Several affinity chromatography
reagents have been proposed for purification of
progesterone receptor (PgR), and significant results have been achieved with some of these. None, however, have approached the results achieved in affinity chromatography of
estrogen receptor. We have therefore synthesized a number of new
19-nortestosterone derivatives capable of chemically stable linkage with
Sepharose beads, and have identified one with very high PgR affinity for further study. We first synthesized the
epoxides of 17 alpha-allyl
nortestosterone, by analogy with the
estradiol derivatization of Greene and Jensen. The relative affinity of these
epoxides for PgR from T47D human
breast cancer cells, however, was only around 5% that of
R5020, and affinity beads prepared from them bound very little PgR. We then reacted appropriately protected 17 alpha-ethynyl-
nortestosterone with a series of diiodo
alkanes, and found that 17 alpha-(6'-iodohex-1'-ynyl)nortestosterone had an affinity of 22% relative to
R5020, equal to the affinity of
progesterone itself. Reaction with
Thiopropyl-Sepharose 6B yielded hexynyl-
nortestosterone-
Sepharose beads with a
ligand density of about 7 micromoles/ml beads. One-hundred microliter of these beads adsorbed 71% of the PgR present in 1 ml of cytosol from T47D cells. This adsorption was inhibited by 10 microM
progesterone but not
cortisol, indicating the specificity of the binding. Comparisons with NADAC and Sterogel, other affinity beads used for PgR purification, show that the former takes up much less receptor, while the latter takes up and releases similar amounts of receptor but more extraneous
protein, and is less stable. We therefore believe that hexynyl-
nortestosterone-
Sepharose, having a high density of a high affinity
ligand, and having chemically and biochemically stable covalent bonds, should be a good
reagent for affinity purification of PgR.