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NMR Studies of Cooperativity in Hemoglobin


Adler, George (1980) NMR Studies of Cooperativity in Hemoglobin. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/089e-bj69.



In this part the binding of ligands, like oxygen and carbonmonoxide to hemoglobin and myoglobin, and the connection between the physiological functions and the structure of these proteins are discussed. The allosteric models, which have been proposed to explain the cooperative binding of oxygen, and the effect of single residue mutations on the structure of hemoglobin are reviewed. Finally, questions which have not yet been satisfactorily answered are raised about the structure and function of hemoglobin.


The binding of carbon monoxide to rabbit hemoglobin and to trifluoroacetonylated rabbit hemoglobin has been studied by ¹³C NMR and by ¹⁹F NMR. The ¹³C NMR studies show that CO binds preferentially to the β chains of rabbit hemoglobin in the absence of effectors such as 2,3-diphosphoglycerate (DPG) and inositol hexaphosphate.

The ¹⁹F spectrum of trifluoroacetonylated human adult hemoglobin, at low CO or O₂ ligation, indicated preferential a chain ligation (Huestis and Raftery, 1973, Biochemistry 12, 2531). There is a linear relationship between the fraction of the ligated signal of trifluoroacetonylated rabbit hemoglobin as monitored by ¹⁹F NMR and the overall ligation detected by optical density measurements at 650 nm. Therefore, the trifluoroacetonyl label does not, in the case of rabbit hemoglobin, distinguish initial β ligation from random ligation.

Both in the absence of and in the presence of DPG, the ¹⁹F spectra of partially ligated trifluoroacetonylated rabbit hemoglobin reveal only two significant resonances. These two resonances, which correspond to the deoxy (tense) and ligated (relaxed) structures of hemoglobin, are joined by two additional resonances when inositol hexaphosphate is added to the solution. The new resonances are presumably due to hemoglobin in intermediate structures between the relaxed and tense states.

The ¹⁹F chemical shifts of the deoxy and ligated TF labelled hemoglobin change due to the addition of organic phosphates. This observation implies that the organic phosphates bind to hemoglobin in both the deoxy and the ligated states.


Titrations of trifluoroacetonyl labelled human deoxy-, oxy-, carbonmonoxy-, nitrosylhemoglobin, aquamethemoglobin and cyanomethemoglobin have been followed by ¹⁹F NMR. The titration curve of trifluoroacetonyl human deoxyhemoglobin (HbTF) in the presence or absence of 2,3-diphosphoglycerate (DPG) is similar to that found by Huestis and Raftery (Proc. Natl. Acad. Sci. USA (1972) 69, 1887). Nitrosyl-, oxy-, and carbonmonoxy-HbTF in the absence of effectors exhibit pH dependence only below pH 5.5. The addition of inositol hexaphosphate (IHP) resulted in large upfield shifts of the fluorine resonance of HbTF(NO)₄, HbTF(O₂)₄. In addition, the fluorine signal of HbTF(CO)₄ and HbTF(O₂) exhibited large linewidths which is evidence for the exchange between two structures. The addition of DPG to HbTF(NO)₄ below pH 7.4 results in the appearance of a second resonance which indicates the presence of two different protein structures.

The titration of the labelled aquarnethemoglobin in the presence or in the absence of DPG supports the results of Huestis and Raftery (1972). In the presence of IHP trifluoroacetonyl Hb⁺(CN⁻)₄ shows a complicated ¹⁹F spectra which suggests that cyanornethernoglobin can have three different structures. The implication of these results on the mechanism of cooperativity and on the two state model is discussed.


The intracellular pH, and the binding of intracellular 2,3-diphosphoglycerate (2,3-DPG) to hemoglobin are studied in AA and SS blood. There is no significant difference in the intracellular pH of AA and SS erythrocytes in the fully oxygenated state. However, in the ³¹P spectrum of deoxygenated AA blood the resonance due to 2-P of 2,3-DPG is downfield, while in the ³¹P spectrum of deoxygenated SS blood this resonance is upfield, from the chemical shift of the same phosphorous signal in oxygenated blood. This difference in ³¹P chemical shifts is likely due to a lower intracellular pH of the SS erythrocytes relative to the AA erythrocytes.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Chemistry
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Dervan, Peter B.
Thesis Committee:
  • Dervan, Peter B. (chair)
  • Richards, John H.
  • Raftery, Michael Augustine
  • Goddard, William A., III
Defense Date:28 April 1980
Record Number:CaltechTHESIS:12112020-012735380
Persistent URL:
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:14025
Deposited By: Mel Ray
Deposited On:11 Dec 2020 20:28
Last Modified:22 Dec 2020 20:54

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