Steroid-protein interactions. XV. Isolation and characterization of corticosteroid-binding globulin from human plasma.

Corticosteroid-binding globulin was isolated from normal human plasma by successive anion exchange chromatography and gel filtration. Ammonium sulfate fractionation was employed as a preliminary step when large volumes were processed. The purification was about 4000-fold. Homogeneity was shown by free boundary, agar and paper electrophoresis, sedimentation velocity, and diffusion. Immunoelectrophoretic behavior against antisera to human serum and serum fractions indicated purity. Other immunoelectrophoretic analyses showed lack of reaction with antisera produced against sera from several species other than human. The molecular weight of the protein was calculated as 51,700 from sedimentation and di&lsion data. Chemical composition was ascertained by amino acid and carbohydrate analyses, nitrogen determination, and biuret. The physicochemical properties described include sedimentation coefficient (&, y = 3.79 S), diffusion coefficient (Daa,W = 6.15 x lo-’ cm2 se@), electrophoretic mobility (u = 4.9 X lo-& cm2 volt-l se&), extinction coefficient (Ei”,, = 6.45), partial specific volume (v = 0.708 ml g-l), and frictional ratio (j/f,, = 1.42). One binding site for cortisol was found by equilibrium dialysis at 4’ and at 37”; the association constants at these temperatures were 5.2 X lo* M-I and 2.4 x 10’ WI-~, respectively. Complete removal of sialic acid did not significantly affect the cortisol-binding affinity. Pure corticosteroid-binding globulin was inactivated by gel tiltration at 45’ or by dialysis against glass-redistilled water.