麩胺酸2,3胺基異位酶(Glutamate 2,3-Aminomutase, G2,3AM)的催化反應機制需要 SAM與連二亞硫酸鹽參與，與離胺酸2,3胺基異位酶(Lysine 2,3-Aminomutase, 2,3-LAM)相同，且兩者也有其他相似之處，PLP、鐵和硫化物含量以及UV-visible都很相似，推測兩者也擁有類似的催化反應機制。
在Frank J. Ruzicka, Perry A. Frey (2007)的論文中已經證明Glutamate 2,3-Aminomutase反應機制中自由基3號的存在，本論文想證明反應機制中自由基1號的存在，實驗我們利用4F-L-glutamate這個受質來幫助我們偵測出自由基1號的訊號。
為得到自由基3的訊號，我們需要純化出更濃更純的麩胺酸2,3胺基異位酶，在修改純化流程後可以經由過三隻膠體管柱分別為Q-FF、Phenyl與S200來純化出更純的蛋白質濃度。在光吸收也得出反應接受4F-L-glutamate。並且在EPR訊號上餵L-glutamate可以順利偵測出自由基訊號，餵4F-L-glutamate可以得到可能為受質自由基訊號。

Glutamate 2,3-Aminomutase (G2,3AM) is a protein with B6 (PLP) and S-adenosyl methionine (SAM) as coenzymes. The PLP-, SAM, iron-, and sulfide-content of Glutamate 2,3-Aminomutase and requirement for SAM are the same as Lysine 2,3-Aminomutase (2,3-LAM). Also that UV-visible spectrum of Glutamate 2,3-Aminomutase is similar to Lysine 2,3-Aminomutase.So we speculate that both have similar catalytic reaction mechanisms.
In the paper of Frank J. Ruzicka, Perry A. Frey (2007), the existence of free radical No. 3 in the reaction mechanism of Glutamate 2,3-Aminomutase has been proved. In this paper, we want to prove the existence of free radical No. 1 in the reaction mechanism. In the experiment, we used 4F-L-glutamate to help us detect the signal of free radical 1.
In order to obtain the signal of free radical NO.3, we need to purify a more concentrated and purer Glutamate 2,3-Aminomutase. After modifying the purification process, it can be purified by three columns: Q-FF, Phenyl and S200. Purify to a purer protein concentration. The light absorption also yields the reaction to accept the substrate of 4F-L-glutamate. And feeding L-glutamate on the EPR signal can successfully detect the free radical signal, and feeding 4F-L-glutamate can get signals that may be host free radicals.

謝誌 i
摘要 ii
Abstract iii
目錄 iv
圖目錄 vi
第一章 緒論 1
1.1 研究背景 1
1.2 研究目的 1
第二章 文獻回顧 3
2.1 蛋白質簡介 3
2.1.1 酶簡介 3
2.1.2 輔酶 4
2.2 麩胺酸2,3胺基異位酶 5
2.3 麩胺酸2,3胺基異位酶知輔酵素 6
2.3.1 輔酶SAM 6
2.3.2 輔酶PLP磷酸吡哆醛 7
2.2.3 G23AM 之反應機制 7
第三章 實驗儀器與原理 11
3.1 層析法 11
3.1.1 快速液相層析儀(FPLC) 11
3.2 電泳膠片分析法 13
3.3 紫外光-可見光分光光度法 14
3.4 電子順磁共振儀(EPR) 16
3.4.1 離曼效應 16
3.4.2 精細結構交互作用(Hyperfine Interactions) 18
第四章 實驗材料與方法 21
4.1 養菌 21
4.1.1 小量培養 21
4.1.2 大量培養 22
4.1.3 收菌 23
4.2 純化 24
4.2.1 破菌 24
4.2.2 純化 25
4.3 UV-vis光吸收 29
4.4 EPR訊號測量 29
第五章 實驗結果與討論 31
5.1 純化 31
5.1.1 Q-FF陰離子交換層析 31
5.1.2 Phyenyl疏水交換層析 32
5.1.3 S200分子大小層析 33
5.2 UV-vis光吸收 35
5.3 EPR訊號 39
5.3.1 自然受質(L-glutamate) 39
5.3.2 4F-L-glutamate 40
第六章 結論與總結 43
參考文獻 45

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