Magnetic Resonance Studies of Human Hemoglobins and Their Implications to the Structure‐function Relationships in Human Normal and Abnormal Hemoglobins *

Ho, Chien; Lindstrom, Ted R.; Baldassare, Joseph J.; Breen, J. J.

doi:10.1111/j.1749-6632.1973.tb15250.x

Cited by 40 publications

(34 citation statements)

References 29 publications

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“…Addition of 11 mM Ins-P6 at 31.6% O2 saturation ( Figure 3) causes a shift of the B peak by about 0.4 ppm downfield [as first reported by Ho et al (1973)l and an unquestionable decrease in the intensity of the 12-ppm a peak. This must be a true decrease in the area of the a peak because the total area (from 10 to 22 ppm) has decreased, whereas no substantial change can be observed in the area of the @ peak.…”

Section: Methodsmentioning

confidence: 75%

Proton nuclear magnetic resonance and biochemical studies of oxygenation of human adult hemoglobin in deuterium oxide

Viggiano¹,

Ho²,

Ho³

1979

Biochemistry

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The proton nuclear magnetic resonance spectrum of human adult deoxyhemoglobin in D2O in the region from 6 to 20 ppm downfield from the proton resonance of residual water shows a number of hyperfine shifted proton resonances that are due to groups on or near the alpha and beta hemes. The sensitivity of these resonances to the ligation of the heme groups and the assignment of these resonances to the alpha and beta chains provide an opportunity to investigate the cooperative oxygenation of an intact hemoglobin molecule in solution. By use of the nuclear magnetic resonance correlation spectroscopy technique, at least two resonances, one at approximately 18 ppm downfield from HDO due to the beta chain and the other at approximately 12 ppm due to the alpha chain, can be used to study the binding of oxygen to the alpha and beta chains of hemoglobin. The present results using approximately 12% hemoglobin concentration in 0.1 M Bistris buffer at pD 7 and 27 degrees C with and without organic phosphate show that there is no significant line broadening on oxygenation (from 0 to 50% saturation) to affect the determination of the intensities or areas of these resonances. It is found that the ratio of the intensity of the alpha-heme resonance at 12 ppm to that of the beta-heme resonance at 18 ppm is constant on oxygenation in the absence of organic phosphate but decreases in the presence of 2,3-diphosphoglycerate or inositol hexaphosphate, with the effect of the latter being the stronger. On oxygenation, the intensities of the alpha-heme resonance at 12 ppm and of the beta-heme resonance at 18 ppm decreases more than the total number of deoxy chains available as measured by the degree of O2 saturation of hemoglobin. This shows the sensitivity of these resonances to structural changes which are believed to occur in the unligated subunits upon the ligation of their neighbors in an intact tetrameric hemoglobin molecule. A comparison of the nuclear magnetic resonance data with the populations of the partially saturated hemoglobin tetramers (i.e., hemoglobin with one, two, or three oxygen molecules bound) leads to the conclusion that in the presence of organic phosphate the hemoglobin molecule with one oxygen bound maintains the beta-heme resonance at 18 ppm but not the alpha-heme resonance at 12 ppm. These resluts suggest that some cooperativity must exist in the deoxy quaternary structure of the hemoglobin molecule during the oxygenation process. Hence, these results are not consistent with the requirements of two-state concerted models for the oxygenation of hemoglobin. In addition, we have investigated the effect of D2O on the oxygenation of hemoglobin by measuring the oxygen dissociation curves of normal adult hemoglobin as a function of pH in D2O andH2O media. We have found that (1) the pH dependence of the oxygen equilibrium of hemoglobin (the Bohr effect) in higher pH in comparison to that in H2O medium and (2) the Hill coefficients are essentially the same in D2O and H2O media over the pH range from 6.0 to 8.2...

