Some practical aspects of the separation of estrogen and progesterone receptors by size exclusion high performance liquid chromatography which are relevant to quantitation

Estrogen and progesterone receptors, prepared from rodent and human tissues, have been separated on a size exclusion column (TSK-G3000SW) using a high performance liquid Chromatograph. Reproducible, minor differences in elution behaviour between various preparations of human estrogen and progesterone receptors were often observed. The trailing edges of receptor profiles obtained by size exclusion HPLC characteristically broadened the elution of individual peaks so that overlap between peaks often occurred. This broadening reduces the feasibility of automated receptor analysis based upon the pre-programmed collection of select peaks. Nonetheless, automated receptor analysis, based upon the collection of all eluant fractions remains practical. Quantitative estimates of estrogen and progesterone receptor concentration were made using the size exclusion HPLC method and compared to estimates obtained with the hydroxylapatite adsorption assay, the dextran-coated charcoal assay and sucrose gradient sedimentation separation. Size exclusion HPLC estimates of rodent and human receptor activity were in agreement with estimates obtained by the other assays. Moreover, receptor estimates obtained by size exclusion HPLC were generally greater than estimates obtained by methodologies where significant ligand dissociation can occur during the application of the assay (i.e. hydroxylapatite adsorption and sucrose gradient sedimentation). Thus, in addition to improved qualitative receptor analysis, the size exclusion HPLC methodology provides receptor quantitiation which is compatible with other techniques.