臺灣博碩士論文加值系統

環氧化酶(cyclooxygenases, 簡稱COX)是生成前列腺素(prostaglandins, 簡稱PGs)的關鍵酵素，COX被發現存在著兩種異構酶，最早發現的COX-1是本質性(consititutive)酵素，在哺乳類動物大部分的組織中都能偵測到，而COX-2則是屬於誘發性的異構酶，COX-1製造的前列腺素被認為與胃腸的保護功能有關，而COX-2製造的前列腺素可能導致發炎、發燒、疼痛等，亦影響排卵及生產過程。
本實驗藉由COX-1與抗發炎藥結合造成蛋白質上tryptophan螢光的衰減，透過螢光儀以及停止流系統(stopped-flow system)觀察螢光quenching的過程，發現對於indomethacin, meclofenamic acid, piroxicam而言，抑制apo-COX-1螢光會先造成立即的螢光強度降低，隨後螢光會隨著時間而衰減；在抑制holo-COX-1螢光動力學實驗中，indomethacin與meclofenamic acid也都是兩階段的結合過程，但是indomethacin的time-dependent螢光衰減kobs值略小於對apo-COX-1的實驗值，meclofenamic acid對兩種酵素螢光的抑制則差別不大，而piroxicam對holo-COX-1則是屬於time-independent螢光quenching行為。由於缺乏heme的apo-COX-1會在active site留下一個空間，使得active site channel變長，造成抗發炎藥對兩種酵素不同的螢光抑制行為；而抗發炎藥的官能基是否與active site中的胺基酸產生作用力，也影響抗發炎藥對兩種酵素的結合行為差異現象。
Apo-COX-1與collisional quenchers混合之後，發現中性quencher─acrylamide對於螢光的quenching效果遠大於陰、陽離子quenchers─I -, Cs+，表示COX-1上的主要螢光團tryptophan residues所處在的位置環境較不帶電荷，造成陰陽離子對於tryptophan影響很小，而且acrylamide所造成的螢光quenching幅度可超過80 % (0.28 M acrylamide)，表示大部份的tryptophan residues未被包埋在蛋白質結構中，使得其易受到溶劑中分子的影響。若apo-COX-1先與diclofenac結合後再與抑制劑混合，發現抑制劑抑制螢光的能力較小，表示diclofenac結合在酵素上，阻擋住部分tryptophan不受collisional quenchers影響。
2,6-TNS (6-p-toluidinylnaphthalene-2-sulfonate )與apo-COX-1產生螢光能量轉移，我們觀察apo-COX-1螢光quenching與2,6-TNS螢光之增強結果，發現2,6-TNS對於apo-COX-1應該只有一個結合位置，而2,6-TNS對於抗發炎藥產生競爭效應，顯示2,6-TNS結合在COX-1的active site中，未來可利用2,6-TNS與酵素結合產生增強螢光的特性，作為一種偵測抗發炎藥與COX-1結合的探針。

Cyclooxygenase or prostaglandin H2 synthase (COX, PGHS) is the key enzyme that catalyzes the conversion of arachidonic acid and O2 to PGH2, the committed step in prostaglandin biosynthesis. Two isoforms of COX are known: One isoform, COX-1, is constitutively produced in most tissues, and appears to be important in maintainance of normal physiological functions of gastrointestinal protection. The second isoform, COX-2, is induced by inflammatory mediators, and the prostaglandins produced by COX-2 lead to inflammation, fever, pain, and are important for process of parturition.
We have studied the inhibition kinetics of several non-steroidal anti-inflammatory drugs (NSAIDs) binding to apo- and holo-COX-1 by fluorescence quenching of tryptophans of enzyme using the stopped-flow system. Addition of NSAIDs (indomethacin, meclofenamic acid, and piroxicam) to apo-COX-1 results in a rapid fluorescence decrease, followed by a slow time-dependent quenching. The fluorescence quenching of holo-COX-1 by indomethacin and meclofenamic acid also occurs in two stages. The time-dependent binding of indomethacin to the enzyme is slower for holo-COX-1 than for apo-COX-1. However, holo- and apo-COX-1 show similar rate of time-dependent binding for meclofenamic acid. In contrast, piroxicam exhibits time-independent inhibition for holo-COX-1. Apo-COX-1 retains an open space at the heme-binding site, creating a deep active channel. NSAIDs might have different binding modes in the active site requiring a multi-step inhibition. The functional groups of NSAIDs would interact with some amino acid residues of the enzyme and influence the binding with COX-1.
The steady state tryptophan fluorescence is quenched by adding collisional quenchers, CsCl, KI, and acrylamide. The quenching efficieny of the quencher decreases in the order of acrylamide >> KI > CsCl, suggesting that the tryptophan residues in COX-1 are located at a hydrophobic environment. More than 80 % of the fluorescence was quenched by acrylamide, indicating that most tryptophan residues are not buried in the interior of the protein. When apo-COX-1 was pre-treated with diclofenac, the collisional quenching becomes less effective. It suggests that binding of diclofenac in the active site may block some tryptophan residues against solvent accessibility.
We have also studied the fluorescence resonance energy transfer analysis of TNS (6-p-toluidinylnaphthalene-2-sulfonate) interacting with apo-COX-1. By measuring the fluorescence decrease of apo-COX-1 and enhancement of TNS emission, it was concluded that there was only one binding site for TNS within apo-COX-1. TNS might bind to apo-COX-1 competitively with NSAIDs. TNS could be used as a probe to determine the inhibition behavior of binding NSAIDs to COX-1.

中文摘要..................................................................................................I
英文摘要.................................................................................................III
目 錄.................................................................................................V
圖 目 錄...............................................................................................VIII
表 目 錄................................................................................................XI


第一章 緒 論

第一節 環氧化酶(COX)之簡介………………………………………...1
第二節 COX-1和COX-2的生物化學以及晶體結構特性……….……..3
第三節 環氧化酶催化反應機制………………………………………..6
第四節 環氧化酶催化反應之中間體…………………………………11
第五節 前列腺素合成酶在生理學疾病理學上的功能………………13
第六節 非類固醇抗發炎藥……………………………………………17
第七節 非類固醇抗發炎藥抑制的機制………………………………18
第八節 非類固醇抗發炎藥抑制動力學的探討………………………21
第九節 實驗目的………………………………………………………24
第二章 實 驗

第一節 實驗藥品………………………………………………………25
第二節 實驗儀器………………………………………………………26
第三節 Holo-COX-1的製備……………………………….…………..29
第四節 實驗方法………………………………………………………32
2-4-1 吸收光譜及螢光光譜之測量……………………………...32
2-4-2 Collisional Quenchers抑制Apo-COX-1螢光實驗………….33
2-4-3 NSAIDs對apo-COX-1螢光抑制動力學……………………35
2-4-4 NSAIDs對holo-COX-1螢光抑制動力學…………………..37
2-4-5 Apo-COX-1與2,6-TNS之Energy Transfer實驗…………....38

第三章 結果與討論

第一節 光譜測量………………………………………………………41
第二節 Collisioinal Quenchering實驗……………………………..…..49
第三節 NSAIDs抑制Apo-COX-1螢光動力學………………….…….58
第四節 NSAIDs抑制Holo-COX-1螢光動力學………………….……71
第五節 Apo-COX-1與2,6-TNS的Energy Transfer實驗……..….…….80
第六節 討論……………………………………………………………86

第四章 結 論……………………….…..……………………89

參考資料………………………………………………………………..96

附 錄
附錄一 NSAIDs光譜圖…………………………………………………i
附錄二 NSAIDs結構圖………………………………………………..vii

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