Nuclear magnetic resonance and spin-label studies of hemoglobin Kempsey

Abstract

Nuclear magnetic resonance and spin-label studies of Hb Kempsey indicate that the substitution of asparagine for aspartate at G 1(99)0 produces widespread structural changes. The structural differences as well as concomitant changes in the functional properties indicate that the * subunit interface has a profound influence on the heme groups as they bind ligands. Comparison of the hyperfine shifted nuclear magnetic resonance (nmr) spectra of deoxyHb A and deoxyHb Kempsey shows that the structure of both a and 0 hemes is altered. Similar comparison of the ring-current shifted proton resonances of HbCO A and HbCO Kempsey X xemoglobin Kempsey is a mutant human hemoglobin which produces erythrocytosis in individuals heterozygous for Hb A1 and Hb Kempsey (Reed et al., 1968). Hb Kempsey differs from Hb A by a single amino acid substitution in the «102 subunit interface where the aspartic acid at 099 is replaced by an asparagine residue (Reed et al., 1968).Normally in deoxyHb A, the carboxy group of the aspartate at G 1(99)0 forms a hydrogen bond with the phenolic hydroxyl group of the tyrosine at C7(42)a and this hydrogen bond is broken in the transformation to the liganded form of Hb A (Perutz and Ten Eyck, 1971). In deoxyHb Kempsey, this important intersubunit hydrogen bond cannot be formed and the deoxy quaternary structure is less stable (Morimoto et al., 1971). In fact, the crystals of deoxyHb Kempsey have been found to have oxy-like quaternary structure (Perutz et al., 1974). This quaternary structural change in Hb Kempsey is thought to be the cause of the increased oxygen affinity and reduced cooperativity. In 0.05 m bis-tris + 0.1 m NaCl at pH 7.2 and 20°, the partial pressure of oxygen necessary to saturate 50% of the hemes {pbo) in Hb Kempsey is only 0.25 Torr whereas the pbi) of Hb A under similar conditions t From the Department of Biophysics and Microbiology and the

Restoring allosterism with compensatory mutations in hemoglobin.

“…The resonances from -1.7 to -1.9 ppm from DSS, which have been assigned to -A-and 'y2-methyl groups of (367EllVal (distal valine) (28,29), merged into one peak in the spectrum of the rHb((3:D99N, a:Y42D). This has also been observed for Hb Kempsey in the CO form (30). The exchangeable and ferrous hyperfine-shifted proton resonances of Hb A, rHb((B:D99N, a:Y42D), and Hb Kempsey in the deoxy form are shown in Fig.…”

“…4), indicating that there are structural alterations among these three Hbs. In contrast to Hb Kempsey, where the addition of IHP can convert its spectrum to one similar to that of deoxy-Hb A in the hyperfine-shifted proton resonance region (30,33), the addition of IHP to our double mutant did not cause any noticeable changes, as shown in Fig. 4 Fig.…”

Chain Inequivalence in Bovine Methemoglobin

“…The slow component of the valence intermediate from bovine blood has similar rate constants to those measured for the human valence intermediate (Tables 3 and 5) and is similar to the rate constants measured by Antonini et al [6].…”

“…The reaction proceeds by a single electron transfer from the heme iron to ferricyanide producing oxidized ferric heme. Although the overall reaction has been studied in great detail [6] less information is available concerning the oxidation of individual chains within the tetramer. It is evident that non-equivalence of the subunits will be observed most clearly when the hemoglobin molecules are either fully in the high-affinity state or all in the lowaffinity state.…”

Hemoglobin Variants: Biochemical Properties and Clinical Correlates

“…Asn) perturbs a1b2 interactions by preventing the formation of a hydrogen bond between b99 Asp and a42 Tyr, which normally stabilizes the deoxygenated low O 2 affinity T state (Fig. 3) (Reed et al 1968;Lindstrom et al 1973;Bunn et al 1974). This structural change shifts quaternary equilibrium toward the oxygenated R form, which impairs O 2 release to peripheral tissues and thus increases erythropoietic drive.…”