We are developing chemical approaches to study the molecular basis of steroid hormone action. The technique of photoaffinity labeling has been selected as the most appropriate method for attaching covalently a radiochemical label to the high-affinity binding protein for estradiol, found in the rat uterus. We have prepared several derivatives of estradiol, estrone, and hexesterol that bear photolabile diazo and azide functional groups. Using a series of assays, we have determined their binding affinity and their photoreactive efficiency with the uterine binding protein. Certain of the more promising azide and diazo derivatives have been prepared in radiolabeled form. Using a method for electrophoretic analysis of uterine estrogen receptors, based on limited trypsinization to disaggregate receptor, we will analyze the photocovalent attachment of these reagents to the receptor from lamb and rat uterus. The labeled receptor preparations will be subjected to detailed physicochemical characterization, and questions concerning transformation and subcellular dynamics will be addressed by in vivo labeling. Finally, other estrogen binding proteins - enzymes regulated by estrogens, enzymes involved in estrogen biosynthesis and metabolism, serum transport proteins and mammary tumor estrogen binding proteins - will be studied. BIBLIOGRAPHIC REFERENCES: J. A. Katzenellenbogen, T. S. Ruh, K. E. Carlson, H. S. Iwamoto and J. Gorski, Ultraviolet Photosensitivity of the Estrogen Binding Protein from Rat Uterus, Wavelength and Ligand Dependence. Photocovalent Attachment of Estrogens to Protein, Biochemistry 14, 2310 (1975).