Properties of hemoglobin G. Ferrara (beta57(E1) Asn replaced by Lys).

Hemoglobin G. Ferrara is an abnormal human hemoglobin in which an asparagine residue is replaced by a lysyl residue at position beta57 (beta57 Asn replaced by Lys). Oxygen equilibria show that cooperativity and alkaline Bohr effect are maintained to normal levels while the acid Bohr effect appears increased; in addition, a smaller effect of diphosphoglycerate is also observed. Flash photolysis experiments performed as a function of protein concentration show that the fraction of quickly reacting form is always higher than that of human hemoglobin A. This fact, together with the increase of the oxygen affinity observed at acid pH values, may be related to an enhanced dissociation of the molecule into dimers. Several attempts to isolate the native chains by treatment of the protein with p-chloromercuribenzoate were unsuccessful due to the great instability of the isolated variant beta-chains, which precipitated completely during incubation with p-chloromercuribenzoate. Therefore, although the substitution is on the surface of the molecule, there are several properties of hemoglobin G. beta Ferrara which are clearly different from hemoglobin A.     

Solution-active structural alterations in liganded hemoglobins C (beta6 Glu --> Lys) and S (beta6 Glu --> Val).

Based upon existing crystallographic evidence, HbS, HbC, and HbA have essentially the same molecular structure. However, important areas of the molecule are not well defined crystallographically (e.g. the N-terminal nonhelical portion of the alpha and beta chains), and conformational constraints differ in solution and in the crystalline state. Over the years, our laboratory and others have provided evidence of conformational changes in HbS and, more recently, in HbC. We now present data based upon allosteric perturbation monitored by front-face fluorescence, ultraviolet resonance Raman spectroscopy, circular dichroism, and oxygen equilibrium studies that confirm and significantly expand previous findings suggesting solution-active structural differences in liganded forms of HbS and HbC distal to the site of mutation and involving the 2,3-diphosphoglycerate binding pocket. The liganded forms of these hemoglobins are of significant interest because HbC crystallizes in the erythrocyte in the oxy form, and oxy HbS exhibits increased mechanical precipitability and a high propensity to oxidize. Specific findings are as follows: 1) differences in the intrinsic fluorescence indicate that the Trp microenvironments are more hydrophobic for HbS > HbC > HbA, 2) ultraviolet resonance Raman spectroscopy detects alterations in Tyr hydrogen bonding, in Trp hydrophobicity at the alpha1beta2 interface (beta37), and in the A-helix (alpha14/beta15) of both chains, 3) displacement by inositol hexaphosphate of the Hb-bound 8-hydroxy-1,3,6-pyrenetrisulfonate (the fluorescent 2,3-diphosphoglycerate analog) follows the order HbA > HbS > HbC, and 4) oxygen equilibria measurements indicate a differential allosteric effect by inositol hexaphosphate for HbC approximately HbS > HbA.     

Identification of hemoglobin G-Philadelphia (alpha 68 Asn replaced by Lys) and hemoglobin Matsue-Oki (alpha 75 Asp replaced by Asn) in a black infant

Two alpha-chain variants, Hb G-Philadelphia and Hb Matsue-Oki, were present in members of a relatively large black family from South Carolina. The four Hb G-Philadelphia heterozygotes averaged 35.6% Hb G, suggesting the presence of an alpha-thalassemia-2 condition in cis to the Hb G mutation, which was confirmed by DNA structural analysis. The seven Hb Matsue-Oki heterozygotes averaged 22.2% Hb MO and likely have four active alpha-chain genes. One infant was a compound heterozygote for the two Hb variants which could not be separated from each other. The quantity of Hb G plus Hb MO was 58% by DEAE-cellulose chromatography and 69% by chain analyses. These results and the family data indicate that this child had three active alpha-chain genes, of which one regulated the synthesis of the normal alpha chain, one was mutated to give the alpha G chain, and one to give the alpha MO chain. The amino acid substitutions in Hb G-Philadelphia and Hb Matsue-Oki are located in the tryptic peptide alpha T-9, which is 29 amino acid residues long. Structural analyses of these abnormalities made use of high-pressure liquid chromatography for the separation of both tryptic and thermolytic peptides and of a highly sensitive ultra-micro sequencing procedure. Although the alpha 68 Asn replaced by Lys substitution is readily demonstrable in Hb G-Philadelphia the elucidation of the alpha 75 Asp replaced by Asn replacement in Hb Matsue-Oki was greatly facilitated by the use of these microprocedures.     

Hemoglobin Athens-Georgia, or alpha 2 beta 2 40(C6)Arg replaced by Lys, a hemoglobin variant with an increased oxygen affinity.

