Affinity Chromatography and the Purification of Estrogen Receptors (1973)

Abstract The preparation, testing, and use of a variety of estradiol-containing agarose supports for affinity chromatography of estrogen receptors from crude extracts of calf uterus are described. Before use the gels must be washed exhaustively with organic solvents over prolonged periods of time. The adequacy of the washing procedures must be tested by examining the effluents of chromatographed uterine extracts for free estradiol as well as for soluble estradiol-receptor complexes. Even complete removal of the non-covalently adsorbed hormone may not safeguard against the release of hormone during the purification procedures, since certain estradiol-containing adsorbents possess chemical linkages which are susceptible to accelerated cleavage by reducing substances and nucleophiles present in the crude uterine extracts. The release of hormone from the gel into the medium does not necessarily exclude effective adsorption of receptors onto the adsorbent; whether this occurs will depend on the relative affinities and concentrations of the gel-bound and free hormone molecules. Receptor molecules specifically extracted by affinity adsorbents can be eluted from the gels in high yield by incubating the gel, in suspension, with estradiol-containing buffers at 30°; the conditions utilized enhance the rate of dissociation of gel-bound receptors and favor an effective exchange of these receptors with the soluble estradiol molecules. Various agarose derivatives containing estradiol covalently bound through linkages with the A ring of the steroid molecules are ineffective as adsorbents for receptors. Similar kinds of adsorbents prepared on glass and polyacrylamide supports proved to be inferior to those which utilize agarose. Although agarose derivatives containing 19-nortestosterone 17-hemisuccinate can selectively bind estradiol receptors, the capacity of these columns is low. The most effective affinity adsorbents were prepared by attaching 17β-estradiol 17-hemisuccinate to agarose derivatives containing albumin or the branched copolymer of poly(l-lysine) (backbone) and poly(dl-alanine) (side arms). These macromolecular leashes are presumably bound to the agarose matrix by multipoint attachments, they possess numerous functional groups which permit a high degree of ligand substitution, and the attached ligand is very distant from the agarose backbone. Some of these adsorbents are so effective that significant extraction of receptors occurs even when the gels are diluted 100-fold with unsubstituted agarose. Adsorbents diluted in this way can be used routinely in purification procedures, since selective adsorption of receptors in excess of 90% can be achieved. Furthermore, the complications of washing the gels are diminished, nonspecific adsorption of non-receptor proteins is minimal, and elution of the adsorbed receptors is facilitated. Uterine estradiol receptors (4.5 S) from crude uterine supernatants can be purified between 10,000- and 100,000-fold in over-all yields of 30 to 50% in a single step. The purified receptors are identical with those described earlier by molecular size (gel filtration), sedimentation coefficient, isoelectric focusing, and heat stability.