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Section: Methodsmentioning

confidence: 75%

Proton nuclear magnetic resonance and biochemical studies of oxygenation of human adult hemoglobin in deuterium oxide

Viggiano¹,

Ho²,

Ho³

1979

Biochemistry

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“…ppm unfield (Ho et al, 1973). The addition of DPG or IHP to deoxy Hb Chesapeake in 0.1 M Bis-Tris at pD 7 results in corresponding shifts in the /3-heme resonance and in the ct-heme resonance at -12.8.…”

Section: Resultsmentioning

confidence: 98%

Nuclear magnetic resonance studies of hemoglobin Chesapeake. α1β2 Mutant

Wiechelman¹,

Charache²,

Ho³

1974

Biochemistry

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Proton nuclear magnetic resonance studies have been used to compare the deoxy and ligand-bound forms of hemoglobins A and Chesapeake («92FG4 Arg -Leu). Partial saturation studies have also allowed us to study the equilibrium binding of oxygen and carbon monoxide to Hb Chesapeake. The hyperfine shifted resonances of the heme protons of hemoglobins Chesapeake and A differ, indicating that the deoxy quaternary structure of hemoglobin Chesapeake has been altered in some way. The ringcurrent shifted proton resonances of both HbCO and Hb02It has been shown that hemoglobins with mutations at the ai/32 subunit interface exhibit decreased heme-heme interactions (Perutz and Lehmann, 1968) indicating that the structural integrity of this region of the hemoglobin (Hb)1 molecule is critical for cooperative interactions in normal Hb A. X-Ray crystallographic studies have shown that the transition from the deoxy to the oxy quaternary structure results in a drastic change in the ai/32 interface. On oxygenation the two subunits shift by 13°relative to each other resulting in the breaking of the hydrogen bond between tyrosine-cv42(C7) and aspartic acid-/399(Gl) which helps stabilize the deoxy structure, and the formation of a new hydrogen bond between aspartate-«94(Gl) and asparagine-/3102(G4) in the oxy quaternary structure (Perutz, 1970;. The deoxy to oxy quaternarystructural change results in a slight loosening of the a\ß\ subunit interface but the two subunits do not shift relative to each other . The a-i/j2 interface is both smaller and smoother than the aidi subunit interface and it is constructed so that the subunits can easily slide past one another. It is closely connected to the heme groups so that any changes in the ß2 subunit interface would be expected to affect the heme environment . Thus studies of hemoglobins with mutations at the a i,d2 interface have proved to be invaluable in the study of structure-function relationships in hemoglobin.Hb Chesapeake is an a chain mutant with a leucine residue replacing the normal arginine residue at a92(FG4). This substitution results in a higher than normal oxygen aff From the

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“…(For recent reviews of our current 1H NMR studies of hemoglobins, see refs. 27 and 28.) MATERIALS AND METHODS Whole blood containing Hb M Milwaukee was drawn from a heterozygous patient and treated with acid-citrate-dextrose to prevent coagulation.…”

mentioning

confidence: 96%

Nuclear magnetic resonance study of heme-heme interaction in hemoglobin M Milwaukee: implications concerning the mechanism of cooperative ligand binding in normal hemoglobin.

Fung¹,

Minton²,

Ho³

1976

Proc. Natl. Acad. Sci. U.S.A.

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Magnetic Resonance Studies of Human Hemoglobins and Their Implications to the Structure‐function Relationships in Human Normal and Abnormal Hemoglobins *

Cited by 40 publications

References 29 publications

Proton nuclear magnetic resonance and biochemical studies of oxygenation of human adult hemoglobin in deuterium oxide

Proton nuclear magnetic resonance and biochemical studies of oxygenation of human adult hemoglobin in deuterium oxide

Nuclear magnetic resonance studies of hemoglobin Chesapeake. α1β2 Mutant

Nuclear magnetic resonance study of heme-heme interaction in hemoglobin M Milwaukee: implications concerning the mechanism of cooperative ligand binding in normal hemoglobin.

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