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研究生:張荏韋
研究生(外文):Jen-Wei Chang
論文名稱:胃蛋白酵素或胰蛋白酵素之蛋白質二級結構變化與其酵素活性之相關性研究
論文名稱(外文):Correlation Between Secondary Conformational Changes and Enzyme Activities of Pepsin or Trypsin
指導教授:林山陽林山陽引用關係段國仁段國仁引用關係
指導教授(外文):Prof. Shan-Yang LinProf. Kow-Jen Duan
學位類別:碩士
校院名稱:大同大學
系所名稱:生物工程學系(所)
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2004
畢業學年度:93
語文別:英文
論文頁數:157
中文關鍵詞:紅外光光譜儀蛋白質二及結構胃蛋白酶胰蛋白酶
外文關鍵詞:FT-IRpepsinprotein secondary structuretrypsin
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由於蛋白質容易受到外在環境,而影響到其原有之結構,為了探討蛋白質結構,除了以X光單晶繞射來得知外,尚可利用紅外光光譜儀來偵測蛋白質,並探討到蛋白質二級結構,且不需經過繁雜的結晶步驟,即可訊速的偵測該狀態的蛋白質樣品。本實驗除了利用紅外光光譜儀來探討蛋白質外,還利用酵素活性偵測方法來測定各種狀態下之蛋白質活性,希望能夠更進一步了解蛋白質之結構改變與酵素活性之相關性。本實驗選擇兩種酵素,分別為胃蛋白酵素以及胰蛋白酵素,分別在不同的pH緩衝溶液或添加鋁離子,及藥物 (enalpril、captopril、lisinopril、以及acetaminophen),來探討蛋白質在不同pH緩衝溶液中或與添加物反應後,蛋白質結構與酵素活性之相關性。其結果顯示當兩酵素處於不同pH緩衝溶液中時,其紅外線光圖譜amide I區域之面積改變與酵素之相對活性之間的相關性,是屬於負相關,以pH 5.0下之胰蛋白酵素的相關性最高,其相關係數為-1.0,而pH 8.0之胃蛋白酵素之相關係數較低-0.78;若以IR圖譜amide I二次微分之相似度與酵素相對活性來進行相關性之比較下,其結果屬於正相關,最高之相關係數以胰蛋白酵素與captopril反應之相關係數最高0.97,而最低的則是胰蛋白酵素內天加鋁離子之相關係數最低0.55;就整體而言,相關係數約有72 %高於0.8,代表著酵素活性的改變與蛋白質結構之改變具有相當高的相關性。
Structure of protein is easily denatured by the environmental condition. Protein structure is usually determined by X-ray diffraction. Infrared spectrum is another method to analyze primary and secondary structure of protein. Crystallization of protein can be avoided using infrared spectrometer to determine the structure of protein. The present study employed the infrared spectrum to determine structure changed of protein under various environmental conditions. Structure of protein determined by infrared spectrometer is related to the trypsin and pepsin activities of the present study. Influence of trypsin and pepsin to protein structure in various pH solutions, in various drugs (enalapril, captopril, lisinopril, and acetaminophen) and in aluminum ion was determined by infrared spectrometer, and related to enzyme activities. The infrared spectrum of trypsin and pepsin in various buffer indicated enzyme activity is negative relation to the area change in amide I region of infrared spectrum. The relation coefficient was –1.0 for trypsin in pH 5, while that for pepsin in pH 8 was –0.78. Secondary differential of infrared spectrum in amide I region is positive relation to the enzyme activity. The relation coefficient of pepsin and captopril was 0.97. While that for trypsin in aluminum ion was 0.55. Over 72 % of data in this study, the relation coefficient is more than 0.8. This indicates enzyme activity is closely relation to the secondary differential of infrared spectrum of protein.
ENGLISH ABSTRACT……….……………….………………………………………i
CHINESE ABSTRACT…….……………...............…………………………………iii
TABLE OF CONTENTS….……………......…….…………..……...………………..v
LIST OF FIGURES………………….......………………..………………………...viii
LIST OF TABLES……………….......………………………………….…………..xiii
CHAPTER I INTRODUCTORY…………………..…..…………………….….1
1.1 Introduction……………..…………..……………….……….………………1
1.2 Literature Reviews.....…………….………………….…….………………...3
1.2.1 Introduction to the pepsin................................................................…...3
1.2.2 Introduction to the trypsin................................................................…..5
1.2.3 Relation between the protein and metal ion...........................................8
1.2.4 Generally commonly used medicine introduction................................11
1.2.4.1 Introductions not becoming addiction pain killer acetaminophen………………………….…………………..11
1.2.4.2 Blood vessels shrink and usually change the ferment inhibitor.............................…................................................13
1.2.5 Utilize FT-IR to analyse the relevant technology of protein...............16
1.2.5.1 Protein structure summary.............................…...................16
1.2.5.2 Principle of FT-IR.............................…................................17
1.2.5.3 Advantage of only composing the appearance of infrared light.............................……………………………………..20
1.2.5.4 Deduct and include water or the method to buffer solution in the protein aqueous solution atlas.............................……....22
1.2.5.5 Curve fitting analyses second structure of protein................25
1.2.5.6 Relation of the protein , FT-IR and activation......................29
CHAPTER II MATERIAL AND METHOD…………………..……………...31
2.1 Material……………..…………..……………………….……..……………31
2.2 Instrument and equipment………………………...……………………….33
2.3 Experiment method…………………………………………………………34
2.3.1 Set up the basic method to analyse the protein....................................34
2.3.2 Protein structure under solid and aqueous solution type attitude…….40
2.3.3 Make protein structure and active behavior in different pH value…...42
2.3.4 Probe into the relation between protein and metal and ion…………..53
2.3.5 The protein and medicine function…………………………………...57
CHAPTER III RESULT AND DISCUSSION…………..………………….....64
3.1 Set up the basic method to analyse the protein………..……..……………64
3.1.1 Set up the basic method to analyse the protein....................................64
3.1.2 Atlas smooth principle.........................................................................72
3.1.3 Little of two times of atlas is judged....................................................75
3.2 Protein structure under solid and aqueous solution type attitude………..……..………………..……..………………..……..……..78
3.3 Structure in the lower pancreas albumen ferment of different pH value and dependence of the activation…………..……..…….82
3.3.1 Dependence of the stomach albumen ferment structure and activation under different pH value......................................................................82
3.3.2 Dependence of the pancreas albumen ferment structure and activation under different pH value........................................................................92
3.4 Function of the protein and aluminum ion…………..……..……103
3.4.1 Pepsin ferment and function of the aluminium ion............................103
3.4.2 Trypsin and function of the aluminium ion........................................111
3.5 The protein and medicine function…………..……..……………...116
3.5.1 Pepsin and medicine function............................................................116
3.5.2 Trypsin and medicine function..........................................................125
CHAPTER IV CONCLUSION…………..…………………..........................133
REFERENCES…………..…………………..........................................................135
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