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    题名: 利用毛細管電泳分離蛋白質及測量其物性
    其它题名: Protein separation and property measurements by capillary electrophoresis
    作者: 蘇稜雅;Su, Leng-ya
    贡献者: 淡江大學化學學系碩士班
    吳俊弘;Wu, Chunhung
    关键词: 毛細管電泳;蛋白質分離;磷酸化;capillary electrophoresis;Protein Separation;phosphorylation
    日期: 2008
    上传时间: 2010-01-11 02:38:12 (UTC+8)
    摘要: 本論文主要分為三個部份,第一個部份是將毛細管電泳技術與Poiseuille’s Law及Taylor-Aris Dispersion方法相結合,用以研究蛋白質的構形變化。從實驗結果顯示,胰島素(Insulin, Ins)在pH8.4的緩衝溶液中,有聚集的行為,且緩衝溶液離子強度增高時會使其聚集行為更明顯。增加緩衝溶液的鋅離子濃度,則所測得的Ins流體動力半徑也隨之增大。當 [Zn2+]/[Ins]≧8.7時,聚合體Ins與單體Ins的體積比值顯示此時Ins已聚集形成六聚體。鋅離子誘發Ins形成聚合體的效應在較高離子強度的緩衝溶液中較為顯著。

    第二個部份是以不同種類的線性親水性高分子作為分離介質,以及在緩衝溶液中添加金屬離子,以期達到高解析度的聚胜肽毛細管電泳分離。我們以F127、dextran、PEG及纖維素等高分子對聚離胺酸(polylysine)混合樣品及經過胰蛋白酶進行消化反應後的蛋白質樣品進行毛細管電泳分離。其中以20%~30%的F127及15%~25%的Dextran有較佳的解析效果,並得到類似的消化樣品電泳圖譜,可見這兩種高分子解析聚胜肽的行為和機制是相似的。另外在緩衝溶液中添加金屬離子會改變聚胜肽的電泳行為。二價金屬離子中,鎂離子(Mg2+)的添加對聚胜肽的電泳解析度較佳。當添加高價金屬離子如鈷胺錯離子 時,會使聚胜肽的電泳圖譜產生較劇烈的改變。

    第三個部份則是利用毛細管電泳技術,開發出新的分析方法,用以偵測磷酸化蛋白上的磷酸化位置。本實驗以牛奶中的β-酪蛋白作為主要的研究對象,將原本具有五個磷酸化位置的β-酪蛋白以酵素進行消化反應以及去磷酸化反應。利用電泳遷移率位移的現象,亦即從消化後的β-酪蛋白胜肽片段在有磷酸化修飾及去磷酸化後的電泳圖譜,可以分辨出具有磷酸化位置的片段。為了驗證結果的正確性,我們利用製備型的HPLC將在β-酪蛋白中具有磷酸化位置的胜肽片段純化分離,以質譜(MALDI-TOF)確定其序列,並進行毛細管電泳分析以確認具磷酸化胜肽之吸收峰。
    This thesis consists of the results of three research subjects. In the first part we combined capillary electrophoresis (CE) technique with Poiseuille’s Law and Taylor-Aris Dispersion method to investigate the conformational changes of proteins. According to the results of this experiment, we found that insulin (Ins) would aggregate in tris/boric acid (TB, pH8.4) buffer and the aggregation of Ins would increase with increasing ionic strength. The hydrodynamic radius of Ins would increase gradually with increasing zinc concentration in TB buffer. When [Zn2+]/[Ins]≧8.7, the volume ratio of associated Ins to monomeric one showed that Ins had aggregated to form the hexameric conformation. The effect of Zn2+ induced polymeric conformation of Ins was more significant in buffer solution with higher ionic strength.
    In the second part linear hydrophilic polymer and metal ion were added into CE buffer so as to enhance the separation resolution of polypeptide mixture. Both samples of polylysine mixtures and peptide fragments of myoglobin digested by trypsin were analyzed by using CE with different concentrations of F127, dextran(70K、500K、2000K), PEG, and celluloses as separation mediums. Higher separation resolutions were achieved when 20~30% F127 and 15~25% dextran were used. Moreover, the peak patterns of myoglobin digest in the electropherograms obtained from the two polymer systems were very similar. Therefore, we considered F127 and dextran to have similar resolving mechanism for the CE separation of polypeptides. We also found that the presence of metal ion in CE buffer would alter the electrophoretic migration behaviors of polypeptides. Among the divalent cations added, Mg2+ would result in better separation resolution. When multivalent cation like Co(NH3)63+ was added, the consequential peak pattern in the electropherogram of myoglobin digest differed very much from that of the other metal ions, which suggested some special binding affinities might occur between metal ion and peptides.
    In the last part we developed a CE-based analytical method for the detection of phosphorylation sites in phosphoproteins. β-casein, the model phosphoprotein with five phosphorylation sites, was digested by trypsin and followed by the treatment of dephosphorylation reaction. According to the electropherograms obtained before and after dephosphorylation reaction for the digested peptide fragments, the peaks with mobility shifts indicated the peptides with phosphorylation sites. In order to confirm the result, we used preparative HPLC to purify the digested β-casein sample, MALDI-TOF to identify the sequence, and CE to differentiate the phosphorylated peptide peaks in the electropherogram.
    显示于类别:[化學學系暨研究所] 學位論文

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