Mary H. Johnson

Hemoglobin presbyterian: β108 (G10) asparagine→lysine. A hemoglobin variant with low oxygen affinity

Characterization of abnormal hemoglobin variants continues to provide important information on the relationship between structural and functional properties of hemoglobin. Correlation of the properties of specific variants with the three-dimensional structure of hemoglobin has clarified which features of the molecule govern particular aspects of its properties and activity at the molecular level. Hemoglobin Presbyterian is a new variant which migrates electrophoretically between Hbs A and F on cellulose acetate (pH 8.4), and in the Hb C position on citrate agar (pH 6.0). The variant was found in a 7-yr-old girl, her father, and paternal grandmother, all of whom have mild anemia. Although the variant hemoglobin exhibited low oxygen affinity, the effect of the allosteric effector 2,3-bisphosphoglycerate on the variant hemoglobin and on Hb A was similar. The degree of cooperativity was unchanged, and there was a slight increase in the Bohr effect. The low oxygen affinity can probably be explained on the basis of the disruption of the hydrogen bond between histidine a103 (GlO) and the carbonyl of asparagine plO8 (GIO), which is now replaced by lysine in Hb Presbyterian. This report presents data on the structural and functional properties of this variant.

Hemoglobins Austin and Waco: Two Hemoglobins with substitutions in the α1β2 contact region

Abstract Hemoglobins (Hbs) Austin and Waco were detected by their electrophoretic migration on cellulose acetate (pH 8.4) and citrate agar (pH 6.2). By these methods, both variants migrated between Hbs A and F. Globin chain analysis at pH 8.6 indicated that the mutant β chain of Hb Austin was faster moving than the βA chain; however, the mutant chain of Hb Waco was indistinguishable from the βA chain by this technique. The two variants were isolated by ion-exchange column chromatography. Sequence studies demonstrated a substitution of serine (Hb Austin) and lysine (Hb Waco) for arginine at position 40 in the β chain. These mutations involve an amino acid residue in the α1β2 contact region, which, before this report, had been considered invariant in all hemoglobin sequences. Hb Austin was found to exist as dimers when oxygenated and as tetramers when deoxygenated. The equilibrium constant (Kd) for the tetramer to dimer transition was approximately 300 × 10−6 m , as calculated from sedimentation velocity studies. This variant also had high oxygen affinity, a much reduced heme-heme interaction, and a normal Bohr effect. The functional properties of Hb Waco were similar to those of Hb A.

A case of hemoglobin Indianapolis [β112(G14)CYS→ARG] in an individual from Cordoba, Spain

Hemoglobin Indianapolis was first described by Adams et al (1,2) as a very unstable variant with a phenotype similar to severe beta-thalassemia. We have also characterized this variant, but there are several differences in the clinical expression of the variant described in our report and those described in the original case. We found Hb Indianapolis to be unstable, but not to the extent that it could not be detected by routine testing. The four family members heterozygous for the variant were not anemic, showed normal hematologic values, and did not exhibit any severe clinical disadvantages, although there was slight reticulocytosis. The variant could not be resolved from Hb A on cellulose acetate (pH 8.4), but isoelectric focusing showed a double band in the region of Hb A that is probably the variant and Hb A. However, the variant chain was clearly evident by globin chain analyses in acid and alkaline buffers. The condition of additional blood samples did not allow us to determine the oxygen dissociation properties of the variant or the rates of globin chain synthesis.

Hemoglobin Tarrant: alpha126(H9) Asp leads to Asn. A new hemoglobin variant in the alpha1beta1 contact region showing high oxygen affinity and reduced cooperativity.

Abstract Hemoglobin (Hb) Tarrant was detected by its electrophoretic mobility on cellulose acetate (pH 8.4) and citrate agar (pH 6.2). On cellulose acetate it moved as a band between hemoglobins F and S, and on citrate agar as a band at hemoglobin S. The test for solubility in 2 M phosphate buffer with Na2S2O4 was negative. The new variant has a substitution of asparagine for aspartic acid in position 126 of the α-chain, one of the sites involved in the α1β1 contact. Furthermore, in deoxyhemoglobin aspartic acid 126 of each α chain also forms a non-covalent electrostatic salt bridge with arginine 141 of the corresponding α chain (Perutz, M. F. and Ten Eyck, L. F. (1972) Cold Spring Harbor Symp. Quant. Biol. 36, 295–310 and Perutz, M. F. (1970) Nature 228, 726–739). As a consequence of this substitution in hemoglobin Tarrant, the deoxy conformation or T state is destabilized because these two bridges cannot be formed. This condition is reflected in high oxygen affinity and low cooperativity.

