Separation of two molecular forms of human estrogen receptor by hydrophobic interaction chromatography. Gradient...

High-performance hydrophobic interaction chromatography (HPHIC) was used to separate and characterize two molecular forms of estrogen receptor with a SynChropak propyl hydrophobic column (300 A pore size). The linear gradient utilized earlier with a polyether-bonded column (2 to 0 M) ammonium sulfate in 40 min, gave poor resolution with the propyl column. However, resolution was maximized with either an initial ammonium sulfate concentration of 1 M (40-min gradient) or with a two-phase gradient (2 to 0.5 M in 10 min, 0.5 to 0 M in 30 min). This indicated that the propyl column was more hydrophobic than the polyether column. Estrogen receptor separated into two isoforms, either in the presence [MI, retention time (tR) = 13-14 min; MII, tR = 20-21 min] or absence (I, tR = 21-23 min; II, tR = 31-33 min) of the estrogen receptor stabilizing reagent, sodium molybdate. Similar isoforms were observed in cytosols from human breast tumors, uterus, and MCF-7 breast cancer cells. Unlike others, MCF-7 estrogen receptor did not show MI. Since MCF-7 cells contain 90,000 dalton heat shock proteins (HSP-90), HSP-90 is probably not directly involved in MI formation. Sodium molybdate selectively interacted with isoform II and converted it to MI. All isoforms appeared to be high-molecular-weight proteins (greater than 60 A) when subsequently analyzed by high-performance size-exclusion chromatography. Interestingly, when estrogen receptor was immobilized on the stationary phase, no change was detected in either hydrophobicity or steroid-binding capacity. After 16-18 h, immobilized receptor was eluted with a slightly longer tR. During incubation on the column, component MI was converted into I and/or II. HPHIC appears to be a rapid, yet gentle procedure for isolating large receptor complexes in significant quantities with high recoveries. This allows one to discern the complicated structure-function relationships of estrogen receptor and associated non-receptor proteins and provides information about the on-column behavior of complex proteins.