Nuclear magnetic resonance studies of hemoglobin Chesapeake. α1β2 Mutant

Abstract

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

Proton nuclear magnetic resonance studies of hemoglobins M Boston (.alpha.58E7 His .fwdarw. Tyr) and M Milwaukee (.beta.67E11 Val .fwdarw. Glu): spectral assignments of hyperfine-shifted proton resonances and of proximal histidine (E7) NH resonances to the .alpha. and .beta. chains of normal human adult hemoglobin

“…This change in the sign of the chemical shift scale should be noted when referring to earlier publications reported by this laboratory. The three main peaks have been assigned to the protons from the ß, a, and a chains, respectively (Davis et al, 1971;, and have been used for the study of ligand binding to the a and ß chains of hemoglobins (Lindstrom et al, 1971;Ho et al, 1973;Johnson & Ho, 1974; Wiechelman et al, 1974;. It should be noted that the resonance at ~17 ppm consists of at least two components, both of which are derived from the ß chains.…”

“…A comparison of the spectra of Hb M Boston and Hb M Milwaukee allows us to assign individual ferrous hyperfine-shifted proton resonances to the a and ß chains of deoxy-Hb A. The *H NMR spectrum of deoxy-Hb A has three major resonances at 17.5, 12.2, and 8.0 ppm and several less intense or broader resonances.The three main peaks have been assigned to the protons from the ß, a, and a chains, respectively(Davis et al, 1971;, and have been used for the study of ligand binding to the a and ß chains of hemoglobins(Lindstrom et al, 1971;Ho et al, 1973;Johnson & Ho, 1974;Wiechelman et al, 1974;. It should be noted that the resonance at ~17 ppm consists of at least two components, both of which are derived from the ß chains.…”

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

“…Stock solutions of [bis(2-hydroxyethyl)imino]tris(hydroxyl)methane (Bistris; obtained from Aldrich), 2,3-diphosphoglycerate (obtained from Calbiochem), and inositol hexaphosphate (obtained from Sigma Chemical Co.) were prepared in D 2 0 as described previously (Wiechelman et al, 1974). All other chemicals were obtained from commercial companies as the reagent grade and were used without further purification.…”

Allosteric interpretation of haemoglobin properties

“…The MWC model introduces two quaternary structures of the haemoglobin molecule, and postulates that the affinity is purely linked to the quaternary structure. This model will be useful only if there exists a set of structure monitors which are observed to have only two values, one when a haemoglobin molecule has high affinity and a second when Kilmartin, and also Castillo (unpublished) Salhany et al (1974) Salhany et al (1975) Greer (1971a) Anderson (1973) Brunori et al (1970 Harbor and Koshland (1969) Maeda et al (1972) Cassoly et al (1971 Cassoly and Gibson (1972 Gibson and Nagel (1974) Wiechelman et al (1974) Nagai et al (1972 it has a low affinity. Table 15 shows that many structure sensitive measurements have this property.…”