Steroid Binding in Polyacrylamide Gels: QUANTITATION AT STEADY STATE CONDITIONS

Abstract

Abstract A method which permits quantitation of steroid-binding proteins and determination of binding constants at state binding conditions is described. This method, which is called steady state polyacrylamide gel has been used to demonstrate and quantitate binding of radioactive steroid hormones to serum testosterone-binding globulin, corticosteroid-binding globulin, albumin, and testicular androgen-binding protein. Steady state gel electrophoresis may be used to study binding of any noncharged molecule. Radioactive steroid is dissolved in the acrylamide solution before polymerization. When the sample migrates into the gel during electrophoresis, steroid is bound by the individual proteins. Gels are sliced and the radioactivity is extracted from each slice and measured. The law of mass action can be applied when the rate of association equals the rate of dissociation. This permits determination of total number of binding sites, and dissociation constant (Kd) for the binding between the protein and the steroid. The presence of several binding components in the same sample can be demonstrated and quantitated in one state electrophoresis, under identical conditions. The precision is satisfactory in the range of 0.01 to 10 pmoles per sample (coefficient of variation l10 %), while the sensitivity is dependent on the concentration of steroid in the gel. If the rate of dissociation of radioactive steroid from the protein is slow, as for the binding of [3H]dihydrotestosterone to the testosterone-binding globulin of human and monkey serum (t1/2 at 0° g 60 min), accurate quantitation can also be performed by saturation of the protein by radioactive ligand before electrophoresis. Using state gel electrophoresis of nonlabeled samples and samples saturated with labeled steroid, concentrations of testicular androgen-binding protein in rat and rabbit, as well as concentration of rabbit, monkey, and human serum testosterone-binding globulin have been measured.