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
References 101

1. Cui, Q. & Karplus, M. Allostery and cooperativity revisited. Protein. Sci. 17, 1295-1307 (2008).

2. Tsuneshige, A., Park, S. & Yonetani, T. Heterotropic effectors control the hemoglobin function by interacting with its T and R states - a new view on the principle of allostery. Biophys. Chem. 98, 49-63 (2002).

3. Takayanagi, M., Kurisaki, I. & Nagaoka, M. Non-site-specific allosteric effect of oxygen on human hemoglobin under high oxygen partial pressure. Sci. Rep-Uk. 4(2014).

4. Park, S.Y., Yokoyama, T., Shibayama, N., Shiro, Y. & Tame, J.R. 1.25 A resolution crystal structures of human haemoglobin in the oxy, deoxy and carbonmonoxy forms. J. Mol. Biol. 360, 690-701 (2006).

5. Monod, J., Wyman, J. & Changeux, J.P. On the nature of allosteric transitions: A plausible model. J. Mol. Biol. 12, 88-118 (1965).

6. Levantino, M., et al. The Monod-Wyman-Changeux allosteric model accounts for the quaternary transition dynamics in wild type and a recombinant mutant human hemoglobin. Proc. Natl. Acad. Sci. U. S. A. 109, 14894-14899 (2012).

7. Koshland, D.E., Nemethy, G. & Filmer, D. Comparison of experimental binding data and theoretical models in proteins containing subunits. Biochemistry 5, 365-385 (1966).

8. Perutz, M.F. Stereochemical mechanism of oxygen transport by haemoglobin. P. Roy. Soc. B-Biol. Sci. 208, 135-162 (1980).

9. Perutz, M.F., Wilkinson, A.J., Paoli, M. & Dodson, G.G. The stereochemical mechanism of the cooperative effects in hemoglobin revisited. Annu. Rev. Biophys. Biomol. Struct. 27, 1-34 (1998).


10. Perutz, M.F. Stereochemistry of Cooperative Effects in Haemoglobin. Nature 228, 726-734 (1970).

11. Yonetani, T., Park, S., Tsuneshige, A., Imai, K. & Kanaori, K. Global allostery model of hemoglobin. Modulation of O(2) affinity, cooperativity, and Bohr effect by heterotropic allosteric effectors. J. Biol. Chem. 277, 34508-34520 (2002).

12. Yoo, B.K., Kruglik, S.G., Lamarre, I., Martin, J.L. & Negrerie, M. Absorption band III kinetics probe the picosecond heme iron motion triggered by nitric oxide binding to hemoglobin and myoglobin. J. Phys. Chem. B 116, 4106-4114 (2012).

13. Kim, S., Park, J., Lee, T. & Lim, M. Direct observation of ligand rebinding pathways in hemoglobin using femtosecond did-IR spectroscopy. J. Phys. Chem. B 116, 6346-6355 (2012).

14. Yamada, K., Ishikawa, H. & Mizutani, Y. Protein dynamics of isolated chains of recombinant human hemoglobin elucidated by time-resolved resonance Raman spectroscopy. J. Phys. Chem. B. 116, 1992-1998 (2012).

15. Balakrishnan, G., et al. Time-resolved absorption and UV resonance Raman spectra reveal stepwise formation of T quaternary contacts in the allosteric pathway of hemoglobin. J. Mol. Biol. 340, 843-856 (2004).

16. Knapp, J.E., Pahl, R., Šrajer, V. & Royer, W.E. Allosteric action in real time: Time-resolved crystallographic studies of a cooperative dimeric hemoglobin. Proc. Natl. Acad. Sci. U. S. A. 103, 7649-7654 (2006).

17. Cammarata, M., Levantino, M., Wulff, M. & Cupane, A. Unveiling the timescale of the R–T transition in human hemoglobin. J. Mol. Biol. 400, 951-962 (2010).

18. Fischer, S., Olsen, K.W., Nam, K. & Karplus, M. Unsuspected pathway of the allosteric transition in hemoglobin. Proc. Natl. Acad. Sci. U. S. A. 108, 5608-5613 (2011).


