臺灣博碩士論文加值系統

嘌呤5’-核苷酸酶 (purine 5’-nucleotidase, p5’-NT) 可以調節各組織器官中具各種生理活性的嘌呤核苷、核苷酸及兩者之類似物的濃度，來維持體內核苷酸代謝的平衡，為重要的磷酸酯水解酶。本研究之目的在於開發一毛細管區帶電泳方法，以同步分析肌核苷單磷酸 (Inosine 5’-monophosphate, IMP) 經酵素催化代謝後之相關產物，包括肌苷 (Inosine, I)、次黃嘌呤 (Hypoxanthine, hXan)、黃嘌呤 (Xanthine, Xan) 與尿酸 (Uric acid)。樣品緩衝液的組成是以p5’-NT之酵素分析緩衝液為主 (50 mM Tris-HCl, 20 mM MgCl2, 2 mM β-glycerol phosphate，pH 8.0)，再另外添加氯化鈉與氰甲烷來造成線上樣品濃縮的效果。在我們所尋找的最佳條件下，可於八分鐘內將四種標準品有效分離，故此分析方法可以用來同步分析IMP及其代謝的相關產物。在此毛細管區帶電泳法中，其線性範圍在5~300和10~400 M之間，I、hXan、Xan 及 Uric acid 檢量線之相關係數分別為0.9999、0.9999、0.9997 和 0.9999，而偵測極限分別為1.4 M、2.1 M、5.4 M、3.1 M。於再現性試驗中，I、hXan、Xan 、Uric acid 和內標準品Adenosine 3’,5’-cyclic monophosphate (cAMP) 面積比再現性的相對標準偏差皆小於 5.48 %，滯留時間的相對標準偏差皆小於 0.99 %。初步的真實樣品分析結果顯示：人類肝癌細胞 Hep G2 的p5’-NT最初反應速度與濃度的關係符合 Michaelis Menten 動力學模式，而最大反應速度與Michaelis常數分別為11.75 nmol/min/mg、5.97 mM。

The purine 5’-nucleotidases (p5’-NT) are involved in the regulation of various physiologically active purine nucleosides, nucleotides, and their analogues in organs. Therefore p5’-NT maintains balanced purine nucleotide pools, so it is one of the important phosphohydrolases. The purpose of the research was to develope a capillary zone electrophoretic method to simultaneously analyze inosine 5’-monophosphate (IMP) and its metabolites, namely inosine (I), hypoxanthine (hXan), xanthine (Xan), and Uric acid. The sample buffer is the p5’-NT assay solution, which contains 50 mM Tris-HCl , 20 mM MgCl2, and 2 mM β-glycerol phosphate at pH 8.0. Addition of acetonitrile and sodium chloride could enhance sample stacking online. In the optimal condition, the four standards, I, hXan, Xan, and Uric acid, were effectively separated in 8 minutes. The method was linear in the range of 5-300 M for I and hXan, and 10-400 M for Xan and uric acid with the correlation coefficients 0.9999, 0.9999, 0.9997, and 0.9999 for I, hXan, Xan, and Uric acid, respectively. The concentration limits of detection of I, hXan, Xan, and Uric acid were 1.4 M, 2.1 M, 5.4 M, and 3.1 M, respectively. In the precision test, all relative standard deviations of migration time and area ratio were less than 0.99 % and 5.48 %, respectively. The method was applied to analyze the p5’-NT in the cellular extract of Hep G2. It suggested that p5’-NT from Hep G2 followed Michaelis Menten kinetics . The maximum catalytic velocity and Michaelis constant of Hep G2 p5’-NT were 11.75 nmol/min/mg and 5.97 mM, respectively.

目錄

中文摘要..........................................................I
英文摘要.........................................................II
目錄............................................................III
圖目錄...........................................................VI
表目錄.......................................................... VII

第一章 緒論......................................................1
1.1 前言.....................................................1
1.2 肌核苷單磷酸及5’-核苷酸酶簡介.............................1
1.2.1 肌核苷單磷酸的構造...................................1
1.2.2 肌核苷單磷酸之重要性.................................2
1.2.2.1 IMP是合成AMP和GMP的前驅物...................2
1.2.2.2 IMP之來源.......................................4
1.2.2.3 IMP之代謝反應...................................5
1.2.3 嘌呤5’-核苷酸酶重要性..................................6
1.2.4 過去分析方法..........................................7
1.3 毛細管電泳簡介............................................10
1.3.1 毛細管電泳技術的發展歷程..............................10
1.3.2 毛細管電泳基本構造....................................11
1.3.3 毛細管電泳原理........................................11
1.3.4 進樣方式..............................................15
1.3.5 偵測方式..............................................15
1.3.6 影響毛細管電泳分離效率的因素..........................17
1.3.6.1 緩衝溶液的選擇...................................17
1.3.6.2 毛細管的尺寸.....................................19
1.3.6.3 分離電壓........................................ 20
1.3.6.4 溫度.............................................21
1.3.6.5 添加劑...........................................21
1.3.7 樣品前濃縮............................................23
1.4 酵素動力學簡介.............................................25
1.5 研究動機與目的.............................................30

