Normal HbA Research Articles

Specific Antibodies to Hemoglobin A1 (anti-Glu) and Hemoglobin S (anti-Val) in the Guinea Pig: Immunologic and Structural Correlations

Like goats and sheep, guinea pigs can produce, in response to human sickle cell hemoglobin (beta6 Glu leads to Val), an antibody population (anti-Val) that will bind sickle cell hemoglobin but not normal hemoglobin HbA. Unlike goats and sheep, guinea pigs can produce in response to human hemoglobin A1 an antibody fraction, anti-Glu, that will not react with human sickle cell hemoglobin. These anti-Glu antibodies have been isolated by affinity chromatography and their specificity confirmed by fluorescence-quenching titrations. The sequence of the first 10 amino acids of the beta-chain of guinea pig hemoglobin has been determined. This sequence differs from those of both hemoglobin HbA and sickle cell hemoglobin by two residues, those at positions 5 and 6. This explains the similarity of the immunogenicity of this site on the two human hemoglobins when administered to guinea pigs. Both goats and sheep are identical to hemoglobin A1 at the beta-6 position.

Competition of normal beta chains and sickle haemoglobin beta chains for alpha chains as a post-translational control mechanism.

ADULT human haemoglobin (HbA) is a tetrameric protein with two α and two β polypeptide chains. Theoretically, individuals heterozygous for a β-chain mutation should have equal amounts of the normal HbA (αA2βA2) and an abnormal variant (αA2βX2) in their red cells. However, these people usually have different amounts of the two haemoglobins. For example, the red cells of sickle cell trait individuals usually contain about 60% HbA and 40% HbS1–5. Those who also have an α-thalassaemia gene(s), which decreases production of α chains, have an even lower proportion of HbS6–12. Each of these two haemoglobin polymers has an α-chain type common to both proteins as well as a specific βA or βS chain type. The individual α, βA, and βS chain types can be isolated in vitro in a functional form so that the haemoglobin tetramers can be reconstituted from appropriate mixtures of α and β chains13. Here we report that more HbA than HbS was formed when a mixture of equal amounts of βA and βS chains was incubated with limiting concentrations of α chains. These results suggest that a similar form of post-translational control of haemoglobin assembly may exist in some erythroid cells in vivo. The accomplishment of these experiments and the interpretation of the results were greatly facilitated by the use of radiolabelled haemoglobin chains of high specific activity.

Interaction Between Hb Hasharon and α-Thalassemia: An Approach to the Problem of the Number of Human α Loci

We report the case of an Italian infant girl from Polesine (Po delta region in northern Italy) who was heterozygous for Hb Hasharon and alpha-thalassemia, did not synthesize any normal HbA, and had 3% HbH on electrophoresis. Hematologic and biosynthetic studies on Hb Hasharon carriers of the propositus' family suggest the possibility that the Hb Hasharon gene is linked to an alpha-thalassemia gene. On the other hand, in the Askenazy carriers of Hb Hasharon, Hb Harsharon is probably linked to a normal alpha gene. In comparing Hb Hasharon's behavior with that of other alpha variants, particularly HbG Philadelphia, frequent recombinations between alpha structural genes were suggested. The possible identity between the single alpha locus and the alpha2-thalassemia genotype is discussed.

Interaction between Hb Hasharon and alpha-thalassemia: an approach to the problem of the number of human alpha loci

We report the case of an Italian infant girl from Polesine (Po delta region in northern Italy) who was heterozygous for Hb Hasharon and alpha-thalassemia, did not synthesize any normal HbA, and had 3% HbH on electrophoresis. Hematologic and biosynthetic studies on Hb Hasharon carriers of the propositus' family suggest the possibility that the Hb Hasharon gene is linked to an alpha-thalassemia gene. On the other hand, in the Askenazy carriers of Hb Hasharon, Hb Harsharon is probably linked to a normal alpha gene. In comparing Hb Hasharon's behavior with that of other alpha variants, particularly HbG Philadelphia, frequent recombinations between alpha structural genes were suggested. The possible identity between the single alpha locus and the alpha2- thalassemia genotype is discussed.

Homozygous State for Hb Constant Spring (Slow-moving Hb X Components)

Abstract A 12-yr-old Malay boy who was studied because his youngest brother and both his parents had the slow-moving Hb X components (earlier reported to lead to Hb H disease when combined with α-thalassemia) was found to be homozygous for the same slow-moving components. He had splenomegaly and a just palpable liver, mild anemia with microcytosis, hypochromia, slight morphologic changes of the red blood cells, and slight reticulocytosis. Of eight children in the family, six had the trait for the abnormality, one was normal, and one, the propositus, was homozygous. Structural studies of the isolated abnormal hemoglobin showed it to be identical to Hb Constant Spring (Hb CoSp), an α-chain variant with 172 residues instead of the usual 141, the additional 31 being attached to the C-terminal end. In addition to the abnormal α variant for which the propositus was homozygous, he also had normal Hb A and normal Hb A2 with normal α-chains. If the theory that Hb CoSp is due to a structural mutation affecting the terminator codon is correct, this case provides evidence for a duplication of the gene for α-chain production. Results of study of several erythrocyte enzymes are also reported.