A new hemoglobin variant, termed hemoglobin Athens-Georgia, has been found in a 23-year-old Caucasian student and three members of her family. The electrophoretic mobility of this variant at pH 9.0 is slightly less than that of hemoglobin-A. Arginyl residue in position 40 of the beta chain, corresponding to position 6 of the C helix, has been replaced by a lysyl residue. This amino acid substitution is at the alpha1-beta2 contact and slightly affects the oxygen binding properties of the hemoglobin molecule. Hemoglobin Athens-Georgia has an increased affinity for oxygen, a normal heme-heme interaction and a normal Bohr effect. Hematological abnormalities are not associated with this variant.     

Effect of the beta6 Glu replaced by Val mutation on the optical activity of hemoglobin S and of its beta subunits

The carbomonoxy derivatives of hemoglobin A and S showed a different optical activity in the Soret region of the spectrum as measured by circular dichroism. Different optical activity was also measured in the carbomonoxy derivatives of the beta subunits of hemoglobin A and S, the respective deoxy derivatives showed different circular dichroism spectra only in the presence of inositol hexaphosphate. Sedimentation velocity experiments showed that the differences in optical activity are not due to a different state of aggregation of the subunits. Modification of the tertiary structure of the beta subunits seems to be responsible for the phenomenon. Speculation based on the work of Hsu and Woody (Hsu, M.C., and Woody, R.W. (1971) J. Am. Chem. Soc. 93, 3515-3525) suggests the involvement of the beta15 tryptophan in the conformational changes produced by the beta6 Glu-Val mutation in hemoglobin S.     

Proton nuclear magnetic resonance studies of hemoglobin Providence (beta82EF6 Lys replaced by Asn or Asp): a residue involved in anion binding

High-resolution proton nuclear magnetic resonance studies of hemoglobins Providence-Asn (beta82EF6 Lys replaced by Asn) and Providence-Asp (beta82EF6 Lys replaced by Asp) show that different amino acid substitutions at the same position in the hemoglobin molecule have different effects on the structure of the protein molecule. Hemoglobin Providence-Asp appears to be in a low-affinity tertiary structure in both the deoxy and carbonmonoxy forms. Deoxyhemoglobin Providence-Asn has its beta heme resonance shifted downfield slightly from its position in normal adult hemoglobin; however, the tertiary structures of the heme pocket of hemoglobins A and Providence-Asn are very similar when both proteins are in the carbonmonoxy form. These results are consistent with the oxygen equilibrium measurements of Bonaventura, J., et al. [(1976) J. Biol. Chem. 251, 7563] which show that both Hb Providence-Asn and Hb Providence-Asp have oxygen affinities lower than normal adult hemoglobin, with Hb Providence-Asp having the lowest. Our studies of the effects of sodium chloride on the hyperfine shifted proton resonances of deoxyhemoglobins A, Providence-Asn, and Providence-Asp indicate that the beta82EF6 lysine is probably one, but not the only binding site for chloride ions.     

Clinicohematologic effects of hemoglobin Altdorf (alpha 2 beta 2 135 Ala replaced by Pro)

Hb Altdorf alpha 2 beta 2 135 Ala leads to Pro is an unstable variant occurring near Lecce in Italy. The abnormal hemoglobin does not separate from Hb A in the electrophoresis. In vitro a marked Heinz body formation is produced with phenylhydrazin. In heterozygous individuals an almost compensated hemolysis and a slight splenomegaly are found. Hemolysis can be aggravated by exogenous factors. A rather severe hemolysis was induced by a viral infection in a 3 years old girl.     

Conformational changes in hemoglobin S (betaE6V) imposed by mutation of the beta Glu7-beta Lys132 salt bridge and detected by UV resonance Raman spectroscopy

The impact upon molecular structure of an additional point mutation adjacent to the existing E6V mutation in sickle cell hemoglobin was probed spectroscopically. The UV resonance Raman results show that the conformational consequences of mutating the salt bridge pair, betaGlu(7)-betaLys(132), are dependent on which residue of the pair is modified. The betaK132A mutants exhibit the spectroscopic signatures of the R --> T state transition in both the "hinge" and "switch" regions of the alpha(1)beta(2) interface. Both singly and doubly mutated hemoglobin (Hb) betaepsilon7Alpha exhibit the switch region signature for the R --> T quaternary state transition but not the hinge signature. The absence of this hinge region-associated quaternary change is the likely origin of the observed increased oxygen binding affinity for the Hb betaepsilon7Alpha mutants. The observed large decrease in the W3 alpha14beta15 band intensity for doubly mutated Hb betaepsilon7Alpha is attributed to an enhanced separation in the A helix-E helix tertiary contact of the beta subunits. The results for the Hb A betaGlu(7)-betaLys(132) salt bridge mutants demonstrate that attaining the T state conformation at the hinge region of the alpha(1)beta(2) dimer interface can be achieved through different intraglobin pathways; these pathways are subject to subtle mutagenic manipulation at sites well removed from the dimer interface.     

