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Resonance Raman Study on a Mechanism of Quanternary Structual Change of Human Hemoglobin A
https://ir.soken.ac.jp/records/329
https://ir.soken.ac.jp/records/329baea526f-ba43-40d9-910d-72c8a02b4ff0
| 名前 / ファイル | ライセンス | アクション |
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要旨・審査要旨 / Abstract, Screening Result (408.1 kB)
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本文 / Thesis (5.2 MB)
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| Item type | 学位論文 / Thesis or Dissertation(1) | |||||||
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| 公開日 | 2010-02-22 | |||||||
| タイトル | ||||||||
| タイトル | Resonance Raman Study on a Mechanism of Quanternary Structual Change of Human Hemoglobin A | |||||||
| タイトル | ||||||||
| タイトル | Resonance Raman Study on a Mechanism of Quanternary Structual Change of Human Hemoglobin A | |||||||
| 言語 | en | |||||||
| 言語 | ||||||||
| 言語 | eng | |||||||
| 資源タイプ | ||||||||
| 資源タイプ識別子 | http://purl.org/coar/resource_type/c_46ec | |||||||
| 資源タイプ | thesis | |||||||
| 著者名 |
長友, 重紀
× 長友, 重紀
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| フリガナ |
ナガトモ, シゲノリ
× ナガトモ, シゲノリ
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| 著者 |
NAGATOMO, Shigenori
× NAGATOMO, Shigenori
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| 学位授与機関 | ||||||||
| 学位授与機関名 | 総合研究大学院大学 | |||||||
| 学位名 | ||||||||
| 学位名 | 博士（理学） | |||||||
| 学位記番号 | ||||||||
| 内容記述タイプ | Other | |||||||
| 内容記述 | 総研大乙第116号 | |||||||
| 研究科 | ||||||||
| 値 | 数物科学研究科 | |||||||
| 専攻 | ||||||||
| 値 | 08 機能分子科学専攻 | |||||||
| 学位授与年月日 | ||||||||
| 学位授与年月日 | 2003-03-24 | |||||||
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| 値 | 2002 | |||||||
| 要旨 | ||||||||
| 内容記述タイプ | Other | |||||||
| 内容記述 | The quaternary structural change of human hemoglobin A (Hb A) was studied by resonance Raman spectroscopy. The quaternary structural change of Hb A occurs upon ligand (oxygen, Carbon monoxide (CO) and nitric oxide (NO)) binding to hemes and has a close correlatuon with oxygen affinity and cooperativity. It is known that no ligand-bound form of Hb A (deoxyHb A) adopts T (tense) structure with the low affinity extreme and that fully ligand-bound form of Hb A (COHb A) adopts R (relaxed) structure with the high affinity extreme. The most important problem to be answered for the quaternary structural change of Hb is when and how it occurs. However, generally it is difficult to detect a partially ligand-bound form of Hb A in a solution condition, because most of Hb A molecules are present in either no ligand-bound form or fully ligand-bound form. Therefore, in this study were selected the following Hbs in which a partially ligand-bound form can be stabilized in solution conditions: NOHb, Ni-Fe hybrid Hb, and Hb M Boston. In Part I the results from ultraviolet resonance Raman (UVRR) studies on NOHb (αNOβdeoxy),Ni-Fe hybrid Hb (αNiβandαCOβNi),and Hb M Boston (αMmetβCO) will be discussed. NOHb has the property that NO binds heme more strongly than CO. As the dissociation rate of NO is quite different between the α-heme and β-heme, it is possible to prepare a stable intermediate in which NO is bound only to α-heme. In this case a condition of the Fe-His bond can be controlled by pH or addition of IHP: the heme can be made five- or six-coordinate state. Therefore the author can investigate the effect of the Fe-His bond of α-heme on the quaternary structural change of tetramer. Ni-Fe hybrid Hb has the property that CO does not bind to the Ni-heme. In this case, the author can investigate the quaternary structure of a half ligand-bound form. Hb M Boston has the property that CO does not bind to ferric α-abnormal chain. Hb M Boston is not exactly the same as Hb A owing to the difference in a distal residue of α-chain. Recently it is reported that Hb M Boston has cooperatively at high pH (pH = 9). This suggests that Hb M Boston may induce a quaternary structural change. The quaternary structure of Hb M Boston in a partially ligand-bound form can be studied. The largest structural differences between the T and R structures revealed by X-ray crystallographic analysis, are located in the α1-β2 subunit interface. In this study an amount of the T and R structures in the intermediate states of quaternary structural change was estimated from UVRR spectral changes of the bands of tyrosine (Tyr) and tryptophan (TIP) residues. The quaternary structural change from T structure (deoxyHb A) to R structure (COHb A) results in the lower frequency shifts of Y8a and Y9a bands of tyrosine and intensity reduction of W3, W16, W18 bands of tryptophan. The results from the measurements of the three Hbs are as follows. The quaternary structures of partially ligand-bound forms in NOHb (αNOβdeoxy), Ni-Fe hybrid Hb (αNiβCO) and (αCOβNi), and Hb M Boston (αMmetβCO) depend on pH and the absence or presence of IHP(inositol-hexakis-phosphate), and cannot be described by superimposition of only T and R structures which are limiting structures. Generally the ligand (CO not NO) binding to α-heme causes both lower frequency shifts of the special bands of tyrosine and intensity reduction of the special bands of tryptophan, but the ligand binding to β heme causes only the intensity reduction of the special bands of tryptophan, although the ligand