Steroid-Protein Interactions: XXVI. STUDIES ON THE POLYMERIC NATURE OF THE CORTICOSTEROID-BINDING GLOBULIN OF THE RABBIT

Abstract

Abstract The rabbit corticosteroid-binding globulin (CBG), fully saturated with corticosteroid, was separated by Sephadex G-200 filtration into a corticoid-binding monomer and a steroid-free tetramer. Polymerization could be enhanced by removal of the steroid from CBG or by prolonged storage. Partial depolymerization and reactivation of the inactive tetramer was obtained by incubation with a relatively large quantity of cortisol; the restored binding affinity was associated with the monomeric species. Equilibrium ultracentrifugation according to Yphantis indicated paucidispersity of the CBG; the predominant component had a molecular weight of 34,700 ± 1,200. The approach to sedimentation equilibrium technique of Archibald gave a weight-average molecular weight of 61,400 ± 1,600, suggesting a mixture of approximately 75% monomer and 25% tetramer. A single broad band was observed in sodium dodecyl sulfate electrophoresis; two closely adjacent bands resulted when mercaptoethanol treatment was included. The corresponding apparent molecular weight, based on calibration with carbohydrate-free proteins was approximately 40% greater than that obtained by the Yphantis method. Similar deviations occurred with α1-acid glycoprotein and ovomucoid. We attribute these deviations to abnormal behavior of glycoproteins in the polyacrylamide electrophoresis. The results are compatible with a molecular weight of approximately 35,000 for the monomeric rabbit CBG.