在人類及其他哺乳類動物中，血紅蛋白扮演輸送氧氣的重要角色。血紅蛋白是由兩個α 球蛋白單體和兩個β 球蛋白單體所組成結構穩定的四聚體蛋白質。α及β 單體在結構上雖然相似，二者卻無法相互取代且缺一不可。當α 及β 單體組成比例或是空間中排列的相對位向不正確時會導致血液疾病的產生，例如：地中海型貧血、鐮刀型貧血等。為深入了解α、β 單體間的交互耦合作用情形以及血紅蛋白如何分離 (disassemble) 成單體的過程，本工作利用有機汞化合物PMB (p-hydroxymercuribenzoate)與血紅蛋白上的半胱胺酸進行反應，使原本穩定的血紅蛋白四聚體結構產生鬆動，進而分離成單體。由於一個血紅蛋白分子包含三種不同的半胱胺酸殘基 (cysteine residue) 分別分佈在β93、α104、β112 位置上，本工作首先利用基質輔助雷射脫附游離飛行時間質譜儀 (Matrix-Assisted Laser
Desorption Ionization Time-Of-Flight Mass spectrometer, MALDI-TOF MS) 觀察血紅蛋白不同位置上的半胱胺酸殘基與PMB 的反應活性、反應順序以及其在單體分離過程中之必要性。為瞭解半胱胺酸殘基與PMB 反應後所導致的結構變化，本工作藉由共振拉曼光譜探討在有氧及無氧環境下，血紅蛋白與不同比例PMB反應所產生的結構轉變。最後結合以上實驗結果探討當PMB 與不同位置上的半胱胺酸殘基反應時可能造成的單體介面間作用力改變，並進而推衍出一血紅蛋白四聚體分離為單體的可能反應機制以及各不同位置上的半胱胺酸殘基在維持血紅蛋白四聚體結構中所扮演的樞鈕角色。

Hemoglobin plays an important role in transporting oxygen in human beings and other mammals. Hemoglobin is a tetrameric protein composed of two alpha and two beta subunits. The α and β subunits are both necessary and the stoichiometric ratio of the two dislike subunits is critical for hemoglobin to perform its oxygen-carrying function properly. To better understand the coupling between the α and β subunits and the subunit disassembly pathway, p-hydroxymercuri-benzoate (PMB) has been used to react with the cysteine residues in hemoglobin. The hemoglobin tetramer becomes unstable and disassembles into α and β subunits when the cysteine sites are perturbed
upon reacting with PMB. There are three kinds of cysteine residues, β93, α104 and β112, in human hemoglobin. The reactivity of different cysteine residues with PMB and their reaction sequence have been studied via the Matrix-assisted laser desorption
ionization time-of-flight mass spectrometry (MALDI-TOF MS). The resonance Raman spectroscopy has been used to investigate the structural changes of hemoglobin accompanying the PMB-modification under the oxygenated and deoxygenated conditions. At last, a hemoglobin subunit disassembly mechanism is proposed and the site-specific roles of cysteine residues in human hemoglobin are discussed in detail.

Chapter 1 Introduction……….........………………………1
1.1. Hemoglobin (Hb) .……………………………………3
1.2. Cysteine (Cys)…....…………………………………...7
1.2.1. The properties of cysteine……………….………..7
1.2.2 . The significance of cysteine in hemoglobin.…..8
1.3. Cysteine-specific reagents………………………...10
1.4. The reaction of hemoglobin with PMB…………....11
1.5. Issues of interest……………………………………14
Chapter 2 Experiments….……………………………....16
2.1. Introduction………………………………..…………16
2.2. Sample preparation…………………..…………….16
2.2.1. Hemoglobin……………………………………….17
2.2.2. Isolation of subunits…………………..………….18
2.3. Experiment techniques…………………………….21
2.3.1. Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS)...21
2.3.2. Resonance Raman spectroscopy…….………..23
Chapter 3 MALDI-TOF Mass Spectrometric Studies of Hb and PMB-modif i ed Hb…...............................…. . . . 26
3.1. Introduction...............................................…………...26
3.2. Number of Reactive Cysteine Residues to PMB..26
3.3. PMB reacts with Different Cysteine sites sequentially.........................................................................29
3.4. Reaction evolutions of PMB-labeled α and β globins…………….........................................................…34
3.5. All three cysteines are necessary to disassemble Hb…………….................................................................….37
3.6. Summary……………………..........…………………41
Chapter 4 Resonance Raman Spectroscopy of PMB-modified Hemoglobin…...................................................42
4.1 Introduction…………………………....……………...42
4.2 . Hemoglobin has strong absorption at 532 nm...42
4.3. Resonance Raman spectroscopy reveals internal structure changes accompanying the PMB-modification.........................................................................44
4.3.1. 1700-1200 cm-1 region…..……………………...46
4.3.2. 1200-600 cm-1 region………………..……….…53
4.4. Summary……………………………………………..58
Chapter 5 A Proposed Hemoglobin Subunit Disassembly Mechanism and the Roles of Cysteine Residues…………………………….......……………..…60
5.1. Introduction……………….…………….……………60
5.2. Inter-subunit salt bridges of hemoglobin………..60
5.3. Preliminary modification on βCys93 first unlocks α1/β2 interface…................................................................63
5.4. Modification on αCys104 partially unlocks the α1/β1 interfacial contacts……….................................……..70
5.5. Final modification on βCys112 completes the breaking of α1/β1 interface…………………….……….73
Chapter 6 Conclusion………….……………………….78
References………………………..……………………...81

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