Improved stokes radius measurement of the glucocorticoid receptor using TSK G4000SW and TSK G3000SW high-performance size-exclusion columns : Analytical and preparative applications

Abstract

The Stokes radius of the rat liver glucocorticoid receptor was determined using TSK G3000SW and TSK G4000SW high-performance size-exclusion columns. The accuracy of the calibration graph for proteins larger than 6 nm on the TSK G4000SW column allowed the resolution of a heterogeneous structure for the cytosolic untransformed receptor, giving two forms with R s values of 8.3 and 7.1 nm, whereas the transformed receptor elutes with an R s value of 4.7–5.3 nm. The 8.3 nm form was not observed for the highly purified untransformed receptor. Parallel analyses of the cytosolic untransformed receptor on conventional gravity-fed Bio-Gel A 1.5-m or Ultrogel AcA-22 size-exclusion columns could not resolve two components. The resolution efficiencies of high-performance size-exclusion chromatography and open-column size-exclusion chromatography were compared. Further, owing to its rapidity, high-performance chromatography allowed the characterization of steroid-receptor complexes having half-lives as short as 90 min and very unstable receptor forms could be detected. Specific applications are considered, such as the resort to a small TSK GSWP guard column for the rapid separation of affinity-purified [ 3H]TA-receptor complexes from free eluting steroid, and to a preparative TSK G4000SW column for the fractionation of significant amounts of the two untransformed receptor forms.