Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/5135
標題: 發展一生物化學法分析雌性激素蛋白質胼合物
Development of a Biochemical Assay to Analyze the Protein Adducts of Estrogen Quinones
作者: 盧秀卿
Lu, Hsiu-Ching
關鍵字: protein adduct;蛋白質胼合物;estrogen;雌性激素
出版社: 環境工程學系
摘要: 
本研究係利用生物性指標分析方法,針對雌性激素之醌類化物與蛋白質所形成共價鍵結產物-蛋白質胼合物,作為推估雌性激素之醌類化物於標的器官之組織劑量。本研究建立一衍生方法,以TFAA (trifluoroacttic acid anhydride) 作為衍生劑,在強酸環境下催化,將雌性激素之醌類化物與蛋白質上之Cysteine之硫原子鍵結產生之胼合物,自蛋白質結構中移除,並經由溶劑萃取濃縮,再利用氣相層析電子撞擊與負離子化學離子化質譜儀 (GC/EI/MS及GC/NICI/MS),進行定性與定量分析。同時利用同位素內標準品,完成對雌性激素醌類化物(estrone-2,3-quinone) 與cysteine形成之胼合物之同分異構物之定性結構確認,並針對雌性激素醌類化物與cysteine及蛋白質形成之胼合物之分析方法進行反應條件最佳化測試。此外運用高效能液相層析儀純化雌性激素之醌類化物之cysteine胼合物,作為定量所需檢量線之標準品。並針對BSA (Bovine serum albumin) 和人類血清白蛋白 (human serum albumin) 之背景值,以及人類乳癌細胞株 (MCF-7 cells/MDA-MB-231 cells),對於雌性激素醌類化物之蛋白質胼合物之背景值進行偵測,並確認雌性激素可於乳癌細胞中代謝活化形成醌類化物並進而與細胞內之蛋白質上之cysteine形成胼合物。實驗結果顯示於人類及小牛血清白蛋白,偵測到4-OHE2-2-Y及2-OHE2-4-Y之生成,對於E1蛋白質胼合物亦具有相似之結果此一結果顯示雌性激素能代謝活生成具活性之醌類化物並能進而與蛋白質反應形成穩定之蛋白質胼合物。對於乳癌細胞MDA-MB-231及MCF-7細胞之分析結果顯示,4-OHE2-2-Y及2-OHE2-4-Y皆存在於此二細胞株內,但並未發現E1醌類化物之蛋白質胼合物。

The objective of this research is to develop a biochemical assay using protein adducts as biomarker of exposure to assess the cumulative body burden of estrogen-derived quinones in target organs. This method ultilizes trifluoroacetic acid (TFAA) and methanesulfonic acid (MSA) as catalysts to cleave cysteinyl adducts of estrogen-derived quinones on proteins. The cleaved adducts are recovered by organic solvent extractions and analyzed by gas chromatography-electron-impact/negative-ion-chemical-ionization-mass spectrometer (GC-EI-MS/GC-NICI-MS). The isomeric forms of cysteinyl adducts of estrogen-2,3-quinones (2-OHE2) are characterized by using isotopically-labelled bound internal standards after adducts cleavage by the TFAA/MSA derivatization procedure. Additionally, we optimized the method by modifying the derivatization procedure to recover the estrogen quinone-derived cysteinyl adducts on proteins. Additionally, synthetic cysteinyl adducts of estrogen quinones were further purified by HPLC-UV and were used as standards to quantify the modified proteins. We applied this derivatization method to analyze the background levels of cysteinyl adducts of estrogen quinones on bovine serum albumin and human serum albumin. Further, we measured the cysteinyl adducts of estrogen quinones on total cellular proteins derived from human breast cancer cells MCF-7 and MDA-MB-231. Results confirmed that E2 was converted to estrogen quinones which subsequently bind to cellular proteins in human breast cnacer cells. The background levels of cysteinyl adducts of estrogen quinones, including 4-OHE2(E1)-2-Y and 2-OHE2(E1)-4-Y, were detected in human and bovine serum albumin. In addition, we detected the presence of 4-OHE2-2-Y and 2-OHE2-4-Y adducts in the total cellular proteins derived from human breast cancer cells MDA-MB-231 and MCF-7 whereas 4-OHE1-2-Y and 2-OHE1-4-Y were not detected. Overall, results from our investigation confirmed the presence of estrogen-2,3 and -3,4-quinones in human and bovine serum albumin and in the cellular proteins derived from human breast cancer cells.
URI: http://hdl.handle.net/11455/5135
Appears in Collections:環境工程學系所

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