Photoaffinity labeling of extracellular and intracellular androgen binding proteins

A method is described for the photoaffinity labeling of human and rabbit serum sex hormone binding globulin (SHBG) in ammonium sulphate precipitates utilizing 3H-delta 6-testosterone as affinity label. The precipitation step diminished albumin contamination and at the same time concentrated the binding globulin. Photolysis was conveniently carried out with an electronic flash. Unbound and non-covalently bound steroids were adsorbed by prolonged dextran-coated charcoal treatment. Sephadex G-200 gel filtration column chromatography showed a single peak of covalently bound radioactivity with the elution volume of SHBG. With some modifications, the method was applied to the affinity labeling of the prostate androgen receptor utilizing 3H-methyltrienolone (R 1881) as affinity label. The receptor was also precipitated from prostate cytosol with ammonium sulphate. After labeling, photolysis and heating at 50 degrees C, non covalently bound 3H-R 1881 was removed by dextran coated charcoal treatment. Sephadex G-25 microcolumn chromatography after heating showed a peak of radioactivity eluting with macromolecules if photolysis had been carried out, while it disappeared in the absence of previous photolysis. However, the photolabeled receptor had a sedimentation coefficient different from the non-irradiated receptor, suggesting that photolysis induced a change in the configuration of the complex.