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研究生:孫思安
研究生(外文):Szu-An Sun
論文名稱:稻米芽鞘胰蛋白"酉每"抑制劑之純化與特性
論文名稱(外文):Purification and Characterization of Rice Coleoptile Trypsin Inhibitor
指導教授:黃福永
指導教授(外文):Fu-Yung Huang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:化學系碩博士班
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:74
中文關鍵詞:胰蛋白"酉每"胰凝乳蛋白"酉每"胰蛋白"酉每"抑制劑
外文關鍵詞:Michaelis-Menten equationchymotrypsintrypsin
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從浸水條件環境下生長的稻米芽鞘,可經由陽離子交換管柱及膠體管柱層析的方式分離出一種分子量大約在18 kDa的胰蛋白"酉每"抑制劑。我們已經進一步探討此抑制劑對於胰蛋白"酉每"及胰凝乳蛋白"酉每"的抑制行為。於研究中發現,此抑制劑對於胰凝乳蛋白"酉每"水解受質N-苯甲醯基-精胺酸-對硝基苯胺(L-BAPNA)的抑制行為並非一個典型的競爭型抑制反應;但是其對於胰蛋白"酉每"水解同一受質的抑制機制與從大豆中萃取出的胰蛋白"酉每"抑制劑相同,都屬於競爭型的抑制反應。
從稻米芽鞘中取得的抑制劑與胰蛋白"酉每"結合之複合物(EI),其解離常數為4.0 ×10-7 M,而從大豆中萃取出的抑制劑與同一酵素結合之複合物(EI),其解離常數為7.4 X 10-7 M。當稻米抑制劑與胰蛋白"酉每"之濃度比為0.3時,可抑制百分之五十的胰蛋白"酉每"對受質L-BAPNA之水解活性,而黃豆抑制劑則需約0.5的比率才能抑制百分之五十的胰蛋白"酉每"對同一受質之水解活性。此研究結果顯示對胰蛋白"酉每"而言,稻米芽鞘所萃取出的抑制劑的抑制能力比大豆所萃取出的抑制劑為佳。因此,對此抑制劑更進一步的研究,在醫藥及食品上之應用,是一個重要且值得去探討之課題。
A rice trypsin inhibitor with molecular of around 18 KDa has been obtained through cationic and gel filtration columns from coleoptiles grown under submerge condition. This trypsin inhibitor was further characterized toward proteases of chymotrpsin and trypsin. It was found that the inhibition mode when competing with substrate L-N-α-benzoyl-arginine-para- nitroanilide (L-BAPNA) toward chymotrypsin was not a typical competitive mode. However, the inhibition mode when competing with L-BAPAN toward trypsin was found a typical competitive mode as that of soybean trypsin inhibitor. The EI complex dissociation constant, Kd, for rice trypsin inhibitor, toward trypsin was 4.0 x 10-7M, while it was 7.4 x 10-7M for soybean trypsin inhibitor. When the molar ratio of Rice trypsin inhibitor to trypsin was about 0.3, 50% of trypsin activity had been inhibited; while it was about 0.5 for soybean trypsin inhibitor. This study shows that rice trypsin inhibitor has better inhibition activity than soybean trypsin inhibitor does toward trypsin. Thus, it would be interesting and important to investigate further in the application of this inhibitor in medicinal and food chemistry.
中文摘要……………………………………………………………i
英文摘要……………………………………………………………ii
致謝…………………………………………………………………iii
表目錄………………………………………………………………vi
圖目錄………………………………………………………………vii
第一章、 前言
一、稻米基本特性…………………………………………………1
二、胰蛋白"酉每"…………………………………………………3
  胰蛋白"酉每"活性區…………………………………………5
  順序式催化機制………………………………………………6
三、胰凝乳蛋白"每酉"……………………………………………7
四、胰蛋白"每酉"抑制劑…………………………………………9
五、Bowman-Birk型抑制劑………………………………………12
六、抑制劑對酵素反應的影響………………………………….14
1. 不可逆的抑制作用……………………………………………14
2. 可逆的抑制作用………………………………………………15
