Kazuhiko Adachi

Structure and function of haemoglobin Philly (Tyr C1 (35) β→Phe)

Abstract We have purified haemoglobin Philly by isoelectric focusing on polyacrylamide gel, and studied its oxygen equilibrium, proton nuclear magnetic resonance spectra, mechanical stability, and pH-dependent u.v. difference spectrum. Stripped haemoglobin Philly binds oxygen non-co-operatively with high affinity. Inorganic phosphate and 2,3-diphosphoglycerate have little effect on the equilibrium curve, but inositol hexaphosphate lowers the affinity and induces co-operativity. These properties are explained by the nuclear magnetic resonance spectra which show that stripped deoxyhaemoglobin Philly has the quaternary oxy structure and that inositol hexaphosphate converts it to the deoxy structure. An exchangeable proton resonance at −8.3 p.p.m. from water, which is present in oxy- and deoxyhaemoglobin A, is absent in both these derivatives of haemoglobin Philly and can therefore be assigned to one of the hydrogen bonds made by tyrosine C1-(35)β, probably the one to aspartate H8(126)α at the α1β1 contact. Haemoglobin Philly shows the same pH-dependent u.v. difference spectrum as haemoglobin A, only weaker, so that a tyrosine other than 35β must be mainly responsible for this.

Mechanical stability of hemoglobin subunits: An abnormality in βS-subunits of sickle hemoglobin

Abstract In order to study the mechanism of the ease of precipitation of oxyhemoglobin S by mechanical shaking, the rates of precipitation of α- and β-subunits of oxyhemoglobin A and oxyhemoglobin S were compared. At pH 8.0, the α A -subunits precipitated rapidly, while the β A -subunits were very stable, although a part of β A -bunits converted to the hemichrome form. At pH 6.0, the β A -subunits precipitated rapidly while the α A -subunits were stable. Similar studies with β S -subunits showed that β S -subunits precipitated rapidly both at acidic and alkaline pHs. The abnormal precipitation of tetrameric oxyhemoglobin S during mechanical shaking may be due to this instability of the β S -subunits.

Image analysis studies of the degree of irreversible deformation of sickle cells in relation to cell density and Hb F level.

Summary We analysed, quantitatively by image analysis, the degree of irreversible deformation of red cells (SS cells) from patients with homozygous sickle cell disease, and studied the relationships among the degree of irreversible cell deformation, cell density, and Hb F level. SS cells from 2 5 patients (aged 1–36 years) whose Hb F levels ranged from 2.5%) to 40.0%. were fully oxygenated and then were separated into four fractions by density centrifugation. Every fraction was studied for morphology and Hb F content. We found that the irreversible deformation of SS cells from the circulation occurred mainly by elongation and that the degree of elongation was extremely variable. We also found that in the cells of patients with Hb F levels < 20% the degree of irreversible elongation increases as cell density increases, suggesting that dehydration occurs concomitantly with irreversible elongation. Statistical analysis (Student t test) indicated that there were significant differences (P= 0.008 or < 0. 001) in the degree of elongation among density‐fractionated SS cells from patients with Hb F < 20%. although there was no significant difference (P>0.1) among those from patients with Hb F ≥20%. We also found that cell density increased as Hb F level of the density fraction decreased in all patients with Hb F < 20% but not always in those with Hb F ≥20%. This suggests that cells with lower Hb F levels are selectively susceptible to dehydration. Furthermore, we found that the mean degree of irreversible elongation decreases linearly with increasing levels of Hb F and reaches the normal range at 21–24%. Since the degree of irreversible deformation of SS cells quantified by image analysis is directly related to cell density, and inversely to Hb F levels, mechanical stress or membrane damage caused by Hb S polymerization may be an important factor in the formation of dense cells in viva

Effects of β6 amino acid hydrophobicity on stability and solubility of hemoglobin tetramers

The relationship between different amino acids at theβ6 position of hemoglobin and tetramer stability was addressed by a site‐directed mutagenesis approach. Precipitation rates during mechanical agitation of oxyhemoglobins with Gln, Ala, Val, Leu and Trp at the β6 position increased 2, 5, 13, 21 and 53 times, respectively, compared with that for Hb A. There was a linear relationship between the log of the precipitation rate constant and amino acid hydrophobicity at the β6 position, suggesting that enhanced precipitation of oxy Hb S during mechanical agitation results in part from increased hydrophobicity of β6 Val. Deoxyhemoglobin solubility increased in the order of β6 Ile, Leu, Val, Trp, Gln, Ala and Glu suggesting that hydrophobic interactions between β6 Val and the acceptor site of another hemoglobin molecule during deoxy‐Hb S polymerization not only depend on hydrophobicity but also on stereospecificity of the amino acid side chain at the β6 position. Furthermore, our results indicate that hydrophobic amino acids at the β6 position which promote tetramer instability in the oxy form do not necessarily promote polymerization in the deoxy form.