Properties of hemoglobin A and hemoglobin Zurich (beta63 histidine replaced by arginine): quantitative evaluation of functional abnormalities in hemoglobins.

The abnormal hemoglobin Zurich (β₆₃ his→arg) exhibits abnormal properties. Thus, ν_CO occurs at 1951 cm^?1 for HbACO while HbZCO shows bands at 1950 cm^?1 and 1958 cm^?1 for CO bound in α and β chains respectively (the βCOs are displaced less readily by O₂). Acid catalyzed reductive displacement of superoxide by azide is slower on the β chain of HbZO₂ than on the α chain under conditions where with HbAO₂ both chains appear equally reactive. The one electron donor hydroquinone produces metHb and peroxide more rapidly from HbZO₂ than from HbAO₂. These property differences can be related to the β₆₃ residue. Such studies provide generally useful probes of the structural basis for hemoglobin diseases.     

Elastic properties of deoxy hemoglobin S (deoxy-HbS) gels

Equilibrium shear moduli for deoxyhemoglobin S(HbS) gels were measured as a function of HbS concentration. The modulus was found to depend approximately on the 18th power of the HbS concentration. Deoxy-HbS gels were found at HbS concentration as low as 10 g/dl.     

Hemoglobin Kariya [alpha 40 (C5) Lys leads to Glu]: a new hemoglobin variant with an increased oxygen affinity

A new abnormal hemoglobin, Hb Kariya [alpha 40 (C5) Lys leads to Glu], with an amino acid substitution at the alpha 1 beta 2 contact was discovered in a young Japanese man. This variant migrated to the anode faster than Hb A, being nearly the same as Hb I in electrophoretic mobility. It amounted to about 6% of the total hemoglobin of the hemolysate. This hemoglobin showed an increased oxygen affinity, decreased heme-heme interaction and a lowered 2,3-DPG effect.     

Allosteric interactions in non-alpha chains isolated from normal human hemoglobin, fetal hemoglobin, and hemoglobin Abruzzo (beta143 (H21) His replaced by Arg).

Oxygen-linked effects of inositol hexaphosphate occur in heme-containing non-alpha chains isolated from normal human hemoglobin, fetal hemoglobin, and the abnormal human hemoglobin Abruzzo, beta143(H21) His leads to Arg. The occurrence of these effects implies that the chains undergo ligand-linked conformational changes. Inositol hexaphosphate lowers the oxygen affinity of isolated beta and gamma chains by differential binding to their deoxy conformations. Neither 2,3-diphosphoglycerate nor inorganic phosphate produces such an effect. In the case of Abruzzo beta chains, the binding of inorganic phosphate and 2,3-diphosphoglycerate is also oxygen-linked. Stripped beta chains isolated from hemoglobin Abruzzo have much higher oxygen affinity than beta chains isolated from HbA. Their higher oxygen affinity and enhanced allosteric interactions with phosphates account, in large part, for the abnormal functional behavior of the hemoglobin Abruzzo tetramer. In this hemoglobin variant the substitution of arginine for histidine at beta143 involves a residue known to interact with anionic allosteric effectors of hemoglobin. It is of interest that the effect of inositol hexaphosphate observed with isolated gamma chains is comparable to the effect observed with isolated beta chains, even though the gamma143 position is occupied by an uncharged serine residue.     

Isolation and functional characterization of hemoglobin Casper: beta106(G8) Leu replaced by Pro.

Hemoglobin Casper (beta106Leu replaced by Pro) can be separated from hemoglobin (Hb) A by isoelectric focusing on polyacrylamide gel. This abnormal hemoglobin was estimated to be 30% of teh total by both isoelectric focusing and heat lability kinetics. Its oxygen equilibrium curves indicate a high oxygen affinity, low degree of subunit interaction, and a decreased Bohr effect. Mixtures of Hb Casper and Hb A appear to bind oxygen as if no hybrid molecules exist.     

Conformation in solution of hemoglobin Osler (alpha 2 A beta 2 145 Tyr replaced by Asp).

