ISOLATION, CHARACTERIZATION AND RADIOIMMUNOASSAY OF CORTICOSTEROID-BINDING GLOBULIN (CBG) IN HUMAN SERUM – CLINICAL SIGNIFICANCE AND COMPARISON TO THYROXINE-BINDING GLOBULIN (TBG)

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Abstract
Isolation of the corticosteroid-binding globulin [CBG] was achieved by 5 chromatographical steps on cortisol Sepharose. QAE-Sephadex A-50, Con [concanavalin] A-Sepharose and hydroxylapatite. The purity of the isolated CBG was demonstrated in polyacrylamide gel electrophoresis, sodium dodecyl sulfate electrophoresis, immunodiffusion and ultracentrifugation. Microheterogeneity was shown in isoelectric focusing by 5 bands in the pH range of 3.7-4.2, which could be reduced to 1 major band after neuraminidase treatment. The equimolar binding of cortisol to CBG was demonstrated by binding studies. The association constant for cortisol was 2.8 .times. 108 M-1, for progesterone 1.7 .times. 106 M-1. From analytical ultracentrifugation, the MW was calculated on 50,700; the sedimentation coefficient was 3.6 S, the partial specific volume 0.690 ml/g, the Stokes radius 38 .ANG. and the frictional coefficient ratio 1.5. A specific radioimmunoassay for CBG was established using the purified CBG for immunization, radioiodination and for calibration standards. The normal range of CBG levels in human serum was 2.4-4.4 mg/100 ml (mean .+-. 2 SD). Studies were performed to compare the levels of CBG and thyroxine-binding globulin (TBG). No sex differences but a significant biphasic age dependence were observed for both proteins. In pregnancy and under estrogen treatment of women and men, CBG was demonstrated to be the more distinct indicator of estrogenic activity as compared with TBG, whereas the sensitivity of TBG was more pronounced to supposedly antiestrogenic substances like Danazol, and in severe disease. No coincidence of genetic CBG and TBG deficiencies have been found so far.