第二章 實驗方法與儀器設備.......................................31
2.1 材料與設備.................................................31
2.1.1 化學試劑..............................................31
2.1.2 細胞來源..............................................32
2.1.3 儀器設備..............................................32
2.2 嘌呤5’-核苷酸酶活性分析之實驗方法..........................34
2.2.1 電泳緩衝溶液的配製....................................34
2.2.2 核苷酸的配製..........................................34
2.2.3 分析標準品的配製......................................34
2.2.4 毛細管處理............................................36
2.2.5 校正曲線的建立........................................36
2.2.6 理論板數的計算........................................37
2.2.7 蛋白質之定量..........................................37
2.2.8 偵測極限(LOD)及定量極限(LOQ)的測定...................37
2.2.9 酵素分析樣品的配置....................................38
第三章 嘌呤5’-核苷酸酶活性分析法之結果與討論.....................39
3.1 尋找最佳化分析條件.........................................39
3.1.1 樣品緩衝液濃度........................................40
3.1.2 電泳緩衝溶液濃度......................................41
3.1.3 電泳緩衝溶液pH值.....................................43
3.1.4 分離電壓..............................................45
3.1.5 Acetonitrile-NaCl的效應..................................46
3.2 檢量線與再現性試驗.........................................50
3.2.1 檢量線................................................50
3.2.2 確效實驗：再現性與準確性測試...........................53
3.3 酵素活性分析的應用.........................................56
3.3.1 真實樣品分析.......................................... 56
3.3.2 與其他嘌呤5’-核苷酸酶活性分析法之比較..................60
3.4 Hep G2 之酵素動力學......................................61
3.5 Hep G2酵素活性與反應溶液pH值的關係......................64

第四章 結論.....................................................65

參考文獻.........................................................66





圖目錄

圖1.1 肌核苷單磷酸結構............................................2
圖1.2 AMP與GMP的合成...........................................4
圖1.3 嘌呤核苷酸的代謝反應........................................5
圖1.4 肌核苷單磷酸的降解圖........................................7
圖1.5 不同電性之分析物電泳方向與電滲流關係圖.....................14
圖1.6 受質濃度[S]對最初酵素催化反應速度的影響.................... 27
圖1.7 以Lineweaver-Burk雙倒數線性關係式計算動力學常數Km和
Vmax.......................................................28
圖2.1 毛細管電泳儀簡圖...........................................33
圖2.2 p5’-NT活性分析樣品與分析標準品的配置流程圖.................35
圖3.1 電泳緩衝液濃度與核苷酸遷移時間關係圖.......................42
圖3.2 電泳緩衝溶液pH與核苷酸遷移時間關係圖......................44
圖3.3 NaCl濃度與理論板數關係圖...................................47
圖3.4 最佳條件分析標準品之電泳圖.................................49
圖3.5-1 (a) I檢量線................................................50
圖3.5-2 (b) hXan檢量線 (c) Xan檢量線..............................51
圖3.5-3 (d) hXan檢量線.............................................52
圖3.6 真實樣品分析電泳圖。Hep G2的5’-NT催化IMP去磷酸化反應(A)
0 min、(B) 15 min、(C) 30 min、(D) 60 min.......................58
圖3.7 [IMP]對源自Hep G2的p5’-NT催化反應速度飽和曲線圖...........61
圖3.8 酵素催化反應速度與反應pH之關係圖..........................64

表目錄

表1.1 常用的CE緩衝液及其pKa值和有效pH值範圍...................18
表1.2 不同內徑毛細管其表面積與體積比值...........................20
表1.3 毛細管電泳中常見的添加劑及其作用...........................22
表3.1 I、hXan、Xan及Uric acid各濃度訊號面積/I.S訊號面積比和遷移
時間之RSD值及其相對誤差..................................54
表3.2 I、hXan、Xan及Uric acid 再現性與準確性試驗..................55
表3.3 真實樣品分析之生成物I及hXan的濃度.........................59
表3.4 催化AMP與IMP去磷酸化之p5’- NT 特性之比較.................63