Effects of heme addition on formation of stable human globin chains and hemoglobin subunit assembly in a cell-free system.

Our previous assembly studies to form hemoglobin hetero-dimers and -tetramers using a coupled transcription/translation cell-free system suggested that alpha-globin chains bind to nascent non-alpha chains during and/or soon after translation to promote hemoglobin formation [Adachi et al., J. Biol Chem. 2002 (277) 13415]. In this report effects of CN-hemin on subunit assembly were studied using this cell-free system. Addition of CN-hemin and excess unlabeled heme-containing partner chains during synthesis leads to formation of radiolabeled heme-containing alpha(h)beta(h) hetero-dimers. In contrast, in the absence of added CN-hemin, unlabeled heme-containing alpha or beta chains can assemble with newly synthesized radiolabeled beta- and alpha-globin chains to form heme-containing alpha(h)beta(h) and semi-alpha (alpha(h)beta(0)) or semi-beta (alpha(0)beta(h)) hetero-dimers, respectively. These results suggest the existence of semi-hemoglobins as intermediates prior to formation of heme-containing alpha(h)beta(h) and indicate transfer of heme from alpha and/or beta chains into semi-hemoglobin hetero-dimers to form heme-containing hetero-dimers.

Role of β112 Cys (G14) in Homo- (β4) and Hetero- (α2β2) Tetramer Hemoglobin Formation

In order to assess the role of β112 Cys in homo- and hetero-tetrameric hemoglobin formation, we expressed four β112 variants (β112Cys→Asp, β112Cys→Ser, β112Cys→Thr, and β112Cys→Val) and studied assembly with α chainsin vitro. β112 Cys is normally present at β1β2 and α1β1interaction sites in homo- (β4) and hetero-tetramers (α2β2). β4 formation in vitro was influenced by the amino acid at β112. β112 Asp completely inhibited formation of homo-tetramers, whereas β112 Ser showed only slight inhibition. In contrast, β112 Thr or Val enhanced homo-tetramer formation compared with βA chains. Association constants for homo-tetramer formation increased in the order of β112Cys→Ser, βA, β112Cys→Thr, and β112Cys→Val, whereas the value for β112Cys→Asp was zero under the same conditions. These β112 changes also affected in vitroα2β2 hetero-tetramer formation. Order of α2β2 formation under limiting α-globin chain conditions showed Hb βC112S > Hb A > Hb S = Hb βC112T = Hb βC112V >>> Hb βC112D. Hb β112D can form tetrameric hemoglobin, but this β112 change promotes dissociation into α and β chains instead of αβ dimer formation upon dilution. These results indicate that amino acids at α1β1 interaction sites such as β112 on the G helix play a key role in stable αβ dimer formation. Our findings suggest, in addition to electrostatic interaction between α and β chains, that dissociation of β4 homo-tetramers to monomers and hydrophobic interactions of the β112 amino acid with α chains governs stable α1β1 interactions, which then results in formation of functional hemoglobin tetramers. Information gained from these studies should increase our understanding of the mechanism of assembly of multi-subunit proteins.

Crystal structure of deoxy-human hemoglobin beta6 Glu --> Trp. Implications for the structure and formation of the sickle cell fiber.

An atomic-level understanding of the interactions between hemoglobin molecules that contribute to the formation of pathological fibers in sickle cell disease remains elusive. By exploring crystal structures of mutant hemoglobins with altered polymerization properties, insight can be gained into sickle cell hemoglobin (HbS) polymerization. We present here the 2.0-Å resolution deoxy crystal structure of human hemoglobin mutated to tryptophan at the β6 position, the site of the glutamate → valine mutation in HbS. Unlike leucine and isoleucine, which promote polymerization relative to HbS, tryptophan inhibits polymerization. Our results provide explanations for the altered polymerization properties and reveal a fundamentally different double strand that may provide a model for interactions within a fiber and/or interactions leading to heterogeneous nucleation.