19. Frier, J.A. & Perutz, M.F. Structure of human foetal deoxyhaemoglobin. J. Mol. Biol. 112, 97-112 (1977).

20. Motlagh, H.N., Wrabl, J.O., Li, J. & Hilser, V.J. The ensemble nature of allostery. Nature 508, 331-339 (2014).

21. Reynolds, K.A., McLaughlin, R.N. & Ranganathan, R. Hot spots for allosteric regulation on protein surfaces. Cell 147, 1564-1575 (2011).

22. Benesch, R., Benesch, R.E. & Enoki, Y. The interaction of hemoglobin and its subunits with 2,3-diphosphoglycerate. Proc. Natl. Acad. Sci. U. S. A. 61, 1102-1106 (1968).

23. Benesch, R., Benesch, R.E. & Yu, C.I. Reciprocal binding of oxygen and diphosphoglycerate by human hemoglobin. Proc. Natl. Acad. Sci. U. S. A. 59, 526-532 (1968).

24. Benesch, R. & Benesch, R.E. Intracellular organic phosphates as regulators of oxygen release by haemoglobin. Nature 221, 618-622 (1969).

25. Metcalfe, J., Dhindsa, D.S., Edwards, M.J. & Mourdjin.A. Decreased affinity of blood for oxygen in patients with low-output heart failure. Circ. Res. 25, 47-51 (1969).

26. Samaja, M., Diprampero, P.E. & Cerretelli, P. The role of 2,3-DPG in the oxygen transport at altitude. Respir. Physiol. 64, 191-202 (1986).

27. Bersin, R.M., Arieff, A.I. & Chatterjee, K. Importance of 2,3-diphosphoglyceric acid to oxygen transport in congestive heart failure. J. Am. Coll. Cardiol. 9, A186-A186 (1987).

28. Kaminsky, Y.G., et al. Age-related defects in erythrocyte 2,3-diphosphoglycerate metabolism in dementia. Aging Dis. 4, 244-255 (2013).

29. Kosenko, E.A., Aliev, G. & Kaminsky, Y.G. Relationship between chronic disturbance of 2,3-diphosphoglycerate metabolism in erythrocytes and Alzheimer disease. CNS Neurol. Disord. Drug 15, 113-123 (2016).

30. Kaminsky, Y.G., et al. Age-related defects in erythrocyte 2,3-diphosphoglycerate metabolism in Dementia. Aging Dis. 4, 244-255 (2013).

31. Elena, A.K., Gjumrakch, A. & Yury, G.K. Relationship between chronic disturbance of 2,3-diphosphoglycerate metabolism in erythrocytes and Alzheimer disease. CNS Neurol. Disord. Drug Targets 15, 113-123 (2016).

32. Fathallah, H. & Atweh, G.F. Induction of fetal hemoglobin in the treatment of sickle cell disease. Hematology Am. Soc. Hematol. Educ. Program, 58-62 (2006).

33. Galanello, R. & Cao, A. Gene test review. Alpha-thalassemia. Genet. Med. 13, 83-88 (2011).

34. Bassil, N. & Mollaei, C. Alzheimer's dementia: a brief review. J. Med. Liban. 60, 192-199 (2012).

35. Hock, C., et al. Decrease in parietal cerebral hemoglobin oxygenation during performance of a verbal fluency task in patients with Alzheimer's disease monitored by means of near-infrared spectroscopy (NIRS)--correlation with simultaneous rCBF-PET measurements. Brain. Res. 755, 293-303 (1997).

36. Arai, H., et al. A quantitative near-infrared spectroscopy study: a decrease in cerebral hemoglobin oxygenation in Alzheimer's disease and mild cognitive impairment. Brain. Cogn. 61, 189-194 (2006).

37. Roder, F. An analysis of Warburg view on the origin of cancer cells. Philos. Sci. 23, 343-347 (1956).

38. Pearl, P.L., Robbins, E.L., Bennett, H.D. & Conry, J.A. Use of complementary and alternative therapies in epilepsy: cause for concern. Arch. Neurol. 62, 1472-1475 (2005).

39. Kamboj, V.P. Herbal medicine. Curr. Sci. India. 78, 35-39 (2000).

40. Wu, Y.C. & Hsieh, C.L. Pharmacological effects of Radix Angelica Sinensis (Danggui) on cerebral infarction. Chin. Med. 6, 32 (2011).

41. Zhou, S.S., Tang, L.H., Sheng, H.F. & Wang, Y. [Malaria situation in the People' s Republic of China in 2004]. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi 24, 1-3 (2006).

