Simultaneous identification of estrogen and progesterone receptors by HPLC using a double isotope assay

Abstract

Polymorphism of estrogen (ER) and progestin receptors (PR) was analyzed simultaneously using high performance hydrophobic interaction chromatography (HPHIC). HPHIC was used previously to characterize four ER isoforms [Hyder et al., J. Chromat. 397 (1987) 251] based on retention times on Synchropak propyl (100 × 6 mm) HPCL columns (Synchrom, Inc.). ER and PR were prepared from human breast cancer. ER was labeled with 3 nM of either [ 3H]estradiol-17β ([ 3H]E) or [ 125I]iodoestradiol-17β ([ 125I]E) while PR was associated with 5 nM of either [ 3H]R5020 ([ 3H]R) or [ 125I]iodovinylnortestosterone ([ 125I]V). ER was resolved by HPHIC into isoforms MI ( R t = 11 min), I( R t = 16 min), and II ( R t = 24 min. Isoforms I and II each accounted for ca 45% of specific binding. PR saparated into isoforms MI ( R t = 14 min) and I ( R t = 21 min, 80% of specific binding) when eluted with the same gradient used for ER chromatography. Upon inclusion of 10 mM molybdate ER resolved into isoforms MI and MII ( R t = 16 min) and PR into isoforms MI and I (here however isoform MI represented 80–95% of specific binding). Elution patterns were preserved with different batches of stationary phase suggesting the integrity of the isoform distribution. HPLC profiles of ER isoforms labeled with earlier [ 125I]E or [ 3H]E were identical as were PR isoform profiles labeled with either [ 3H]R or [ 125I]V. Pairs of 125I- and 3H-labeled ligands were used in either combination to monitor ER and PR profiles simultaneously. Isoforms analyzed in 50 biopsies gave reproducible retention times, however the ratio between I and II for ER and MI and I for PR varied. This method allows rapid, simultaneous monitoring of the chromatographic behavior of ER and PR isoforms or other associating proteins or nucleotides. One may now better elucidate their interrelationship as it relates to the hormone-response mechanism.