Affinity Labeling of Steroid Binding Sites

Abstract

Abstract To extend the application of affinity labeling for the characterization of macromolecular steroid binding sites, we synthesized two cortisone derivatives (cortisone 21-iodoacetate and 21-iododeoxycortisone) and studied their reactions with the use of 20β-hydroxysteroid dehydrogenase from Streptomyces hydrogenans in 0.05 m phosphate buffer, pH 7.0, as the model protein. Both derivatives are capable of the reversible binding step at the active site, as both serve as substrates. Cortisone 21-iodoacetate inactivates the enzyme in a time-dependent and irreversible manner, cortisone slows the rate of this inactivation, and excess 2-mercaptoethanol stops it. Iodoacetic acid did not inactivate the enzyme (with or without added cortisone), nor did 21-iododeoxycortisone. Nevertheless, both steroid derivatives easily react with model sulfhydryl compounds. Radioactive cortisone 21-iodoacetate was then synthesized with 2-3H-iodoacetic acid. Inactivation of the enzyme by this steroid was accompanied by radiolabeling; neither was seen with 2-3H-iodoacetic acid alone. After inactivation and acid hydrolysis, a single major fraction of radioactivity was seen on paper chromatography. Amino acid analysis of the hydrolysate revealed a major radioactive peak (containing 86% of total radioactivity) with mobility identical with standard 1,3-dicarboxymethyl histidine and a minor peak (containing 5% of total radioactivity) with mobility identical with standard 3-carboxymethyl histidine. These observations are compatible with a mechanism whereby the steroid moiety of cortisone 21-iodoacetate delivers the reagent group to the binding site of 20β-hydroxysteroid dehydrogenase, where it reacts with a histidine residue at that site. These derivatives are offered as compounds which may possibly be used to study certain steroid binding sites of high affinity present in receptor proteins of target organs.