector is added to the background electrolyte. The analyses are performed in an electric field and the migration of the steroids in response to the molar amount of protein is monitored. This is the first report of evaluation of association constants between neutral endogenous steroids (androstenedione, epitestosterone and testosterone) and BSA and HSA by ACE. Notable differences in the association constants were observed.