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.

A variant hemoglobin assicated with α-Thalassemia-2

Abstract Hb Evanston (α14 Trp → Arg) was detected on cellulose acetate at pH 8.4 as a band with an electrophoretic mobility similar to that of Hb S. In addition, a band migrating cathodic to Hb A2 suggested the presence of a variant Hb A2 with a substitution in the α-chain, a fact that was later confirmed by structural analysis. An unusual feature of Hb Evanston is its low percentage; less than 10% occurs in the hemolysate. Studies indicate that the variant is not unstable, but there appears to be a defect in globin-chain synthesis. Gene mapping also shows that it is associated with the α-thalassemia-2 gene. The variant has high oxygen affinity with normal cooperativity and a normal Bohr effect. The combination of Hb Evanston with α-thalassemia-2 produced anemia in this black family.

Hemoglobin Dunn: α6 (A4) Aspartic Acid→ASPARAGINE

Hemglobin Dunn, α6 (A4) Asp→Asn was found in a 39-year-old black woman and her daughter. The hematological data on the propositus were normal and there were no apparent clinical or pathological findings associated with this abnormal hemoglobin. The structural characterization of this variant is described.

Hemoglobin Cheverly: an Unstable Hemoglobin Associated with Chronic Mild Anemia

Summary: The evaluation of a family with chronic mild anemia led to the identification of a new unstable hemoglobin (Hemoglobin Cheverly). Modest anemia and reticulocytosis, normal to slightly increased mean corpuscular volume (MCV), and normal mean corpuscular hemoglobin concentration (MCHC) were present in the affected family members. Electrophoresis of blood samples on cellulose acetate and on citrate agar revealed normal patterns. Globin chain analysis and isoelectric focusing data were also normal. After incubation for 3 h at 41°C, Heinz bodies were detected in 95–100% of erythrocytes from affected individuals. Positive heat and isopropanol tests confirmed the initial observation of the Heinz body preparation and indicated that an unstable hemoglobin was present. Structural analysis showed an amino acid substitution of Phe-Ser at position 45 (CD4) in the β chain. Hemoglobin Cheverly has a reduced affinity for oxygen and a reduced Bohr effect, properties that can be rationalized on the basis of the x-ray crystallographic structure of normal hemoglobin.Despite structural and functional similarities between Hb Cheverly and Hb Hammersmith, β42 (CD1) Phe-Ser, the clinical manifestations of Hb Cheverly are mild in contrast to the severe disease observed with Hb Hammersmith. Reasons for the apparently silent clinical expression of Hb Cheverly are not known. We discuss the implications of unstable hemoglobins in the evaluation of chronic anemia in pediatric patients.

Hemoglobin Shelby [β131(H9) GLU→LYS] a Correction to the Structure of Hemoglobin Deaconess and Hekoglobib Leslie

Hemoglobin Shelby, detected in two unrelated black families, has an electrophoretic mobility like Hb F on cellulose acetate (pH 8.4) and a mobility between Hbs S and C on citrate agar (pH 6.2). Globin chain analysis in acid and alkaline buffers revealed an abnormal chain migrating between beta A and beta S. Tests for unstable hemoglobins were positive. Hematologic data on both families indicated carriers have mild anemia. The variant showed a slightly lower affinity for oxygen with normal cooperativity and Bohr effect, and its reactions with 2,3-diphosphoglycerate and inositol hexaphosphate were similar to those of Hb A. Sequence analysis indicated the substitution of lysine for glutamine at position 131 in the beta-chain. In a previous report (1) we described a variant, Hb Deaconess, in which this residue was deleted. On reexamination of the data, we find that Hb Deaconess is identical to Hb Shelby.

Hemoglobin deaconess a new deletion mutant: β131 (H9) glutamine deleted

Summary Hemoglobin Deaconess was detected as a band migrating in the position of fetal hemoglobin when an hemolyzate was electrophoresed on cellulose acetate at pH 8.4. This abnormal hemoglobin also migrates between Hb S and C on citrate agar electrophoresis at pH 6.2. Chemical characterization of this mutant hemoglobin shows glutamine is deleted at position 131 in the β-chain. Initial data indicates that the stripped hemoglobin has a reduced oxygen affinity with a Hill constant of n=2.0.