The effect of organic compounds on the crystal habit and crystallization of normal and sickle cell hemoglobin in phosphate buffer

Comparative studies were made on the effect of numerous organic compounds in promoting the crystallization of human hemoglobin in 1.9 m phosphate, pH 7.0. It was found that alicyclic or benzenoid structures are essential for promoting crystallization of hemoglobin under these conditions. Hemoglobin crystals prepared in the presence of toluene differed in habit from crystals prepared in its absence. It is suggested that steric factors determine the effectiveness of organic substances in promoting the crystallization of hemoglobin and that the heme group is the binding site involved in the complex formation. The solubility of homozygous sickle cell hemoglobin HbS was found to be less than the heterozygous hemoglobins AS and AC or normal hemoglobin HbA in the presence of organic substances promoting the crystallization of hemoglobin.

Studies on the heterogeneity of hemoglobin: XIV. Chromatography of normal and abnormal human hemoglobin types on CM-Sephadex

A new chromatographic procedure for the separation and quantitation of human hemoglobin components is described. The method utilizes columns of carboxymethyl-Sephadex G-50 and 0.05M Tris-maleic acid buffers as developers; application of a simple pH gradient is preferred over stepwise elution. The procedure allows the separation of Hb-A2 from the hemoglobins S and C, and of Hb-D2 from the slow moving hybrid component SDα. Fetal hemoglobin is eluted in front of the normal Hb-A. The minor hemoglobins of normal red cell hemolysates were fractionated into at least four components, well separated from the major hemoglobin component. The procedure has the disadvantage of requiring several days to complete the chromatographic separations.

PHYSIOLOGIC DIFFERENCES IN HEMOGLOBIN VARIANTS.

Studies were carried out on whole blood, concentrated hemoglobin solutions, and isolated hemoglobin fractions as related to oxygen association. In hemoglobins Ao, A1, Dalpha, GPhiladelphia, and C, no difference was found in the oxygen association curve. Hemoglobins A1, B2, and Lepore showed a 2½ fold increased oxygen association over normal Hb-A. Hemoglobin solutions from cord blood showed no difference from Hb-A, whereas cord blood showed a twofold increase. Hb-H and Hb-Barts showed identical oxygen association curves with a 10- to 12-fold increase over Hb-A; no heme-heme interaction and no Bohr was found with Hb-H or Barts. Hb-E and Hb-S showed a slight decrease in oxygen equilibria over hemoglobin A.

OXYGEN EQUILIBRIA OF RED CELL HAEMOLYSATES OF ADULT INDIVIDUALS, HETEROZYGOUS FOR DIFFERENT RARE ABNORMAL HAEMOGLOBINS.

IN the past few years, oxygen equilibria of various normal and abnormal adult human haemoglobin types have been studied. No differences were observed between normal Hb-A (&α2β2) and the β-chain abnormalities Hb-S (ref. 1), Hb-C (ref. 2) and Hb-E (ref. 3). Investigations of the isolated Hb-A2′ (α2δ2) and its abnormal variant Hb-A2 revealed a considerable increase in oxygen affinity when compared with normal Hb-A (ref. 4). Hb-Lepore, which is composed of normal α-chains and ‘β’ chains being related to the β-chain of Hb-A as well as to the δ-chain of Hb-A2 (ref. 5), showed a high affinity for molecular oxygen similar to that of Hb-A2 (ref. 1). The affinities of the α-chain lacking abnormal haemoglobin types Hb-H (β4) and Hb-Bart's (γ4) for molecular oxygen were found to be extremely high, while no Bohr effect or haem–haem interactions were detectable6,7.

The oxygen equilibria of some “slow-moving” human hemoglobin types

The equilibria between oxygen and four different kinds of abnormal hemoglobin, Hb-DPunjb, Hb-D with another unknown abnormality in the β-chain, Hb-S and Hb-Lepore have been determined. Total red blood cell hemolysates as well as isolated components (DEAE-cellulose chromatography) were studied.The two Hb-D abnormalities in isolated forms showed a slight but distinct increase in oxygen affinities, while their Bohr effects were not altered. Red blood cell hemolysates of sickle-cell anemia patients failed to show a significant change in the affinity for oxygen. The similarity of the oxygen equilibria of Hb-S and Hb-A containing hemolysates was demonstrated with the use of potassium phosphate buffer of varying molarities.A marked increase in oxygen affinity of pure Hb-Lepore as compared to normal Hb-A was found. The equilibrium between oxygen and this Hb-type was comparable with those found for Hb-A2 and its variant Hb-A2′. No change in the Bohr effect was note. A small but distinct increase in the oxygen affinity of a red blood cell hemolysates of a Hb-Lepore trait carrier was also demonstrable.