1.Y. Moriwaki, T. Yamamoto, K. Higashino, Histol. Histopathol., 14 (1999) 1321
2.S.N. Rylova, F. Albertioni, G. Flygh, S. Eriksson, Biochem. Pharmacol. 69(2005) 951.
3.J.D. Carbeck, R.S. Negin, J. Am. Chem. Soc., 123 (2001) 1252
4.S.D. Mendonsa, M.T. Bowser, J. Am. Chem. Soc., 126 (2004) 20
5.O. B. Kashlan, C. P. Scott, J. D. Lear, B. S. Cooperman, Biochemistry, 41 (2002) 462.
6.A. Y. Jong, K. Yu, B. Zhou, T. Frgala, C. P. Reynolds, Y. Yen, J. Biomed. Sci., 5 (1998) 62.
7.S.P. Hendricks, C.K. Mathews, J. Biol. Chem., 272 (1997) 2861.
8.林順富、葉東柏、陳師瑩、顏瑞鴻、蕭慧美，「生物化學」，偉明圖出版社，台北市，第738-767頁
9.林穎正、周坤達、陳甫倫、莊文博、楊明杰、藍敏瑛，「生物化學」，合記圖書版社，台北市，第181-204頁
10.郭婧婧、彭贵华、武四云, 中國醫學工程, 14 (2006) 71.
11.S.A. Hunsucker, B.S. Mitchell, J. Spychala, Phamacol. Ther. 107 (2005) 1
12.J. Avruch, D.F. Wallach, Biochim. Biophys. Acta, 233 (1971) 334.
13.N.L. Edwards, D.B. Magilavy, J.T. Cassidy, I.H. Fox, Science, 201 (1978) 628.
14.J.P van Laarhoven, G.T. Spierenburg, C.H de Bruyn, J. Immunol. Methods, 39 (1980) 47.
15.C.H. Fiske, Y. Subbarow, J. Biol. Chem., 66 (1925) 375.
16.P.S Chen, Jr., T.Y. Toribara, H. Warner, Anal. Chem., 28 (1956) 1756.
17.C.C. Widnell, Methods Enzymol., 32 (1974) 368.
18.R.A. Hartwick, S.P. Assenza, P.R. Brown, J. Chromatogr., 186 (1979) 647.
19.J.N. Stolk, R.A. de Abreu, A.M. Boerbooms, D.G. de Koning, R. de Graaf, P.J. Kerstens, L.B. van de Putte, J. Chromatogr. B Biomed. Appl., 666 (1995) 33.
20.A. Tiselius, Trans Faraday Soc., 33 (1937) 524.
21.李建武、蕭能 、余瑞元、陳麗蓉、陳雅蕙、陳來同、袁明秀合著，「生物化學實驗原理和方法」，藝軒圖書出版社，台北市，第114-117頁 (1999)。
22.J. W. Jorgenson, K. D. Lukacs, Anal. Chem., 53 (1981) 1298.
23.J. W. Jorgenson, K. D. Lukacs, J. Chromatogr., 218 (1981) 209.
24. J. W. Jorgenson, K. D. Lukacs, Science, 222 (1983) 266.
25.R. Kuhn, S. Hoffstetter- Kuhn. Capillary Electrophoresis: Principle and practice. CRC, London. (1993).
26.王鳳玉，「以毛細管電泳/雷射激發螢光偵測應用於微量藥物的分析」，碩士論文，朝陽科技大學應用化學研究所，台中 (2002)。
27.I. S. Lurie, R. F. X. Klein, T. A. D. Cason, M. J. Lebelle, R. Brenneisen and R. E. Weinberger, Anal. Chem., 66 (1994) 4019.
28.Z. K. Shihabi, J. Chromatogr. A, 853 (1999) 349.
29.C. A. Kuffner, E. Marchi, J. M. Morgado and C. R. Rubio, Anal. Chem., 68 (1996) 241A
30.B. F. Kenney, J. Chromatogr., 546 (1991) 423.
31.S. P. D. Lalljie, J. Vindevogel, P. Sandra, J. Chromatogr. A, 652 (1993) 563.
32.D. Lagoutte, G. Lombard, S. Nisseron, M. P. Paper, Y. Saint-Jalm, J. Chromatogr. A, 684 (1994) 251.
33.C. H. Wu, Y. S. Lo, Y. H. Lee, T. I. Lin, J. Chromatogr. A, 704 (1995) 291.
34.K. Bächmann, J. Boden and I. Haumann, J. Chromatogr., 626 (1992) 259.
