Fluorescence Polarization Measurement of the Hormone-Binding Site Interaction

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Abstract
Fluorescence polarization methodology was applied to the binding of fluorescent-labeled prolactin, growth hormone and estradiol to subcellular fractions prepared from rabbit mammary and uterine tissue. Equilibrium measurements treated by Scatchard plots have shown that there are high affinity sites (K .apprx. 109 l mol-1) and lower affinity sites (K .apprx. 108 l mol-1) for both hormones. The binding of the fluorescent labeled hormone to microsomal or cytosol fractions was inhibited by the prior addition of native, unlabeled hormone. Kinetic results on the interaction of prolactin with the microsomal fraction are consistent with a bimolecular reaction involving significant structural rearrangements during the reaction (not diffusion controlled). The forward rate constant calculated from data on initial rates was 1.7 .times. 105 l mol-1 s-1. Stopped flow kinetic measurements on the reaction between fluorescent-labeled estradiol and cytosol binding sites show that at low temperatures, the reaction goes in 2 distinct steps separable in time. The 2nd step may be the reaction found by others (utilizing sedimentation velocity methods) which precedes translocation of the hormone-binding site complex to the nucleus. Fluorescence polarization makes it possible to observe both the formation and dissociation of hormone-binding site complexes over a time scale down to a fraction of a second and at concentrations down to the nanogram per ml range.