Bennett F. Horton

STUDIES ON THE HETEROGENEITY OF HAEMOGLOBIN. VII. MINOR HAEMOGLOBIN COMPONENTS IN HAEMATOLOGICAL DISEASES.

ANY STUDIES have revealed the heterogeneity of the haemoglobin of the normal adult human red blood cells (Singer, Chernoff and Singer, 1951; Kunkel and Wallenius, 1955; Allen, Schroeder and Balog, 1958; Clegg and Schroeder, 1959; Huisman and Meyering, 1960; Schnek and Schroeder, 1961; Jones and Schroeder, 1963a). Of the different minor haemoglobin components, which are present in addition to the major Hb-A. component, the quantities of Hb-A2 are affected by a variety of pathological states, such as thalassaemia, iron deficiency anaemia and megaloblastic anaemia (Josephson, Masri, Singer, Dworlun and Singer, 1958; Ingram and Stretton, 1961). It seems almost certain that small amounts of foetal haemoglobin are present in erythrocytic haemolysates of normal human adults (Singer et al., 1951; Betke, 1954; Huisman, Jonxis and Dozy, 1955; Huisman, 1963) although there is no universal agreement on this (Beaven, Ellis and White, 1960). Convincing evidence has recently been presented by Sturgeon, Schroeder, Jones and Bergren (1963) that the alkali-resistant haemoglobin fraction which is present in increased quantities in adults with either thalassaemia major or sickle-cell anaemia is identical to the foetal haemoglobin of the newborn. Additional minor haemoglobin fractions have been observed using different types of chromatography such as carboxymethylcellulose chromatography (Huisman and Meyering, 1960; Meyering, Israels, Sebens and Huisman, 1960)~ DEAE-cekdose chromatography (Huisman and Dozy, 1962) and Amberlite IRC-50 chromatography (Allen et al., 1958; Clegg and Schroeder, 1959; Schnek and Schroeder, 1961; Jones and Schroeder, 1963a). At least three distinct fast-moving minor haemoglobin fractions have been demonstrated with the Amberlite IRC-50 chromatographic procedure; these components have been designated as Hb-AI,, Hb-An, and Hb-AIc, respectively. In this paper the results of studies are reported in which possible variations in the quantities of these minor haemoglobin fractions, and of Hb-F and Hb-A2, in various haematological diseases have been investigated.

Studies on an abnormal minor hemoglobin component (Hb-B2)

Abstract 1. 1. The occurrence among the Negro population of the State of Georgia of the minor Abnormal hemoglobin fraction B2, 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-B2 carrier and an individual heterozygous for Hb-B2 as well as for Hb-S was made through a series of family studies. 3. 3. Since in the homozygous Hb-B2 carrier the Hb-A2 was completely substituted by Hb-B2, a definite relationship of allelism of Hb-A2 and Hb-B2 was proved. 4. 4. Clinically and hematologically the heterozygous Hb-B2 carrier and the homozygous Hb-B2 carrier are normal. 5. 5. With the use of the “fingerprint” technique three differences between Hb-A2 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-B2. A difference between Hb-A2 and Hb-B2 is located in peptide 12 in which arginine is present in Hb-B2 but absent in Hb-A2 and with a chromatographic mobility greater in Hb-B2 than in Hb-A2. 6. 6. The genetic significance of these findings is discussed.

Studies on the heterogeneity of hemoglobin : VIII. Chromatographic and electrophoretic investigations of various minor hemoglobin fractons present in normal and in vitro modified red blood cell hemolysates

Abstract The results of studies into the nature of the minor hemoglobin components of normal red blood cell hemolysates as obtained by Amberlite IRC-50 chromatography have been reported. All three minor hemoglobin fractions (AIa, ATh and AIc) were found to be inhomogeneous in starch gel electrophoresis. AIa was composed of two fast moving components. The fractions AIb and AIc consisted of fast moving hemoglobin components with specific electrophoretic mobilities, while at times components with the electrophoretic mobilities of Hb-FI and Hb-FII, respectively, were also present. No additional minor hemoglobin fractions were found in freshly prepared hemolysates. No significant differences between the quantities of the AIa, AIb and AIc fractions in old and young red cells could be demonstrated. Incubation of either hemolysates or sterile blood samples at 37° produced notable amounts of an additional fraction (AIc), while the quantities of the fractions AIa+b were also increased. The formation of the AIe fraction was primarily temperature dependent while the increase in the amounts of AIa+b was mainly dependent upon the time of incubation. Incubation of hemolysates with oxidized glutathione produced a component with a unique chromatographic mobility; this component is probably identical with the AId component, occasionally seen in aged red cell hemolysates. Incubation of hemolysates with either oxidized or reduced glutathione also increased the amount of the AIa+b fractions, while no change in the amounts of AIe was observed.

Studies on a fast hemoglobin variant found in a negro family in association with thalassemia

1. (1) A fast moving hemoglobin component was seen in a Negro family together with thalassemia minor. 2. (2) Electrophoretic and Chromatographie properties of the fast moving Hb have made its identity with Hb-J likely. Due to the confusion in literature about electrophoretic properities of Hb-J the tentative name of Hb-J (Georgia) is preposed. The abnormal component is not resistant to alkali and showed the same solubility (in reduced state) as Hb-A. 3. (3) In heterozygous individuals the percentages of Hb-J (Georgia) is higher than that of Hb-A. In the double heterozygous condition (Hb-J (Georgia)-thalassemia) the concentration of Hb-J is increased to 80%; a similar increase of Hb-A2 is present as found in thalassemia minor, while an increased production of Hb-F is hardly detectable in these cases. Clinically these individuals are normal, while the hematologie data (except for the total Hb, which is in the normal range) are comparable with those of thalassemia trait. 4. (4) The genetics of the Hb-J (Georgia) and in particular the interaction of the gene responsible for the Hb-J production and of the thalassemia gene are discussed.

The quantities of various minor hemoglobin components in old and young human red blood cells

Abstract The red blood cells from several normal healthy adult individuals have been separated into young and old cells using a density layer centrifugation technique. The quantities of the following minor hemoglobin components were determined: the hemoglobins A1a, A1b and A1c by Amberlite IRC-50 chromatography; the Hb-A2 by DEAE-cellulose chromatography; and Hb-F by an alkali denaturation procedure. No differences were observed in the percentages of each of these hemoglobin fractions as present in the two types of cell layers. It has, therefore, been concluded that differences in oxygen affinities of the two cell types are not due to differences in the quantities of minor hemoglobin fractions but should probably be sought in salt-labile changes in the hemoglobin molecules.

A new abnormal human hemoglobin Hb: Zurich

Abstract 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.

Erythropoietin and Haemoglobin Studies on AKR Mice

Summary. DEAE‐Sephadex column chromatography has been used to study the haemoglobins of AKR untreated mice, polycythaemic mice injected with saline and polycythaemic mice treated with an extract of human erythropoietin. All elution patterns of the haemolysates showed four haemoglobins. The haemolysates of each group had an elution pattern different from the other groups. Chromatographic studies of an ageing haemolysate indicated the formation of a minor haemoglobin from a major haemoglobin to be linear for a period of about 18 days.