Criteria for Affinity Chromatography of Steroid-binding Macromolecules

When soluble fractions of rat kidney homogenates were exposed to affinity chromatography columns containing deoxycorticosterone covalently linked to an insoluble agarose matrix, aldosterone-binding activity was reduced by 92%. Similarly, when renal homogenates were exposed to affinity chromatography columns containing estriol linked to agarose, renal estradiol-binding activity was reduced by 93%. Various attempts to recover macromolecules capable of binding steroid from columns of these agarose derivatives previously exposed to cytosol have failed. Moreover, the use of competitive protein-binding techniques to examine 0.3 m KCl or renal homogenate eluted from these columns suggests that most of the reduction in steroid-binding activity which occurred when cytosol was exposed to deoxycorticosterone-agarose or estriol-agarose can be accounted for by the solubilization of steroid from the agarose by the action of cytosol on the steroid-agarose complex. The rate of release of free steroid from the agarose matrix into the eluate was increased between 5- and 7-fold when cytosol rather than 0.3 m KCl was added to the agarose derivatives. The competitive protein-binding technique provides a test for this artifact in the form of a new criterion to be examined when affinity chromatography is used with steroid-binding macromolecules or receptors.