A Novel Affinity Column for Isolation of Androgen Binding Protein from Rat Epididymis

Abstract

An androgen affinity column was synthesized by covalently linking 3-oxo-17β-hydroxy-5α-androstan-17α-(6-hexanoic acid) to cyanogen bromide activated Sepharose through a dipropyldiamine side arm. This column was designed to recover androphilic proteins from homogenates rich in nonspecific esterases. An extract of rat epididymis was adsorbed on the affinity column after partial purification by ammonium sulfate precipitation. The column was washed with 1 M KCl and the androgen binding protein eluted with 17β-hydroxy-5α-androstan-3-one resulting in a 1,100-fold increase in specific activity. This protein had the same mobility on polyacrylamide gels and the same estimated molecular weight (135,000 daltons by gel filtration) as androgen binding protein in the original extract. By contrast, electrophoresis on sodium dodecyl sulfate containing gels yielded 2 bands with estimated molecular weights of 42,000 and 47,000 daltons. These observations are consistent with a subunit structure for rat epididymal androgen binding protein.