Computer simulations of Gelin and Karplus ((1977) Proc. Natl. Acad. Sci. U.S.A. 74, 801-805) suggest that in hemoglobin upon ligation the penultimate tyrosyl residues of the subunits are not expelled from the hydrophobic pockets described in the crystals between the helices E and F (Perutz, M.F. (1970) Nature 228, 726-737). This implies that both the liganded and unliganded conformations of hemoglobin may be affected by mutations involving such residues. Investigation of the conformational behavior of liganded and unliganded hemoglobin Osler was conducted measuring the functional properties, the subunits dissociation, the CD and electronic spectra, the protons absorption upon interaction with polyanions, and the reactivity of the -SH groups of the protein. The results suggest that both the liganded and unliganded conformations of the system are affected by the mutation, confirming the anticipations of Gelin and Karplus on the relevance of tyrosine at beta 145 for both allosteric states of hemoglobin.     

Effect of amino acid at the beta 6 position on surface hydrophobicity, stability, solubility, and the kinetics of polymerization of hemoglobin. Comparisons among Hb A (Glu beta 6), Hb C (Lys beta 6), Hb Machida (Gln beta 6), and Hb S (Val beta 6)

Surface hydrophobicity, stability, solubility, and kinetics of polymerization were studied using hemoglobins with four different amino acids at the beta 6 position: Hb A (Glu beta 6), Hb C (Lys beta 6), Hb Machida (Gln beta 6), and Hb S (Val beta 6). The surface hydrophobicity increased in the order of Hb C, Hb A, Hb Machida, and Hb S, coinciding with the hydrophobicity of the amino acid at the beta 6 position. Solubility of the oxy-form of these hemoglobins decreased in relation to increases in their surface hydrophobicity, suggesting that the solubility is controlled by the strength of hydrophobicity of the amino acid at the beta 6 position. The solubility of the oxy-form of these hemoglobins is always higher than that of the deoxy-form. There is a similar linear relationship between the solubility and surface hydrophobicity among deoxyhemoglobins A, C, and Machida. However, the solubility of deoxy-Hb S deviated significantly from the expected value, indicating that the extremely low solubility of deoxy-Hb S is not directly related to the hydrophobicity of the beta 6 valine. Kinetic studies on the polymerization of deoxy-Hb Machida revealed a distinct delay time prior to polymerization. This confirms our previous hypothesis that beta 6 valine is not responsible for the delay time prior to gelation. The kinetics of the polymerization of 1:1 mixtures of sickle and non-sickle hemoglobins were similar to those of pure Hb S, suggesting that only one of the two beta 6 valines is involved in an intermolecular contact. In mixtures of equal amounts of Hb S and Hb A, Hb C, or Hb Machida, half of the asymmetrical AS, SC, and S-Machida hybrid hemoglobins behaved like Hb S during nucleation, while the other half behaved like the non-sickle hemoglobin.     

Proton nuclear magnetic resonance studies of hemoglobins Osler (beta145HC2 Tyr replaced by Asp) and McKee Rocks (beta145HC2 Tyr replaced by term): an assignment for an important tertiary structural probe in hemoglobin

High-resolution proton nuclear magnetic resonance studies of deoxyhemoglobins Osler (beta145HC2 Tyr replaced by Asp) and McKees Rocks (beta 145HC2 Tyr replaced by term) indicate that these hemoglobins are predominately in the oxy quaternary structure in 0.1 M [bis(2-hydroxyethyl)imino]-tris(hydroxymethyl) methane buffer at pH 7. Upon the addition of inositol hexaphosphate, the proton nuclear magnetic resonance spectra of these hemoglobins become similar to those characteristic of a hemoglobin molecule in the deoxy quaternary structure. The exchangeable proton resonance which is found at -6.4 ppm from H2O in the spectrum of normal human adult deoxyhemoglobin is absent in the spectra of these two mutant hemoglobins. Consequently we believe the hydrogen bond between the hydroxyl group of tyrosine-beta145HC2 and the carboxyl oxygen of valine-beta98FG5 gives rise to this resonance. This assignment allows us to use the -6.4ppm resonance as an important tertiary structural probe in the investigation of the cooperative oxygenation of hemoglobin.     

Hemoglobin Wood beta97(FG4) His replaced by Leu. A new high-oxygen-affinity hemoglobin associated with familial erythrocytosis.

The characterization of hemoglobin Wood (beta97(FG4) His replaced by Leu), a high oxygen affinity hemoglobin with reduced Hill constant is described. The amino acid substitution occurs at the alpha1beta2 interface, in the same position as in hemoglobin Malm? (beta97(FG4) His replaced by Gln) and in an homologous position when compared with hemoglobins Chesapeake (alpha92(FG4) Arg replaced by Leu) and J. Capetown (alpha92(fg4) arg replaced by Gln).