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Effect of thiol reductants on estrogen and estrogen receptor: revisiting previous assays
Author(s) -
Pullela Phani Kumar,
Alby Kevin,
Varela Hector,
Sem Daniel S
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a74
Subject(s) - estrone , thiol , chemistry , dithiothreitol , estrogen receptor , cysteine , estrogen , ethanethiol , biochemistry , breast cancer , organic chemistry , hormone , medicine , endocrinology , biology , enzyme , cancer
The disulfide bridges in proteins play an essential role in maintaining protein structural integrity, function and dynamics. It is common to use thiol reductants like DTT (dithiothreitol) and BME (2‐mercapto ethanol) to prevent air oxidation of cysteine residues. Though extensive studies were done over the past forty years, using thiol reductants in protein biochemistry, our recent findings have provided a surprising observation while using these reductants in the estrone‐estrogen receptor system. Both BME and DTT were shown to react with estrone in dichloromethane/HCl and the thiohemiketals produced are reasonably stable. Even in the buffered aqueous medium at pH 7.4, the thiohemiketals are stable, making them relevant in protein biochemical studies. We choose, estrogen receptor as model system, due to its importance in breast cancer research and endocrine disruptors. ER‐LBD was expressed in E.coli and the protein was obtained in its active conformation by adding estrone during the protein expression. The effect of different thiol reductants was studied by incubating the thiol and protein for three hours. The thiol interaction with estrogen in the binding site is possibly mediated by the His 524 or entropy driven due to large volume of ER‐LBD binding site. The possible reaction of thiol reductants with the ketone of D‐ring on estrone raises concerns for their use in relevant protein biochemistry. This work is being funded by the US Army Medical Research and Materiel Command’s “Breast Cancer Research Program (BCRP) # BC046402.