  a.競爭型抑制……………………………………………….15
  b.非競爭型抑制…………………………………………….16
  c.不競爭型抑制…………………………………………….17
七、Michaelis-Menten equation………………………………19
八、胰蛋白"酉每"抑制劑的應用……………………………….21
九、研究目的…………………………………………………….23
第二章、 實驗
一、儀器………………………………………………………….24
二、藥品………………………………………………………….26
三、實驗材料…………………………………………………….28
四、實驗步驟…………………………………………………….29
樣品收集………………………………………………………….29
芽鞘蛋白質之萃取……………………………………………….29
陽離子交換管柱層析…………………………………………….30
膠體管柱層析…………………………………………………….31
蛋白質純化分析………………………………………………….32
酵素活性測試…………………………………………………….32
第三章、 結果與討論………………………………………….35
參考文獻………………………………………………………….64
表一、經由CM-FF陽離子交換管柱後各部分蛋白質比例…………36
表二、N-terminal sequence定序及LC-MS質譜鑑定報告中符合於Bowman-Birk trypsin inhibitor片段的部分…………………38
表三、同源性比對後與soybean Bowman-Birk trypsin inhibitor(SBBI)相同之處………………………………………44
附表一、貴儀中心蛋白質身份鑑定質譜分析結果………………69
圖一、chymotrypsin對受值催化的反應機制及活性部位相關位 置………………………………………………………………5
圖二、競爭型抑制行為的抑制機制以及依其反應速率對基質濃度作圖及雙倒數做圖……………………………………….16
圖三、非競爭型抑制行為的抑制機制以及依其反應速率對基質濃度作圖其做圖…………………………………………….17
圖四、反競爭型抑制行為的抑制機制以及依其反應速率對基質濃度作圖其做圖…………………………………………….18
圖五、藉由CM FF陽離子交換管柱分離在不同梯度下純化稻米牙鞘蛋白質……………………………………………….46
圖六、藉由CM FF陽離子交換管柱分離純化稻米牙鞘蛋白質……46
圖七、透析後沈澱之蛋白質經由100mM醋酸鈉回溶後在相同條件下注入CM-FF陽離子交換管柱…………………………47
圖八、經由CM FF陽離子交換管柱後各部份蛋白質組成…………47
圖九、經由CM FF陽離子交換管柱後的第二部分再由膠體層析管柱 Sephadex G-50 superfine……………………….48
圖十、經由Sephadex G-50 superfine管柱後第二部份以SDS-PAGE鑑定蛋白質成份………………………………..48
圖十一、L-BAPNA 受胰蛋白"酉每"水解後會產生p-nitroanilide….49
圖十二、L-BAPNA加入胰蛋白"酉每"後做全波長掃瞄…………………49
圖十三、固定受質與胰凝蛋白"酉每"濃度,改變rice inhibitor濃度,以吸收度對時間作圖…………………………….50
圖十四、固定chymotrypsin濃度為140μM,改變基質濃度將吸收度對時間作圖……………………………………….50
圖十五、固定[chymotrypsin] = 140μM和[soybean inhibitor] = 100μM,於不同基質濃度下反應,將吸收度對時間作圖…………………………………….51
圖十六、固定[chymotrypsin] = 140μM和[rice inhibitor]= 50μM,改變基質濃度將吸收度對時間作圖…………51
圖十七、固定chymotrypsin和黃豆抑制劑濃度,求出t = 10 s之瞬時速度,將反應速率對基質濃度作圖…………….52
圖十八、反應速率對基質濃度雙倒數(double-reciprocal)做圖,Y軸截距 = 1/Vmax,X軸截距 = (-1/KM)……52
圖十九、競爭性抑制作用,抑制劑與基質競爭相同的酵素活性區示意圖………………………………………………….53
圖二十、固定trypsin濃度為1.17μM,改變基質濃度將吸收度對時間作圖……………………………………………….53
圖二十一、固定[trypsin] = 1.17μM,[soybean inhibitor] =0.42μM,改變基質濃度將吸收度對時間作圖…….54
圖二十二、固定[trypsin] = 1.17μM,[soybean inhibitor] =0.42μM,改變基質濃度將吸收度對時間作圖…….54
圖二十三、固定胰蛋白"酉每"和稻米抑制劑濃度,求出t = 10 s之瞬時速度,將反應率對基質濃度作圖…………………….55
圖二十四、固定[trypsin] = 1.17μM,[soybean inhibitor]=0.42μM反應率對基質濃度雙倒數做圖…………….55
圖二十五、不同黃豆抑制劑濃度將1/V對1/[S]做圖…………….56
圖二十六、不同稻米抑制劑濃度將1/V對1/[S]做圖…………….56
圖二十七、以圖二十五之各斜率對黃豆抑制劑濃度作圖……….57
圖二十八、以圖二十六之各斜率對稻米抑制劑濃度作圖……….57
圖二十九、在酵素與不同抑制劑混和10分鐘後,以酵素活性對[I]/[E]做圖…………………………………………58
圖三十、胰蛋白"酉每"一級結構圖…………………………………….59
圖三十一、胰蛋白"酉每"三級結構圖………………………………….60
圖三十二、胰凝蛋白"酉每"三級結構圖……………………………….61
圖三十三、trypsin-trypsin inhibitor complex三級結構圖…62
圖三十四、胰蛋白"酉每"及胰凝蛋白"酉每"活性抑制區………………….63
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