HEXESTROL DIAZIRINE PHOTOAFFINITY-LABELING REAGENT FOR THE ESTROGEN-RECEPTOR

3-Azibutyl (2R,3S*)-2,3-bis(4-hydroxyphenyl)pentyl sulfide (1), a pho toaffinity labeling reagent for the estrogen receptor (ER), has been p repared in unlabeled and in high specific activity tritium-labeled for m (32 Ci/mmol) and has been shown to undergo selective and efficient p hotocovalent attachment to rat uterine ER. Diazirine 1 demonstrates hi gh binding affinity for ER, as determined by both a competitive bindin g assay and a direct binding assay (relative binding: estradiol = 100; (1) = 17. K(d): estradiol = 0.19 nM; (1) = 0.98 nM, respectively). It is efficient in site-specific photoinactivation of ER, reaching the l evel of 31% after 5 min of irradiation at >315 nm. The tritium-labeled diazirine [H-3]-1 undergoes specific photocovalent attachment to ER w ith an attachment efficiency of 29 % and a selectivity of 90%. Both of these values are quite high for a photoaffinity reagent. SDS-polyacry lamide gel electrophoretic analysis of the photolabeled proteins shows specific labeling of a major species at M(r) 65 000, the same species that is labeled by [H-3]tamoxifen aziridine, a well-characterized aff inity label for ER. Hexestrol diazirine 1 is the first carbene-generat ing photoaffinity label that covalently labels ER with high efficiency and selectivity, and it should be useful in further studies on the ho rmone-binding domain of ER.