Polymerization of Three Hemoglobin A2 Variants Containing Valδ6 and Inhibition of Hemoglobin S Polymerization by Hemoglobin A2

To understand determinants for hemoglobin (Hb) stability and Hb A2 inhibition of Hb S polymerization, three Valδ6 Hb A2 variants (Hb A2 δE6V, Hb A2 δE6V,δQ87T, and Hb A2 δE6V,δA22E,δQ87T) were expressed in yeast, and stability to mechanical agitation and polymerization properties were assessed. Oxy forms of Hb A2 δE6V and Hb A2 δE6V,δQ87T were 2- and 1.6-fold, respectively, less stable than oxy-Hb S, while the stability of Hb A2 δE6V,δA22E,δQ87T was similar to that of Hb S, suggesting that Alaδ22 and Glnδ87 contribute to the surface hydrophobicity of Hb A2. Deoxy Hb A2 δE6V polymerized without a delay time, like deoxy Hb F γE6V, while deoxy Hb A2 δE6V,δQ87T and deoxy Hb A2 δE6V,δA22E,δQ87T polymerized after a delay time, like deoxy Hb S, suggesting that β87 Thr is required for the formation of nuclei. Deoxy Hb F γE6V,γQ87T showed no delay time and required a 3.5-fold higher concentration than deoxy Hb S for polymerization, suggesting that Thr effects on Valδ6 Hb A2 and Valγ6 Hb F variants are different. Mixtures of deoxy Hb S/Hb A2 δE6V,δQ87T polymerized, like deoxy Hb S, while polymerization of Hb S/Hb A2 δE6V mixtures was inhibited, like Hb S/Hb F γE6V mixtures. These results suggest α2βSδ6 Val, 87 Thr hybrids and Hb A2 δE6V,δQ87T participate in Hb S nucleation, while only 50% of α2βSδ6 Val hybrids and none of the Hb A2 δE6V participate. These findings are in contrast to those of mixtures of Hb S with Hb F γE6V or Hb F γE6V,Q87T, which both inhibit Hb S polymerization. Our results also suggest participation in nucleation of some α2βSδ hybrids in A2S mixtures but not α2βSγ hybrids in FS mixtures.

Polymerization and Solubility of Recombinant Hemoglobins α2β26VAL (HB S) and α2β26LEU (HB LEU)

In an effort to clarify the role of amino acid hydrophobicity at the β6 position in sickling we have made recombinant hemoglobin tetramers containing β6 Val (Hb S) and β6 Leu (Hb Leu). Recombinant Hb S and Hb Leu had the same electrophoretic mobility, chromatographic behavior, and absorption spectrum. The deoxy form of both tetramers polymerized in high phosphate buffer (1.8 M) and exhibited distinct delay times prior to polymerization. The kinetics of polymerization.for recombinant and native Hb S were similar, while recombinant Hb Leu polymerized more readily. The solubility of deoxy Hb Leu was less than deoxy Hb S, indicating that rapid polymerization and decreased solubility of deoxyhemoglobin is accelerated with increasing hydrophobicity at the β6 position.

HB Osler [β145(HC2)TYRàASP] Results from Posttranslational Modification

We studied two members of an African American family with eryth-rocytosis. An abnormal hemoglobin variant with an electrophoretic pattern on cellulose acetate similar to Hb J was identified. The oxygen dissociation curve using whole blood was biphasic, dramatically left-shifted, and hyperbolic. Sequence analysis of DNA from the proband showed heterozygosity for a TaA change at the first position of codon 145 in the β-globin gene which results in the substitution of an asparagine residue for normal tyrosine. The second cycle of C-terminal amino acid sequence analysis of a mixture of a- and β-globin chains showed tyrosine, aspartic acid, and small amounts of asparagine. Collectively, these results indicate the existence of a mutation at codon 145 of the β-globin gene which encodes for asparagine instead of tyrosine, and that asparagine then undergoes a partial posttranslational deamidation to aspartic acid. This amino acid substitution corresponds to Hb Osler, which is a high oxygen affinity hemoglobin vari...