臺灣博碩士論文加值系統

人體的動態平衡 (homeostasis) 係仰賴充足的氧氣傳輸至各器官及組織細胞。一旦此動態平衡受到破壞，將可能導致人體細胞缺氧 (hypoxia)、器官功能障礙，並且衍生為多種氧缺陷導致之相關疾病，甚至危及到生命。人體的氧氣運輸系統基本上包含作為氧氣載體-血紅蛋白及藉由降低血紅蛋白對氧親和力以引導其適當地釋放氧氣的血紅蛋白內調因子-2,3-二磷酸甘油酸 (2,3-bisphosphorglycerate，2,3-BPG)。然而，隨著年紀增長，個體的2,3-BPG代謝會慢性退化，導致氧氣運輸缺陷之相關疾病的發病率增加，包含阿茲海默症及癌症。本論文之主要目的即係希望找出當人體2,3-BPG的含量無法有效幫助血紅蛋白釋放出充足的氧氣時，能夠有效替代2,3-BPG行使變構調控血紅蛋白之2,3-BPG功能性替代物 (2,3-BPG functional substitutes) 協助血紅蛋白更容易釋放氧氣至各器官及組織細胞，並了解其調控機制。本工作結合了共振拉曼光譜、紫外-可見光吸收光譜及氧氣平衡曲線測定等實驗技術以及分子對接計算模擬找出數種可能成為2,3-BPG功能性替代物之苯酞類化合物。研究結果顯示苯酞類化合物可能藉由與血紅蛋白的α1/α2之界面產生相互作用來穩定帶氧血紅蛋白於低氧親和力之緊張Tense (T) state的四級結構，並藉此促使血紅蛋白提高釋氧率。另外，本工作也發現苯酞類可以與2,3-BPG進行協同作用，補足2,3-BPG因含量不足所造成之釋氧能力受損。綜合上述結果，本工作提出了一苯酞類調控血紅蛋白的可能機制。除了正常成人之血紅蛋白外，本工作也探討苯酞類對於其他血紅蛋白變異物之變構效應，包含胎盤血紅蛋白 (fetal hemoglobin，HbF) 及β-thalassemia患者之血紅蛋白。實驗結果顯示，苯酞類化合物亦能幫助一些血紅蛋白變構物有效地釋放氧氣。此結果更進一步揭示苯酞類化合物對於胎盤血紅蛋白優越的變構調控能力，使其可望成為一個更有效率的血液代用品用於相關血液疾病的治療。本論文亦探討苯酞類化合物於治療多種氧損傷疾病之生醫應用。

The homeostasis of human body critically relies on the sufficient amount of oxygen delivered to organs and tissue cells. Failures to do so may lead to cell hypoxia, organ dysfunction, development of numerous oxygen defect related diseases or even premature death. The oxygen transport machinery for human beings essentially include the oxygen carrier- hemoglobin (Hb) and the endogenous Hb modulator- 2,3-bisphosphorglycerate (2,3-BPG) that guides Hb to properly release oxygen by lowering its oxygen affinity. However, the 2,3-BPG metabolism may degrade chronically with aging, resulting into higher incidence rates of oxygen transport defect related diseases, including Alzheimer’s disease (AD) and cancers. The major goal of this thesis is to identify potential species capable of substituting the function of 2,3-BPG to aid hemoglobin to better release oxygen when the 2,3-BPG metabolism is impaired. By combining the resonance Raman spectroscopy, UV-visible absorption spectroscopy and oxygen equilibrium experiments, and the computational molecular docking modeling, several phthalide derivatives are identified as the potential 2,3-BPG functional substitutes. It is reveals that phthalide species lower Hb’s oxygen affinity by interacting with its α1/α2 interface and stabilizing the oxygenated Hb in the low oxygen affinity tensed (T) state. Phthalides appear capable of cooperating synergetically with 2,3-BPG to fix the impaired oxygen delivery at defect 2,3-BPG levels. A mechanism of action underlying the Hb modulating ability of phthalides is proposed. The allosteric modulating ability of phthalides on Hb variants, including fetal hemoglobin (HbF) and β-thalassemia are also investigated. The results show that are also capable of modulating HbF to facilitate its oxygen delivery efficiency, helping to make HbF a more satisfactory promising blood substitute for treating numerous blood diseases. The biomedical implications of phthalides in treating several oxygen delivery defect related diseases and syndromes are also discussed.

Chapter 1. Introduction 1
1.1. Hemoglobin (Hb) 3
1.1.1. Normal adult hemoglobin (HbA) 3
1.1.2. Fetal hemoglobin (HbF) 7
1.2. Hb allosteric effectors 8
1.2.1. 2,3-BPG as endogenous Hb modulator 9
1.2.2. Other Hb coeffectors (pH value, CO2, temperature) 11
1.3. Common Hb structural-disorder induced blood 13
1.4. Oxygen transport defect related diseases 15
1.5. Blood-nourishing herbal medicines 17
1.5.1. Angelica sinensis (AS) 18
1.5.2. Pharmacological effects of AS 19
1.5.3. The major components of AS 19
1.6. Issues of interest 21

Chapter 2. Experiments 23
2.1. Introduction 23
2.2. Sample preparations 23
2.2.1. Hemoglobin purification 23
2.2.2. Material 24
2.2.3. Preparation of treated Hb 25
2.3. Experimental approaches 26
2.3.1. Resonance Raman spectroscopy 26
2.3.2. Ultraviolet-Visible (UV-Vis) absorption spectroscopy 28
2.3.3. Oxygen equilibrium experiments 30
2.3.4. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) 33
2.4. Molecular docking modeling 35

Chapter 3. The Effects of Angelica Sinensis on the Structure and Function of
Hemoglobin 38
3.1. Introduction 38
3.2. RR spectroscopy of hemoglobin (HbA) treated with AS plant extract
39
3.3. UV-Vis absorption spectra of AS treated with adult hemoglobin 42
3.4. The effect of AS on stabilizing the low affinity T state of oxyHb 44
3.2. Summary 46
Chapter 4. The Hb Modulatory Capability of Bioactive Components in AS and Their Potentials to Serve as 2,3-BPG Functional Substitutes 48
4.1. Introduction 48
4.2. RR spectroscopic studies of phthalide treated hemoglobin 50
4.3. UV-Visible absorption spectra of phthalide-treated hemoglobin 55
4.4. Bioactive phthalides of AS stabilize the low affinity T state of oxyHb 56
4.5. Oxygen equilibrium curve measurements for phthalides-treated
hemoglobin 58
4.6. Phthalide derivatives cooperate with 2,3-BPG synergetically to lower Hb oxygen affinity 63
4.7. Phthalide derivatives are effective for RBC 68
4.8. The intermolecular interactions between phthalides and Hb 69
4.9. Phthalide derivatives strengthen the T state stabilizing salt bridges associated with αArg141 77
4.10. Hb α1/α2 interface could be an efficient route to modulate Hb allostery
82
4.11. Summary 83

Chapter 5. The Capacities of Bioactive Components in AS in Modulating Hb Variants as the 2,3-BPG Functional Substitutes 85
5.1. Introduction 85
5.2. MALDI-TOF mass spectrometric study of cross-linker glutaraldehyde treated HbF 87
5.3. RR spectroscopy of HbF treated with phthalides 88
5.4. Oxygen equilibrium curve measurement for phthalides treated-HbF
91
5.5. Summary 97

Chapter 6. Conclusion 98
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