binding to either α□, or β□ heme at lower pH (pH 6.3～6.7) in the presence of IHP apparently causes no spectral change. This suggests that the roles of α-heme and β-heme (their Fe-His bonds) in the quaternary structural change are different. Bindung of NO and CO to α-heme yields clear difference between the two ligands. Althounh the special bands of both tyrosine and tryptophan changed in the case of CO, the special bands of neither tyrosine nor tryptophan changed by NO even at pH 8.8 in the absence of IHP. This suggests that the difference in coordination ability between CO and NO influences the proximal His-Fe bond in α-heme, which reflects the large difference in the quaternary structural change. On the other hand, the ligand binding to β-heme can be discussed from another views when αNIβCO and Hb M Boston (αMmetβCO) are compared with αNOβNO, because αNOβdeoxy showed T structure at even higher pH (pH = 8.8) in the absence of IHP. But αNOβNO showed the R characteristics including both the lower frequency shifts of special bands of tyrosine and intensity reduction of special bands of tryptophan. It is different from the case of αNiβCO (or αMmetβCO) that the NO (or CO) binding to β-heme in αNOβdeoxy causes the lower frequency shifts of the special bands of tyrosine at lower pH and in the presence of IHP. The most important difference between αNiβCO (or αMmetβCO) and αNOβNO is whether the sixth coordination site of α-heme is occupied by a ligand (NO) or not. This suggests that the quaternary structural change caused by the CO (NO) binding to β-heme depends on the coordination state in α-heme. The network involving the distal histidine such as Fe-NO---His in α-heme may also have a close connection with the change of tyrosune. In conclusion of Part I, the change of tryptophan and tyrosine upon the quaternary structural change due to ligand (CO) binding to α-heme or β-heme can be summarized in the following way. CO binding to α-heme causes changes of both tryptophan and tyrosine and the changes do not depend on the state of β-heme. On the other hand, CO binding to β-heme causes a change of tryptophan only, but the change of tyrosine strongly depends on the state of a-heme. Thus CO binding to a-heme seems to induce the quaternary structural change more strongly than that to β-heme. In Part II the relation between the function and structure of Hb which has very low affinity and apparently no cooperativity is treated. This type of Hb can be prepared under low pH in the presence of strong allosteric effector such as bezafibrate (BZF). Generally it has been considered that binding of ligands to Hb causes a quaternary structural change. However it is reported that ligand binding of Hb occurs with no cooperativity but that judging from the 1H NMR signal, the quaternary structural change takes place. To investigate the relation between quaternary structure and cooperativity, this type of Hb is examined with resonance Raman spectroscopy. The quaternary structural change upon ligand (CO) binding was also observed by resonace Raman spectroscopy for Hb which has very low affinity and apparently no cooperativity due to the strong allosteric effector. The R structure in the presence of the strong allosteric effector was not spectrally different from the R structure of normal HbA. The effects of strong allosteric effector appeared in a rate of structural relaxation after CO photodissociation, which is usually of microsecond order. In the presence of allosteric effector, the structural change from R-structure to T-structure becomes faster. The Fe-His stretching frequency at 13 μs after CO photodissociation at pH 6.4 in the presence of IHP and BZF was lower by 5 cm-1 than that observed in the absence of the effectors, for which the number of CO molecules remaining on hemes was estimated to be 2.8. When the number of CO molecules bound to hemes was changed, the degree of the quaternary structural change from R-structure to T-structure was also changed. At pH 6.4 in the presence of IHP and BZF the quaternary structural change from R-structure to T-structure has finished at 4 μs after CO photodissociation, even if the number of CO molecules remaining on hemes is 3.5. However, at pH 8.8 in the absence of the effectors the quaternary structural change from R-structure to T-structure has not been completed at 13 μs, even if the number of CO molecules bound to hemes is 2.8. This suggests that the quaternary structural change from R-structure to T-structure occurs between R4 and T3 at pH 6.4 in the presence of IHP and BZF, and that at pH 8.8 in the absence of the effectors quaternary structural change from R-structure to T-structure has not been completed yet in 13 μs after CO photodissociation or that T structure cannot be maintained when the number of CO molecules bound to hemes is 2.8. This indicates that mixed allosteric effector, IHP and BZF, shifts the transition point of the quaternary structure from T2 → R3 to T3 → R4. |
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| 値 | 有 | |||||||
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| 内容記述タイプ | Other | |||||||
| 内容記述 | application/pdf | |||||||
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| 出版タイプ | AM | |||||||
| 出版タイプResource | http://purl.org/coar/version/c_ab4af688f83e57aa | |||||||
