Steroid-Protein Interactions: XVI. Isolation and Characterization of the Corticosteroid-Binding Globulin of The Rabbit

Abstract

Abstract Comparative determination of high affinity binding activity for cortisol and corticosterone in the sera of various species by equilibrium dialysis indicated considerable differences, which were independent of the ratio of cortisol to corticosterone present in the blood plasma. Corticosteroid-binding globulin (CBG) was isolated from pooled rabbit serum by chromatography on diethylaminoethyl Sephadex, gel filtration over Sephadex G-200, and hydroxylapatite chromatography. Ammonium sulfate fractionation was found useful as a preliminary purification step. A quantity of 13.5 mg of pure CBG was obtained from 2 liters of serum, with an over-all 6200-fold purification and 50 to 60% yield. The protein was homogeneous by free boundary and paper electrophoresis, sedimentation velocity at different concentrations, and diffusion. Immunoelectrophoretic analysis with antiserum to rabbit serum indicated a pure α-globulin; no precipitation was obtained with antisera to human serum, to rat serum, or to guinea pig serum. Amino acid and carbohydrate composition showed a general similarity to human CBG, in spite of distinct differences in certain residues. The extrapolated sedimentation coefficient was found to be s020,w = 3.55 S; diffusion coefficient, D20,w = 7.02 x 10-7 cm2 sec-1; electrophoretic mobility at pH 8.6, Γ/2 = 5.1 x 10-5 cm2 volt-1 sec-1; extinction coefficient at 279 mµ, E1%1 cm = 8.4; partial specific volume, v = 0.695 ml per g; and frictional ratio, f/f0 = 1.37. A molecular weight of 40,700 was calculated from sedimentation velocity and diffusion data. The pure CBG, isolated under conditions of saturation with endogenous corticosterone and added cortisol-4-14C, contained 1 mole of corticosteroid per 40,700 g of CBG; the number of high affinity binding sites was determined to be n = 1. The association constants at 4° and 37° were k = 9.0 x 108 m-1 and 4.7 x 107 m-1, respectively. Thermodynamic calculations gave negative enthalpy change and negative entropy change for the interaction. Removal of corticosteroid from the isolated pure CBG by gel filtration at 45° resulted in loss of binding activity.