Fluorescein-labeled estradiol: a probe for anti-estradiol antibody.

A fluorescent estradiol derivative binds strongly to antiestradiol antibody. The binding, measured by fluorescence polarization, is inhibited by estradiol and by diethylstilbestrol. Tentatively characterized as N-(estradiol-6-iminooxyacetyl) fluorescein amine, the derivative was prepared from estradiol-6-iminooxyacetic acid, dicyclohexylcarbodiimide, and fluorescein amine, and isolated by TLC. It has a fluorescence emission similar to that of fluorescein and an absorption spectrum consistent with a fluorescein: estradiol molar ratio of 1:1.

Fluorescence polarization measurement of the hormone-binding site interaction.

Fluorescence polarization methodology has been 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 approximately 10(9) 1 mol-1), as well as lower affinity sites (K approximately 10(8) 1 mol-1) for both hormones. The binding of the fluorescent labeled hormone to microsomal or cytosol fractions has been shown to be 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 found to be 1.7 X 10(5) 1 mol-1 sec-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 two distinct steps separable in time. The second 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 nl range.

Hormone binding by cells and cell fragments as visualized by fluorescence microscopy.

Fluorescent-labeled hormones offer an alternative approach to radio-labeling in studying the binding of hormones to intact cells or cell fragments. The binding of fluorescent-labeled hormones may be followed quantitatively by measurement of the polarization or the binding may be directly visualized in the fluorescence microscope. The binding of both fluorescein labeled prolactin and estradiol to a variety of whole cells or to microsomal fragments has been observed by fluorescence microscopy. No staining was observed with fresh cells whereas all cell types investigated, after freeze-thawing, stained at physiological levels (10-9M) of either hormone. Microsomal preparations from the mammary tissue of mid-pregnant rabbits likewise stained at low levels of prolactin. Inhibition of staining was not produced even by 10-6 M unlabeled hormone.

Fluorescence polarization and intensity kinetic studies of antifluorescein antibody obtained at different stages of the immune response.

Kinetic studies of reactions between fluorescein and antifluorescein antibody produced during early, intermediate, and late stages of the immune response have been carried out utilizing both fluorescence intensity and polarization measurements in the static (time constant similar to 5 sec) and in the stopped-flow modes (time constant similar to 5 msec). During maturation of the immune response, it was found that the "on" second-order association rate constant increased its value only by a factor of three, whereas the "off" dissociation first-order rate constant decreased by a factor of over 1000. Hence, it is the rate of dissociation which largely determines the stability of the hapten-antihapten complex. Furthermore, since second-order rate behavior was found for even heterogeneous antibody, most of the heterogeneity with respect to binding affinity occurs as a result of the heterogeneity in the rate of dissociation of the hapten-antihapten complex and not from the primary combination of hapten and antibody. Antifluorescein antibody which exhibits both high binding affinity (K similar to 5 x 10(11) M-1) and homogeneity with respect to equilibrium binding has been shown to obey second-order association kinetics over wide ranges in concentration. Despite the fact that the value of the second-order rate constant for this fluorescein-antifluorescein reaction is as large as that for most other hapten-antihapten reactions (1.4 x 10(8) M-1 sec-1), the binding reaction has an appreciable activation energy (7 kcal/mol). This is true for both divalent and univalent antibody. Furthermore, the reaction rate parameters are markedly affected by specific anions. The value of the second-order rate constant (18.5 degrees) increases according to the following scheme: salicylate less than trichloroacetate less than SCN- less than ClO4- less than Cl- less than F- less than phosphate. The activation energy increases as follows: trichloroacetate less than phosphate less than F- less than Cl- less than ClO4- less than SCN- less than salicylate, whereas estimates of the entropy of activation indicate that deltaS++ increases as follows: tricholroacetate less than phosphate similar to F- less than Cl- less than ClO4- less than SCN- less than salicylate. The same mechanism which was previously proposed by us for the antigen-antibody reaction is also consistent with the kinetics of the fluorescein-antifluorescein reaction. This mechanism postulates a bimolecular process with structural rearrangements (conformational changes and/or the loss of water) in the formation of the transition state complex. The reaction between the fluorescein hapten and its antibody hence is not diffusion limited.