42. Mei, Q.B., Tao, J.Y. & Cui, B. Advances in the pharmacological studies of radix Angelica sinensis (Oliv) diels (Chinese Danggui). Chin. Med. J-Peking 104, 776-781 (1991).

43. Lin, L.-Z., He, X.-G., Lian, L.-Z., King, W. & Elliott, J. Liquid chromatographic–electrospray mass spectrometric study of the phthalides of Angelica sinensis and chemical changes of Z-ligustilide. J. Chromatogr. A 810, 71-79 (1998).

44. Huang, L.F., Li, B.Y., Liang, Y.Z., Guo, F.Q. & Wang, Y.L. Application of combined approach to analyze the constituents of essential oil from Dong quai. Anal. Bioanal. Chem. 378, 510-517 (2004).

45. Teng, C.M., Chen, W.Y., Ko, W.C. & Ouyang, C. Antiplatelet effect of butylidenephthalide. Biochim. Biophys. Acta. 924, 375-382 (1987).

46. Tao, J.Y., et al. [Studies on the antiasthmatic action of ligustilide of dang-gui, Angelica sinensis (Oliv.) Diels]. Yao Xue Xue Bao 19, 561-565 (1984).

47. Mao, X.Q., Kong, L., Luo, Q.Z., Li, X. & Zou, H.F. Screening and analysis of permeable compounds in Radix Angelica Sinensis with immobilized liposome chromatography. J. Chromatogr. B 779, 331-339 (2002).

48. Kan, W.L.T., Cho, C.H., Rudd, J.A. & Lin, G. Study of the anti-proliferative effects and synergy of phthalides from Angelica sinensis on colon cancer cells. J. Ethnopharmacol. 120, 36-43 (2008).

49. Tsai, N.-M., et al. The natural compound n-butylidenephthalide derived from Angelica sinensis inhibits malignant brain tumor growth in vitro and in vivo(3). J. Neurochem. 99, 1251-1262 (2006).

50. Zijlstra, W.G., Buursma, A. & Meeuwsen-van der Roest, W.P. Absorption spectra of human fetal and adult oxyhemoglobin, de-oxyhemoglobin, carboxyhemoglobin, and methemoglobin. Clin. Chem. 37, 1633-1638 (1991).
51. Guarnone, R., Centenara, E. & Barosi, G. Performance-characteristics of Hemox-Analyzer for assessment of the hemoglobin dissociation curve. Haematologica 80, 426-430 (1995).

52. M.J. Frisch et al. Gaussian 03, . Gaussian, Inc., Pittsburgh, PA, 2003.

53. Fermi, G., Perutz, M.F., Shaanan, B. & Fourme, R. The crystal structure of human deoxyhaemoglobin at 1.74 Å resolution. J. Mol. Biol. 175, 159-174 (1984).

54. Morris, G.M., et al. Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J. Comput. Chem. 19, 1639-1662 (1998).

55. Arnone, A. X-ray-diffraction study of binding of 2,3-diphosphoglycerate to human deoxyhemoglobin. Nature 237, 146-149 (1972).

56. Erickson, J.A., Jalaie, M., Robertson, D.H., Lewis, R.A. & Vieth, M. Lessons in molecular recognition: The effects of ligand and protein flexibility on molecular docking accuracy. J. Med. Chem. 47, 45-55 (2004).

57. W.L. De Lano, D.L.S., San Carlos, CA, USA, . The PyMOL Molecular Graphics System. (2002).

58. Spiro, T.G. & Strekas, T.C. Resonance Raman spectra of heme proteins effects of oxidation and spin state. J. Am. Chem. Soc. 96, 338-345 (1974).

59. Abe, M., Kitagawa, T. & Kyogoku, Y. Resonance Raman spectra of octaethylporphyrinato-Ni(II) and meso-deuterated and 15N substituted derivatives. II. A normal coordinate analysis. J. Chem. Phys. 69, 4526-4534 (1978).

60. Torres Filho, I.P., Terner, J., Pittman, R.N., Proffitt, E. & Ward, K.R. Measurement of hemoglobin oxygen saturation using Raman microspectroscopy and 532-nm excitation. J. Appl. Physiol. 104, 1809-1817 (2008).


