臺灣博碩士論文加值系統

Haptoglobin（Hp）是一個血紅素結合蛋白，當血液中有游離的血紅素（Hemoglobin, Hb）時，Hb內Heme所帶的鐵離子會對於組織造成氧化壓力及細胞毒性﹐Hp便擔任血紅素清除及鐵離子再利用的重要角色，以避免血管及腎臟等組織受到損傷。
Hp在人體中有三種不同的型態，各是Hp 1-1，Hp 2-1，Hp 2-2。在先前的研究指出，Hp可以藉由和Hb結合，進而抑制由Hb所引發的LDL氧化反應。而Hp和Hb結合的能力又和Hp的型態有關係，在三種型態中Hp 1-1被指出具有最強的Hb結合能力及抗氧化能力。為了了解三種型態Hp的結構對於Hb的結合能力、Hb過氧化氫酶活性及抗氧化力的影響，因此首先需要獲得大量各種型態的Hp，再經化學修飾進行研究。我們利用本實驗室製備的Hp單株抗體製作免疫親和性層析管柱，用以純化三種型態的Hp，與以往方法（Hb親和性層析法）相較最大的好處便是可以減少Hb的污染。接著再利用化學修飾的方式改變Hp的結構，我們先使用還原劑將Hp的雙硫鍵還原，再使用carboxymethylation（CM）固定SH group，使得三種型態的Hp皆以單體的型式存在。另外利用acetylation（AC）和支鏈末端的amino group（-NH2，-NH3+）反應以改變Hp的電性，觀察電性對Hp-Hb分子間作用的影響。在Hp-Hb結合的測試發現，經CM及AC之後，Hp失去和Hb結合的能力，無法形成complex。在影響Hb過氧化氫酶的活性上，也由於修飾後的Hp無法和Hb結合而失去對Hb過氧化氫酶活性的影響力。令人意外的是，經CM及AC後的Hp雖然無法和Hb結合，卻仍保有抗氧化的能力，CM Hp甚至較原本Hp的抗氧化能力高出五倍之多，表示Hp的抗氧化能力和Hb結合特性並無絕對相關性，提供了另一種Hp抗氧化機制思考與研究方向。

Intravascular hemolysis is a physiologic phenomenon as well as a severe pathological complication when accelerated in various autoimmune, infection and inherited disorders. Efficient removal of free hemoglobin (Hb) is essential, because of the oxidative and toxic properties of iron-containing heme in Hb. The hepatic “clearance” of free Hb is mediated through plasma haptoglobin (Hp) by forming a Hp-Hb complex with an extremely high affinity (>1010 M-1).
In previous studies, the LDL oxidation induced by free Hb (such as free radicals) could be avoided by forming Hp-Hb complex. There are three different phenotypes (Hp1-1, 2-1, and 2-2 ) of Hp in human plasma (such as ABO blood type) and the binding affinity between Hp-Hb complex is dependant on the genetic polymorphism of Hp. In which Hp1-1 has shown to be superior to the other. The mechanism involved in the antioxidant characteristic of Hp phenotypes, however, is not clear. In this study, we used immunoaffinity column to purify these phenotypes and then determined the roles of disulfide-linkage and positively charged groups respectively by chemical modifications with carboxymethylation and acetylation. We found that carboxymethylated Hp and acetylated Hp lost Hb binding activity. In addition, carboxymethylated Hp and acetylated Hp did not alter the peroxidase activity of Hb. Excitingly, the antioxidant activity of Hp was enhanced substantially after carboxymethylation, even though carboxymethylated Hp lost its Hb binding capacity.

摘要……….………………………………………………………………i
英文摘要………………………………………………………………....ii
目錄……………………………………………………………………...iii
圖目錄…………………………………………………………………...vi
表目錄…………………………………………………...……………...viii
第一章 緒論……………………………………………………………..1
1.1 文獻回顧…………………………………………………………….1
1.1.1 Haptoglobin 的分子結構與表現型……………………………….1
1.1.2 Haptoglobin-Hemoglobin Complex ……...………………………..2
1.1.3 Haptoglobin 的抗氧化能力……………………………………….5
1.1.4 Haptoglobin 的其他功能………………………………………….7
1.1.5 Haptoglobin 的表現型與臨床疾病的關係……………………….7
1.2 研究目的…………………………………………………………….8
第二章 材料……………………………………………………………..9
2.1抗體……………..……………………………………………..……..9
2.2人類血漿……………………………………………………………..9
2.3化學試劑……………………………………………………………..9
2.4分析材料……………………………………………………………10
2.5 Marker………………………………………………………………12
2.6蛋白質定量…………………………………………………………12
2.7 親和性管柱基質…………………………………………………...12
2.8緩衝溶液(buffer)……………………………………………………12
第三章 方法…………………………………………….………………19
3.1製作免疫親和性層析管柱(Immunoaffinity chromatography column)…...19
3.2純化人類Haptoglobin（Hp）...…………………….……………...22
3.3 SDS-PAGE..............................................................…................…....23
3.4 西方墨點法………………………………………..………………..25
3.5 Carboxymethylation……………………………….………...……....27
3.6 Acetylation……………………………………….………………….28
3.7準備血紅素…………………………………….…………………....28
3.8 Hp-Hb結合能力測試……………………………………………….29
3.9準備人類低密度脂蛋白（LDL）…………….……………………30
3.10 Hp抗氧化能力測試……………………………………………….31
3.11 過氧化酶活性測試…………………………….………………….33
第四章 結果與討論…………………………………….……….……..34
4.1純化人類Haptoglobin（Hp）…………………..………….……...34
4.2化學修飾Hp………………………………………………………..44
4.3 Hp與血紅素結合能力測試………………………………………...47
4.4 Hp對血紅素過氧化酶活性的影響………………………...….…...50
4.5 Hp抗氧化能力……………………………………………...….…...56
第五章 總結………………………………………………………….…59
第六章 參考文獻…………………………………………………….…62
圖目錄
圖1.三種Hp結構圖……………………………………………………3
圖2.三種型態之Hp亞單元結構排列圖………………………………4
圖3. Hp-Hb結合方式假設圖………………………………………5
圖4. TBRs標準曲線趨勢圖…………………………………………32
圖5.免疫親和性管柱層析法純化Hp之光譜吸收圖…………………36
圖6. 12% SDS-PAGE分析免疫親和性管柱層析法純化Hp各階段.37
圖7. 西方墨點法檢測免疫親和性管柱層析法純化Hp各階段……38
圖8. 三種表現型Hp利用膠體過濾層析法純化Hp之光譜吸收圖…41
圖9. 15% SDS-PAGE分析Hp經膠體過濾層析前後之差異………42
圖10. 12% SDS-PAGE分析Hp經carboxymethylation前後之差異…45
圖11. Native PAGE檢測acetylation前後Hp之差異………………46
圖12. Native PAGE分析Hp-Hb結合能力…………………………48
圖13. Hp-Hb ELISA分析Hp-Hb結合能力…………………………49
圖14. 三種carboxymethyled Hp增強Hb過氧化酶活性的能力比較.52
圖15. 三種acetylated Hp增強Hb過氧化酶活性的能力比較………53
圖16. 三種carboxymethyled Hp抑制Hb過氧化酶活性的能力比較.54
圖17. 三種acetylated Hp抑制Hb 過氧化酶活性的能力比較力…55
圖18. Native Hp與carboxymethylated Hp抗氧化能力之比較圖…56
圖19. Native Hp與acetylated Hp抗氧化能力之比較圖……………57
表目錄
表1. Haptoglobin於純化各階段回收量分析表……………………43

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