METHODOLOGIES FOR ISOLATING ESTROGEN-RESPONSIVE PROTEINS AS MARKERS OF ENVIRONMENTAL TOXICANTS

New and refined methods for the purification of two major estrogen-res ponsive proteins in murine uterine luminal fluid (ULF) are described. Lactoferrin (LF; approx. 70 kDa), was purified using modifications of procedures previously reported. Using column chromatography and electr oelution, the protein was purified to a single band demonstrated by ge l electrophoresis. An antibody to the purified protein was then raised in rabbits. Rabbit serum was IgG purified and used in Western blottin g and immunocytochemical techniques, The serum IgG purified LF antibod y demonstrated only one band in Western blotting procedures using enha nced chemiluminescence (ECL) as a detection method. Another estrogen-i nducible protein with an approximate molecular weight of 63 kDa in mou se ULF, purified to a single band by gel electrophoresis and electroel ution, was determined to be complement C3 through sequence analysis. S imilar to LF, C3 has been previously described as an estrogen-responsi ve protein in the rodent and human reproductive tract. C3 antibody was purchased commercially and was used for Western blotting and immunohi stochemistry. Since interest is increasing in identifying biological m arkers of hormone-disrupting chemicals, the purification techniques de scribed in this report can be useful in identifying proteins that are potential markers of estrogen action. Antibodies to these proteins can then be used as markers of environmental estrogenic toxicants. In fac t, LF and C3 are currently being used to screen for sensitive markers of estrogenicity. Following the methods described in this report, othe r proteins can be identified and used to screen for possible effects o f environmental agents.