Hemoglobin Fort Gordon or alpha2beta2145 Tyr replaced by Asp, a new high-oxygen-affinity hemoglobin variant.

Hemoglobin Fort Gordon, alpha2beta2145 Tyr replaced by Asp (HC2), has been observed in a 20-year-old black male with compensatory erythrocytosis. The variant was readily identified by electrophoresis and chromatography, and comprised about 30% of the red cell hemoglobin. The substitution was identified through analyses of tryptic peptides of various digests of the isolated beta chain. The oxygen affinity of whole blood was increased; two components were observed one of which had a greatly increased affinity for oxygen and a markedly reduced subunit cooperativity. It appears that the Tyr replaced by Asp substitution resembles the Tyr replaced by His substitution in hemoglobin Bethesda (Bunn, H. F. et al. (1972) J. Clin. Invest. 51, 2299-2309; Olson, J. S. and Gibson, G. H. (1972) J Biol. Chem. 247, 3662-3670; Adamson et al. (1972) J. Clin. Invest. 51, 2883-2888) in that both inhibit the quarternary change of the oxy to the deoxy conformation, resulting in greatly altered functional properties. Studies of a few members of the family were negative.     

Hemoglobin New Mexico: beta 100 (G2) Pro----Arg. A variant hemoglobin associated with erythrocytosis

Hemoglobin New Mexico beta 100 Pro----Arg was found in a 4-year-old black male and represents a new mutation. The propositus is also heterozygous for Hb S. The variant shows high oxygen affinity, reduced cooperatively, and a lowered alkaline Bohr effect. Addition of allosteric effectors leads to improved cooperativity and a Bohr effect that is similar to that of Hb A. The high percentage of the variant (53.5%) and its increased oxygen affinity result in erythrocytosis in this patient. The hemoglobin level and packed cell volume values are elevated. In spite of these factors the patient appears healthy and shows no discomfort. The altered oxygen-linked properties of this variant can be related to the fact that the substituted residue contributes to the alpha 2 beta 1/alpha 1 beta 2 subunit interface, an area that is critical not only to the allosteric transitions between the oxy and deoxy states but also to stabilizing the hemoglobin tetrameer.     

A new hemoglobin variant. HB Yatsushiro alpha 2 A beta 2 60 Val replaced by Leu

This study was performed to establish the structural abnormality of a new hemoglobin variant discover-d in a Japanese patient with angina pectoris. The hybridization of the separated hemoglobin with canine hemoglobin revealed a beta-chain anomaly. Peptide betaTp-6 was found to be abnormally located on the peptide map of tryptic digests of the S-carboxymethylated beta-chain from the variant hemoglobin. A structural study on the abnormal betaTp-6 revealed that the variant hemoglobin differs from hemoglobin A by substitution of leucine for valine at residue 60 of the beta-chain. This new variant hemoglobin is designated as hemoglobin Yatsushiro after the name of the city where the propositus lived. The patient is hematologically healthy and his clinical history has nothing to do with this abnormal hemoglobin.     

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.     

Hemoglobin Fannin-Lubbock [alpha2 beta 2 119 (GH2) Gly replaced by Asp]. A new hemoglobin variant at the alpha1 beta 1 contact.

Hemoglobin Fannin-Lubbock was found in a 9-year-old Mexican-American female. The abnormal hemoglobin was detected as a fast-moving variant by electrophoresis on cellulose acetate at pH 8.4. Structural analysis indicated a substitution in the beta-chain of aspartic acid for glycine at position 119, a position involved in the alpha1beta1 contact of the hemoglobin tetramer. This contact between unlike chains is larger and undergoes a smaller shift during the process of oxygenation and deoxygenation that the alpha1beta2 contact (Perutz, M.F., Muirhead, H., Cox, J.M. and Goaman, L.C.G. (1968) Nature 219, 131-139). Mutations in this contact tend to cause slight or no changes in functional behavior. Apart from a mild anemia, the propositus did not exhibit any obvious clinical symptoms.     

Hemoglobin Abruzzo (beta143 (H21) His replaced by Arg). Consequences of altering the 2,3-diphosphoglycerate binding site.