A new abnormal human hemoglobin Hb: Zurich

1. 1. An abnormal slow moving hemoglobin, found in a white Swiss Family, is described. The abnormal hemoglobin is characterized by its electrophoretic and Chromatographic mobilities. It was found to be different from the known abnormal hemoglobin types. The component is alkali non-resistant and, in the reduced state, possesses the same solubility in concentrated phosphate solutions as found for Hb-A. 2. 2. The amounts of the abnormal component present in the blood of the father and his daughter were 28.4 and 22.3% respectively. Similar quantities were present in other members of the family of the father. 3. 3. Hybridization experiments revealed no abnormality in the α-chain of the protein. 4. 4. The fingerprints of the digests of the total hemoglobin and of its β-chain have shown multiple differences as compared with normal Hb-A. The fingerprints of the α-chains of the abnormal component and of Hb-A were identical. The genetic implications of these results are discussed. 5. 5. The tentative name Hb-“Zurich” is proposed.

Studies on an abnormal minor hemoglobin component (Hb-B 2)

1. 1. The occurrence among the Negro population of the State of Georgia of the minor Abnormal hemoglobin fraction B 2, already discovered by Ceppellini, Kunkel and Dunn, is reported. The incidence was found to be 2–3%. 2. 2. The discovery of a homozygous Hb-B 2 carrier and an individual heterozygous for Hb-B 2 as well as for Hb-S was made through a series of family studies. 3. 3. Since in the homozygous Hb-B 2 carrier the Hb-A 2 was completely substituted by Hb-B 2, a definite relationship of allelism of Hb-A 2 and Hb-B 2 was proved. 4. 4. Clinically and hematologically the heterozygous Hb-B 2 carrier and the homozygous Hb-B 2 carrier are normal. 5. 5. With the use of the “fingerprint” technique three differences between Hb-A 2 and normal Hb-A were demonstrated. They are located in peptides 26 and 12, and a new peptide was found on the negative side of peptide 11. Similar differences were found to be present in Hb-B 2. A difference between Hb-A 2 and Hb-B 2 is located in peptide 12 in which arginine is present in Hb-B 2 but absent in Hb-A 2 and with a chromatographic mobility greater in Hb-B 2 than in Hb-A 2. 6. 6. The genetic significance of these findings is discussed.

Properties and inheritance of the new fast hemoglobin type found in umbilical cord blood samples of Negro babies

1. 1. The discovery of a new fast minor Hb component (called “Augusta I”) in cord blood samples of Negro babies, present next to Hb-F, Hb-A and Hb-S, by starch-gel electrophoresis is reported. 2. 2. The amount of the abnormal fraction was found to be about 3% of the total hemoglobin (CMC-chromatography). 3. 3. The component was isolated by CMC-chromatography and studied using different techniques. It showed a slightly increased resistance to denaturation by alkali and an ultraviolet spectrum typical for normal Hb-A. Using starch-gel electrophoresis, the isolated Hb preparation was found to consist of over 90% of the new component next to small amounts of Hb-A and a still faster moving Hb-fraction. 4. 4. It was demonstrated both with Amberlite IRC-50 chromatography and with starch-gel electrophoresis at pH 1.9 that the globin, prepared from the isolated Hbfraction, was composed almost entirely of β polypeptide chains. The formation of Hb-S from a mixture of the new Hb component and Hb-A in hybridization experiments offered strong evidence that this β chain was identical with the abnormal β chain of Hb-S. 5. 5. In the blood of some family members two inherited Hb-abnormalities were demonstrable : a heterozygosity for sickle-cell Hb and the presence of small amounts of a fast moving Hb-fraction, probably identical with Hb-H. These abnormalities were found to be inherited independently.

The heterogeneity of normal haemoglobin in human adults

1. 1. The course of propanol denaturation of haemiglobin and oxyhaemoglobin at pH 10 may be expressed by curves which indicate that normal haemoglobin of human adult is a mixture of fractions. This method is not open to the objections which have been raised against alkaline denaturation. 2. 2. Paper and also cellulose column chromatography yield a separation of normal human HbA into fractions. Propanol denaturation at pH of the most mobile fraction gives a curve different from that of chromatographically unseparated haemoglobin. The curve indicates the homogenous nature of this fraction. 3. 3. The homogeneity of haemoglobin was investigated in 27 individuals. Wiht one exception the results indicate the existence of three fractions with different rates of denaturation. In one individual the haemoglobin apparently contained only two fractions; this was repeatedly demonstrated after various intervals of time.