Isolation of cortisol binding globulin (CBG) from porcine follicular fluid by affinity chromatography

Abstract

To identify further the cortisol binding macromolecule of the follicular fluid of the pig the cortisol binding component was isolated by affinity column chromatography. [ 3H]-Cortisol hemisuccinate was synthesized and attached to AH-sepharose-4B, which was tested for its binding of cortisol binding globulin (CBG) by using [ 125I]-hCBG and by determining the moles of cortisol/ml of gel bound. The indigenous steroids in porcine follicular fluid were removed by charcoal which was then treated with ammonium sulfate. Following centrifugation the supernatant was dialyzed and applied to the affinity column. After washing the column extensively, the column was eluted with PBS buffer solution (0.001 M phosphate buffer pH 6.5 + 0.1 M NaCl) which contained 200 μg cortisol per ml. The cortisol binding protein was eluted, dialyzed and characterized. Porcine blood serum was also analyzed in similar manner as follicular fluid. The cortisol binding component isolated by affinity column chromatography from both the follicular fluid and serum gave a single protein-staining band in polyacrylamide gel electrophoresis, and its mobility was the same as hCBG and [ 125I]-hCBG. This fraction bound cortisol specifically with an association constant of 1.96 × 10 9 M −1 and a binding capacity of 1.2 × 10 8M −1 per liter. It is concluded that the cortisol binding protein of the porcine ovarian follicular fluid is the same or very similar to CBG.