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Hemoglobin (Hb) plays an important role in human bodies by serving as the oxygen-carrying protein. The oxygenation and deoxygenation processes are mainly achieved via the allosteric regulation between the high oxygen affinity, relaxed (R) state and the low oxygen affinity, tense (T) state. This thesis aims to investigate the influences of various allosteric effectors on the structures and functions of Hb by combining resonance Raman spectroscopy, UV-Vis absorption spectroscopy and computational docking approaches. The influences of several biologically important allosteric effectors have been investigated, including two homotropic effectors O2 and NO, and two heterotropic effectors, 2,3-bisphosphoglycerate (2,3-BPG) and inositol hexaphosphate (IHP). In addition, the influences of a prevalently used traditional Chinese medicine, Angelica sinensis (AS) on the structure and function of Hb have also been studied in this thesis due to its pharmacological significance in promoting the blood circulation and in treating numerous blood circulation dysfunction related syndromes and diseases. From the resonance Raman spectroscopic investigation, the bioactive components in AS responsible for the therapeutic efficacy of AS in altering the structure and function of Hb have been identified. With the aid of computational docking modeling, the structural character of the bioactive constituents and the active site on Hb has been discussed, from which a molecular mechanism was proposed for the first time to explain the therapeutic significance of AS.

Chapter 1. Introduction ..………………………………………..………………….. 1
1.1. Hemoglobin (Hb)……………….………............................................... 3
1.1.1. The Structure of Human Hemoglobin …..…………………...… 4
1.1.2. Hemoglobin Function ..………....……....…...…………………. 6
1.2. Allosteric Regulation ……..……….…………………..…….……….... 8
1.2.1. Allosteric States of Hb ……….…......……....…......…………... 9
1.2.2. Models of Allosteric Transition States ...……......……………. 11
1.2.3. Types of Allosteric Effectors ….…….…...…………...….…… 13
1.2.4. Pharmacological Effect of Allosteric Binding Sites …..…...… 15
1.3. Traditional Chinese Medicine ………...............………………….…. 16
1.3.1. Angelica sinensis (AS) .……………………………...…......... 17
1.3.2. Pharmacological Effects of AS .………...………………......... 18
1.3.3. The Major Components of AS ……….……………...…..….... 19
1.4. Spectroscopic Techniques …….….....……….………...…….……..... 22
1.4.1. Resonance Raman (RR) Spectroscopy ……..….…..…………. 22
1.4.2. Ultraviolet-Visible (UV-Vis) Absorption Spectroscopy …....... 23
1.5. Ligand-Protein Docking Modeling ………………………..…..…….. 24
1.6. Issues of Interest ….………..……....……..…..……………...……..... 25
Chapter 2. Experiments and Theoretical Modeling …….……….………………. 27
2.1. Introduction …….……..……...……..…...………………………...... 27
2.2. Sample Preparations ………...……….…………………..…….…..… 27
2.2.1. Hemoglobin Purification ………...……………………………. 27
2.2.2. Preparation of NO-Hb and SNO-Hb .….………………..…….. 28
2.2.3. Preparation of 2,3-BPG-treated Hb and IHP-treated Hb ..……. 29
2.2.4. Preparation of AS Plant Extract ……….……………………… 29
2.2.5. Preparation of AS-treated Hb and Phyto-treated Hb …....……. 30
2.3. Spectroscopic Measurements ……….....................................……….. 31
2.3.1. Resonance Raman (RR) Spectroscopy …...…..………….….... 31
2.3.2. Ultraviolet-Visible (UV-Vis) Absorption Spectroscopy …...… 32
2.4. Protein-Ligand Docking Modeling .…...…………......…....……….... 33
Chapter 3. The Effects of Homotropic and Heterotropic Effectors on Allosteric Properties of Hb ..…………………………….....……………………. 35
3.1. Introduction ………………..……………...….…………………….... 35
3.2. O2 and N2 Have the Reciprocal Effect in Hb Conformation ................. 36
3.3. Inter-subunit Interactions Changes Upon the Hb Conformational Transition …......................................................................................... 41
3.4. The Interaction of NO with Hb …..…………………...……............... 43
3.5. The Effects of Endogenous Effector 2,3-BPG in Hb .…....................... 51
3.6. IHP Also Stabilizes T State of Hb ……………….………………….. 54
3.7. Summary …..….…….………………………….………...………….. 56
Chapter 4. Angelica sinensis Stabilizes the Oxygenated Hb in the T State ….…. 58
4.1. Introduction ….…..……………………………….……….…….…… 58
4.2. Raman Spectral Features of AS-treated Hb ….….……………...……. 59
4.3. The Capacities of AS to Suppress the R State under the Oxygen Atmosphere ………………………………………………..……..….. 61
4.4. UV-Vis Absorption Spectra of AS-treated Hb ……….….….……….. 62
4.5. Summary ………..…………………………....………………….…... 65
Chapter 5. The Bioactive Components of AS Responsible for Inhibiting the R State of Oxygenated Hb ……………….………...…..….…….…….... 67
5.1. Introduction ….……………………………………..………………... 67
5.2. The Characteristics of AS’s Bioactive Phytochemical Components ... 67
5.3. RR Spectra of Phyto-treated Hb ….………...……….......…………… 70
5.4. The R State Inhibition Efficiency of Phyto-treated Hb under the Oxygen Atmosphere ……….…...…………………….…………………..…... 74
5.5. UV-Vis Absorption Spectra of Phyto-treated Hb ……..….…………. 76
5.6. Summary ……..…..……………………….......…………….…….…. 80
Chapter 6. The Structural Characters of 2,3-BPG and Bioactive Phytochemical Components of AS in Binding to the Active Sites of Hb ………..… 81
6.1. Introduction .......………………………....……………………...…... 81
6.2. The Intermolecular Interactions Between 2,3-BPG and Hb ……....... 82
6.3. The Intermolecular Interactions Between the Bioactive Components of AS and Hb ….……………..……..……………………..…............ 84
6.4. The Structural Significance of the Active Binding Sites upon
Interacting with Bioactive Phytochemical Components of AS …....... 91
6.5. Comparison between Phyto-Compounds of AS and 2,3-BPG on the Efficacy of Oxygen Transport Function ..……...………..…...………. 95
6.6. Summary …...………...……….………..………………………….... 97
Chapter 7. Conclusion .………………………………………………...………….. 98
References ………………………………………………………………………...… 102

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