High-performance chromatofocusing and size-exclusion chromatography: separation of human uterine estrogen-binding...

We have employed high-performance chromatofocusing (HPCF) and high-performance size exclusion chromatography (HPSEC) to separate and identify radiolabelled estrogen binding proteins present in human uterine cytosol. Results obtained using these high-performance methods are compared to results of similar analyses by conventional isoelectric focusing procedures and open-column size exclusion chromatography. By HPCF, descending pH gradients (pH 8-4) allow us to discern four to five estrogen binding proteins with elution pH values typically between pH 4.5 and 7.2. However, when using HPCF, significant quantities of estrogen binding proteins are rarely detected between pH 7.2 and 8.0. This observation has been confirmed by open-column chromatofocusing of these proteins on Polybuffer Exchanger 94. In contrast, preparative isoelectric focusing by electrophoresis in polyampholytes reveals substantial quantities of estrogen binding activity eluted between pH 7.5 and 8.0. Several possible explanations for this disparity are discussed. Apparent differences are also observed when the size heterogeneity of estrogen binding proteins is analyzed simultaneously by size exclusion chromatography in open-column (Sephacryl S-300) and high-performance (TSK-3000SW) modes. HPSEC of these estrogen binding proteins on TSK-3000SW columns demonstrates a predominant 80-85 A species, whereas size exclusion chromatography on conventional Sephacryl S-300 columns reveals two to three distinct regions of estrogen binding proteins with Stokes radii of ca. 85, 60 and 30 A (major species). The larger form of receptor, whether a non-specific aggregate or a multisubunit complex, is stable in unfractionated cytosol and becomes more labile only during size exclusion chromatography.