FLUORESCENCE INVESTIGATION OF THE SEX STEROID BINDING-PROTEIN OF RABBIT SERUM - STEROID BINDING AND SUBUNIT DISSOCIATION

The relationship between steroid binding and protein subunit interactions of rabbit sex steroid binding protein (rSBP) has been studied by steady-state and time-resolved fluorescence spectroscopy. The high-affinity (Ka ~ 108 M−1 at 4 °C), fluorescent estrogen d-1,3,5(10),6,8-estrapentaene-3,17β-diol [dihydroequilenin (DHE)] was used as a fluorescent probe of the steroid-binding site. Perturbation of the binding site with guanidinium chloride (Gdm-Cl) was monitored by changes in the steady-state fluorescence anisotropy of DHE as well as by changes in fluorescence quenching of DHE with acrylamide. The results of acrylamide quenching at 11 °C show that, while between 0 and 1 M Gdm·Cl the steroid-binding site is completely shielded from bulk solvent, there is decreased DHE binding. To study the subunit-subunit interactions, rSBP was covalently labeled with dansyl chloride in the presence of saturating 5α-dihydrotestosterone (DHT), which yielded a dansyl-conjugated protein that retained full steroid-binding activity. The protein subunit perturbation was monitored by changes in the steady-state fluorescence anisotropy of the dansyl group. At 11 °C, the dansyl anisotropy perturbation, reflecting changes in global and segmental motions of the dimer protein, occurs at concentrations of Gdm-Cl above 1 M. The Gdm·Cl titration in the presence of steroids with equilibrium association constants less than 108 M−1 shows a plateau near 3 M Gdm·Cl at 11 °C; at this Gdm·Cl concentration, no DHE is bound. No plateau is observed at 21 °C. At higher Gdm·Cl concentrations, the dansyl fluorescence anisotropy decreases further and shows no steroid dependence. Recovery of steroid-binding activity (assayed by saturation binding with [3H]DHT), under renaturation conditions, is dependent on both steroid concentration and affinity. Both unlabeled and dansyl-labeled protein recovery the same amount of activity, and according to fluorescence anisotropy, dansyl-labeled rSBP re-forms a dimer upon dilution below 1 M or removal of Gdm·Cl. From the steroid requirement for recovery of steroid-binding activity, it appears that a conformational template is required for the dimeric protein to re-form a steroid-binding site with native-like properties. © 1990, American Chemical Society. All rights reserved.