Hemoglobin Abruzzo is an abnormal human hemoglobin with a substitution at a residue known to be involved in the binding of 2,3-diphosphoglyceric acid. It has increased oxygen affinity and reduced heme-heme interaction in the absence of organic or inorganic phosphate cofactors. In inorganic phosphate buffers the Bohr effect and heme-heme interaction are normal, but the oxygen affinity remains higher than that of hemoglobin A. CO combination in inorganic phosphate is more strongly autocatalytic than in normal hemoglobin and a slower rate of oxygen dissociation is observed. Although many of the functional differences of this variant may be attributed to the high oxygen affinity of the mutant beta chains, the interactions between subunits are also affected by the histidine to arginine substitution at beta143. Stripped hemoglobin Abruzzo appears to be significantly more dissociated than hemoglobin A. Kinetic studies indicate that interaction with organic or inorganic phosphates decreases its subunit dissociation. In all of the functional properties examined, hemoglobin Abruzzo is more sensitive to the allosteric influence of organic and inorganic anions than is hemoglobin A.     

Hemoglobin Deer Lodge (beta 2 His replaced by Arg). Consequences of altering the 2,3-diphosphoglycerate binding site.

Hemoglobin Deer Lodge is an abnormal human hemoglobin with arginine substituted for histidine at the beta 2 position. X-ray crystallography of normal human hemoglobin has shown that the beta 2 residue is normally part of the binding site for 2,3-diphosphoglycerate. The substitution of arginine for histidine at beta 2 affects both the kinetics and equilibria of ligand binding. When stripped of anions, Hb Deer Lodge has an increased oxygen affinity and a decreased degree of cooperativity relative to Hb A. The alkaline Bohr effect is slightly increased and there are marked increases in oxygen affinity below pH 6 and above pH 8. In the presence of 2,3-diphosphoglycerate the cooperativity in increases to nromal and the pH dependence of oxygen binding is reduced. This contrasts with the enhanced Bohr effect seen for Hb A in the presence of organic phosphates. Due to enhanced anion binding at high pH, Hb Deer Lodge has a slightly lower oxygen affinity than Hb A at pH 9 in the presence of 2,3-diphosphoglycerate or inositol hexaphosphate. Kinetic studies at neutral pH in the absence of organic phosphates revealed biphasicity in the rate of oxygen dissociation from Hb Deer Lodge, while approximately linear time courses were observed for Hb A. The fast phase of the oxygen dissociation kinetics shows great pH sensitivity, and organic phosphates increase the rate and percentage of the fast phase without greatly affecting the slow phase. The two phases are not resolvable at high pH. CO combination kinetics are much like those of Hb A except that "fast" and "slow" phases were apparent at wavelengths near the deoxy-CO isobestic point. We suggest that functional differences between the alpha and beta chains are enhanced in Hb Deer Lodge. After flash photolysis of the CO derivative, the percentage of quickly reacting material was slightly greater for Hb Deer Lodge than for Hb A. This may imply a somewhat greater tendency to dissociate into high affinity subunits. The substitution of arginine for histidine at beta 2 thus results in a macromolecule whose ligand-binding properties are significantly altered, the primary differences being expressed at high pH where Hb Deer Lodge binds anions more strongly than Hb A. The properties of Hb Deer Lodge are compared to those of other hemoglobin variants with substitutions at residues involved in binding of 2,3-diphosphoglycerate.     

Electron paramagnetic resonance of Hb St Louis beta28 (B10) Leu replaced by Gln.

Hemoglobin St Louis beta28 (B10) Leu replaced by Gln is a new mutant which occurs as a natural valency hybrid (alpha2beta+2), or hemoglobin M (Cohen-Solal, M., Seligmann, M., Thillet, J. and Rosa, J. (1973) FEBS Lett. 33, 37-41). The electron paramagnetic resonance (EPR) spectrum of native Hb St Louis at pH 6.2 shows a mixture of three species. Two are high spin, one with tetragonal symmetry, like Hb+ A, the other with rhombic distortion. The third is a low-spin form corresponding to a hemichrome with the distal (E7) histidine as the sixth ligand of the ferric iron. The hemichrome is also found in red blood cells. After oxidation to the alpha+2beta+2 form, three EPR species are seen. Surprisingly, there remains only one high-spin signal, with almost tetragonal symmetry. Besides the low-spin hemichrome, a hydroxy signal is observed even at pH 6.2. These observations imply interactions between the alpha and beta hemes.     

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.     

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.     

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.     

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.     

A novel transthyretin mutation at position 30 (Leu for Val) associated with familial amyloidotic polyneuropathy.

A novel transthyretin (TTR) mutation associated with familial amyloidotic polyneuropathy was detected in a Japanese patient. Single-strand conformation polymorphism analysis and sequence analysis of polymerase chain reaction (PCR)-amplified exons of the patient's TTR gene revealed a point mutation resulting in a substitution of leucine for valine at position 30. As the mutation creates a Cfr13I site, it was confirmed by PCR and restriction analysis. Our finding indicates the importance of position 30 in TTR-derived amyloid fibril formation.