Hexestrol diazirine photoaffinity labeling reagent for the estrogen receptor.
3-Azibutyl (2R*,3S*)-2,3-bis(4-hydroxyphenyl)pentyl sulfide (1), a photoaffinity labeling reagent for the estrogen receptor (ER), has been prepared in unlabeled and in high specific activity tritium-labeled form (32 Ci/mmol) and has been shown to undergo selective and efficient photocovalent attachment to rat uterine ER. Diazirine 1 demonstrates high binding affinity for ER, as determined by both a competitive binding assay and a direct binding assay (relative binding: estradiol = 100; (1) = 17. Kd: estradiol = 0.19 nM; (1) = 0.98 nM, respectively). It is efficient in site-specific photoinactivation of ER, reaching the level of 31% after 5 min of irradiation at > 315 nm. The tritium-labeled diazirine [3H]-1 undergoes specific photocovalent attachment to ER with an attachment efficiency of 29% and a selectivity of 90%. Both of these values are quite high for a photoaffinity reagent. SDS-polyacrylamide 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 [3H]tamoxifen aziridine, a well-characterized affinity label for ER. Hexestrol diazirine 1 is the first carbene-generating photoaffinity label that covalently labels ER with high efficiency and selectivity, and it should be useful in further studies on the hormone-binding domain of ER.
Bergmann
K E
KE
Department of Chemistry, University of Illinois, Urbana 61801.
Carlson
K E
KE
Katzenellenbogen
J A
JA
D000345:Affinity Labels / Q000138:chemical synthesis* / Q000493:pharmacokinetics; D000818:Animals; D001389:Azirines / Q000138:chemical synthesis* / Q000493:pharmacokinetics; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006589:Hexestrol / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000493:pharmacokinetics; D066298:In Vitro Techniques; D007553:Isotope Labeling; D010782:Photolysis; D051381:Rats; D011960:Receptors, Estrogen / Q000187:drug effects*; D012756:Sheep; D014599:Uterus / Q000187:drug effects / Q000378:metabolism
D000345:Affinity Labels; D001389:Azirines; D011960:Receptors, Estrogen; D006589:Hexestrol; C088350:hexestrol diazirine