Steroid interactions with progesterone-binding globulin

Abstract

The progesterone-binding globulin (PBG) of the pregnant guinea pig has been purified on sulfopropyl Sephadex and subsequent affinitychroma tography on 19-nortestosterone-Sepharose. A pure glycoprotein of molecular weight 88,000 was obtained that contained about 70% carbohydrate. PBG is polydisperse and has a polypeptide core of about 27,000 daltons. It has one binding site for progesterone ( K a 37° = 4 × 10 8 M −1). Studies on chemical modification of PBG show the involvement of tryptophan, lysine, and tyrosine in steroid binding. In typical reactions, the binding sites were inactivated by the chemical substitutions; complex formation with progesterone protected the binding site from the modification. Association of progesterone or other steroids with PBG results in conformational changes as seen from circular dichroism and ultraviolet difference spectra. The intrinsic tryptophan fluorescence of PBG is quenched by more than 80% when one mol progesterone is bound. This is the basis for a fluorescence quenching titration method to determine binding site concentrations and association constants. Association and dissociation rate constants were measured by stopped-flow fluorescence techniques; it was found that the affinity of the steroid-PBG complexes is controlled by the rate of dissociation. Steroid complexes of high-affinity binders from serum have generally higher dissociation rates than those of cellular receptor proteins. This is in harmony with the physiological functions of the two types of steroid-binding proteins.