35.Y. C. Chao and C. W. Whang, J. Chromatogr. A, 663 (1994) 229.
36.E. D. Zlotorzynska and J. F. Dlouhy, J. Chromatogr. A, 685 (1994) 145.
37.C. E. Lin, C. C. Chang, W. C. Lin and E. C. Lin, J. Chromatogr. A, 753 (1996) 133.
38.D. F. Swaile and M. J. Sepaniak, Anal. Chem., 63 (1991) 179.
39.B. L. Hogan and E. S. Yeung, Anal. Chem., 64 (1992) 2841.
40.V. Dolnĺk and J. Dolnĺková, J. Chromatogr. A, 716 (1995) 269.
41.J. Schneede and P. M. Ueland, Anal. Chem., 67 (1995) 812.
42.J.W. Jorgenson, K.D. Lukacs, Anal. Chem., 53 (1981) 1298.
43.J.W. Jorgenson, K.D. Lukacs, J. Chromatogr., 281 (1981) 209.
44.S.F.Y. Li, Capillary Electrophoresis: Principle and Application, Elsevier, Amsterdam. (1992).
45.J.P. Landers, Handbook of Capillary Electrophoresis, 2nd ed., CRC Press, New York, USA (1997)
46.S.E. Geldart, P.R. Brown, J. Chromatogr. A, 828 (1998) 317.
47.D.A. Skoog, F.J. Holler, T.A. Nieman, Principles of instrumental analysis, 5th ed., BROOKS/COLE, Thomson Learning, USA, (1998), pp. 778-795.J. A. Taylor and E. S. Yeung, J. Chromatogr., 550 (1991) 831.
48.J. P. Chervet, R. E. J. Van Soest and M. Ursem, J. Chromatogr., 543 (1991) 439.
49.A. Hiraoka, J. Akai, I. Tominaga, M. Hattori, H. Sasaki and T. Arato, J. Chromatogr. A, 680 (1994) 243.
50.Joxzef L.Beckers and Petr Bocek, Electrophoresis, 24 (2003) 518.
51.James P. Landers, Handbook of Capillary Electrophoresis, second edition.
52.G. Hempel, Electrophoresis, 21 (2000) 691.
53.Z.K. Shihabi, J. Chromatogr. A, 807 (1998) 27.
54.Z.K. Shihabi, J. Chromatogr. A, 744 (1996) 231
55.Z.K. Shihabi, J. Chromatogr. A, 902 (2000) 107.
56.Tagliaro, F., Manetto, G., Crivellente, F., Scarcella, D., Marigo, M., Forens. Sci. Int., 92 (1998) 201.
57. Wey, A.B., Zhang, C.-X., Thormann, W., J. Chromatogr. A, 853 (1999) 95
58.Chien, R.-L., Burgi, D. S., Anal. Chem., 64 (1992) 1046.
59.McGrath, G., Smyth, W. F., J. Chromatogr. B, 681 (1996) 125.
60. Harland, G. B., McGrath, G., McClean, S., Smyth, W. F., Anal. Commun. 34 (1997) 9.
61.Hadwiger, M. E., Torchia, S. R., Park, S., Biggin, M. E., Lunte, C. E., J. Chromatogr. B, 681 (1996) 241.
62.Z.K. Shihabi, Electrophoresis, 23 (2002) 1612.
63.趙世彬、鄒煦杕、蔡東璣、陳河吉、周大中、李文齡，「生物化學」，藝軒圖書出版社，台北市，第134-172頁 (2001)。
64.李建雄、翁郁嘉、端木梁、黃淑姿，「生物化學」，藝軒圖書出版社，台北市，第139-149頁 (1996)。
65.M.M. Bradford, Anal. Biochem., 72 (1976) 248.
66.周志宏, 「毛細管區帶電泳應用於嘌呤核苷酸之同步分析研究」, 國立暨南國際大學應用化學所碩士論文, 南投埔里, 台灣 (2005)
67.Tzeng, H.-F., Hung, C.-H., Wang, J.-Y., Chou, C.-H., Hung, H.-P., Journal of Chromatography A 1129 (2006) 149.
68.洪照先, 「人類肝細胞株Hep G2及昆蟲細胞株Sf9嘌呤5'-核苷酸酶的特性分析」, 國立暨南國際大學應用化學所碩士論文, 南投埔里, 台灣 (2006)