Katherine C. Bechtel

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.

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.

Characterization of the lac repressor species produced by limited tryptic cleavage.

Abstract Tryptic cleavage of native lac repressor under very mild conditions has been found to yield preparations suitable for detailed physical and chemical analysis. Sephadex G-200 chromatography of the digest produces one main protein peak followed by small peptides. The protein from the main peak was analyzed by automated Edman degradation and revealed two unique cleavage sites, one at residue 51 and the other at 59. The tryptic core protein under native conditions is tetrameric and exhibits a circular dichroism spectrum similar to that of native lac repressor.

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.

Hemoglobin Lufkin: β29 (B11) GLY→ASP An Unstable Hemoglobin Variant Involving an Internal Ahino Acid Residue

Hemoglobin Lufkin was found in a Black-American family. Structural analysis of the abnormal hemoglobin indicates a substitution of aspartic acid for glycine at position 29 in the beta chain. Marked instability of the variant hemoglobin is demonstrated by the rapid formation of inclusion bodies upon exposure of the red cells to redox dyes and by the large percentage of precipitated hemoglobin at 65 degrees C. The oxygen affinity, the Bohr effect, and the degree of cooperativity of Hb Lufkin and Hb A are similar over the physiologic pH range. However, at acid pH the oxygen affinity of the variant is increased. Unlike several other reported variants in the B helix, Hb Lufkin is not associated with methemoglobinemia.

Hemoglobin Detroit: β95 (FG2) lysine → asparagine

Abstract Hb Detroit is a mutant which migrates between Hb A and Hb J Baltimore on cellulose acetate (pH 8.5),and with Hb A on citrate agar (pH 6.0). Globin chain analyses in alkaline and acid buffers reveal an abnormal β chain with a mobility between the βA and βJ Baltimore chains. Structural characterization of this abnormal chain shows that lysine at position 95 is replaced by asparagine. No hematological abnormalities could be attributed to the presence of the mutant, and the oxygen affinity properties of the stripped hemoglobin are similar to those of Hb A. The β95 residue which is substituted in Hb Detroit and also in Hb N Baltimore (β95 Lys → Glu) does not appear to be in a critical functional area of the molecule.

The Cyanogen Bromide and MAI EYL Peptides of the α and β Chains of Human Hemoglobin

Procedures are described for the preparation of cyanogen bromide and maleyl peptides of the alpha and beta chains of human hemoglobin. These relatively large peptides have facilitated application of automated sequencing techniques to determine mutations in the primary structure of abnormal hemoglobins.