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 hexestrol that bear photolabile diazo and azide functional groups. Using a series of assays, we can determine their binding affinity and their photoreactive efficiency with the uterine binding protein. Certain of the more promising azide and diazo derivatives, as well as other photoreactive derivatives of the arylketone or triarylethylene type, will be prepared in radiolabeled form, and sensitive methods will be devised to determine the degree to which they attach covalently to the estrogen binding sites. Four new functional groups will be investigated as photo attaching functions, and reagents will be prepared in which those groups are located at positions analogous to the B- and C-rings of estradiol, remote from the oxygenated functions. Using the best techniques for selective labeling, we will conduct a detailed physical and chemical characterization of the rat uterine estrogen binding protein. 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 (particularly the cancer-associated fetal alpha globulin, alpha-fetoprotein) and mammary tumor estrogen binding proteins - will be studied.