61. Terner, J., Voss, D.F., Paddock, C., Miles, R.B. & Spiro, T.G. Picosecond resonance Raman spectrum of the oxyhemoglobin photoproduct. Evidence for an electronically excited state. J. Phys. Chem. 86, 859-861 (1982).

62. Lao, S.C., et al. Identification and quantification of 13 components in Angelica sinensis (Danggui) by gas chromatography-mass spectrometry coupled with pressurized liquid extraction. Anal. Chim. Acta. 526, 131-137 (2004).

63. Carreau, A., El Hafny-Rahbi, B., Matejuk, A., Grillon, C. & Kieda, C. Why is the partial oxygen pressure of human tissues a crucial parameter? Small molecules and hypoxia. J. Cell. Mol. Med. 15, 1239-1253 (2011).

64. Johnson, M.E. & Ho, C. Effects of ligands and organic phosphates on functional properties of human adult hemoglobin. Biochemistry 13, 3653-3661 (1974).

65. Viggiano, G. & Ho, C. Proton nuclear magnetic resonance investigation of structural changes associated with cooperative oxygenation of human adult hemoglobin. Proc. Natl. Acad. Sci. U. S. A. 76, 3673-3677 (1979).

66. Kavanaugh, J.S., et al. Structure and oxygen affinity of crystalline desArg141 alpha human hemoglobin A in the T state. J. Mol. Biol. 248, 136-150 (1995).

67. Wang, D.J. & Spiro, T.G. Structure changes in hemoglobin upon deletion of C-terminal residues, monitored by resonance Raman spectroscopy. Biochemistry 37, 9940-9951 (1998).

68. Perutz, M.F., Fermi, G., Abraham, D.J., Poyart, C. & Bursaux, E. Hemoglobin as a receptor of drugs and peptides: x-ray studies of the stereochemistry of binding. J. Am. Chem. Soc. 108, 1064-1078 (1986).

69. Lalezari, I., Rahbar, S., Lalezari, P., Fermi, G. & Perutz, M.F. LR16, a compound with potent effects on the oxygen affinity of hemoglobin, on blood cholesterol, and on low density lipoprotein. Proc. Natl. Acad. Sci. U. S. A. 85, 6117-6121 (1988).

70. Papassotiriou, I., et al. Modulating the oxygen affinity of human fetal haemoglobin with synthetic allosteric modulators. Br. J. Haematol. 102, 1165-1171 (1998).
71. Chen, Q., Lalezari, I., Nagel, R.L. & Hirsch, R.E. Liganded hemoglobin structural perturbations by the allosteric effector L35. Biophys. J. 88, 2057-2067 (2005).

72. Barrick, D., Ho, N.T., Simplaceanu, V., Dahlquist, F.W. & Ho, C. A test of the role of the proximal histidines in the Perutz model for cooperativity in haemoglobin. Nat. Struct. Biol. 4, 78-83 (1997).

73. Kilmartin, J.V., Fogg, J.H. & Perutz, M.F. RRole of C-terminal histidine in the alkaline Bohr effect of human hemoglobin. Biochemistry 19, 3189-3193 (1980).

74. Bettati, S., et al. Structure and oxygen affinity of crystalline des-His-146 beta human hemoglobin in the T state. J. Biol. Chem. 272, 33077-33084 (1997).

75. Akinsheye, I., et al. Fetal hemoglobin in sickle cell anemia. Blood 118, 19-27 (2011).

76. Fathallah, H., Taher, A., Bazarbachi, A. & Atweh, G.F. Differences in response to fetal hemoglobin induction therapy in beta-thalassemia and sickle cell disease. Blood Cells Mol. Dis. 43, 58-62 (2009).

77. Faruqi, M. Blood disorders: Epigenetically enhancing haemoglobin production. Nat. Rev. Drug Discov. 12, 262-263 (2013).

78. Bhattacharya, N. A study of placental umbilical cord whole blood transfusion in 72 patients with anemia and emaciation in the background of cancer. Eur. J. Gynaecol. Oncol. 27, 155-161 (2006).

79. Schroeder, W.A., Shelton, J.R., Jones, R.T., Cormick, J. & Shelton, J.B. Amino acid sequence of gamma chain of human fetal hemoglobin. Biochemistry 2, 992-1008 (1963).