Non-α Chains Research Articles

A novel double heterozygous, HbD Punjab/HbQ India, hemoglobinopathy

Hemoglobinopathies and thalassemias together form the most common genetic disease in the world. Double heterozygosity, in which there is a hemoglobin variant, in both the α- and non-α globin chains, is very unusual. A novel double heterozygosity of the α chain variant HbQ India with the non-α chain HbD Punjab is described. The index case is a 39 year old female of Indian origin. HPLC analysis using the Bio Rad β thalassemia method and electrophoresis at both alkaline and acid pH were performed. HPLC shows four major bands and electrophoresis at alkaline pH shows 3 bands and 2 bands at acid pH. Both the HPLC and electrophoresis at alkaline and acid pH are consistent for the double heterozygous hemoglobin variants HbQ India and HbD Punjab. This is the first literature report of the double heterozygosity of HbQ India/HbD Punjab.

Effects of heme addition on formation of stable human globin chains and hemoglobin subunit assembly in a cell-free system

Our previous assembly studies to form hemoglobin hetero-dimers and -tetramers using a coupled transcription/translation cell-free system suggested that α-globin chains bind to nascent non-α chains during and/or soon after translation to promote hemoglobin formation [Adachi et al., J. Biol Chem. 2002 (277) 13415]. In this report effects of CN–hemin on subunit assembly were studied using this cell-free system. Addition of CN–hemin and excess unlabeled heme-containing partner chains during synthesis leads to formation of radiolabeled heme-containing α hβ h hetero-dimers. In contrast, in the absence of added CN–hemin, unlabeled heme-containing α or β chains can assemble with newly synthesized radiolabeled β- and α-globin chains to form heme-containing α hβ h and semi-α (α hβ 0) or semi-β (α 0β h) hetero-dimers, respectively. These results suggest the existence of semi-hemoglobins as intermediates prior to formation of heme-containing α hβ h and indicate transfer of heme from α and/or β chains into semi-hemoglobin hetero-dimers to form heme-containing hetero-dimers.

What should we measure in the diabetic patient and how does this respond to therapy?

Diabetes is characterized by hyperglycaemia and by the development of complications. The criteria for diagnosis have been revised and published by the World Health Organization (WHO). In a symptomatic patient, a random plasma glucose concentration of ≥11.1 mmol l−1 is diagnostic. For asymptomatic patients, two diagnostic tests are required. A fasting glucose level ≥7 mmol l−1 confirms the diagnosis (providing this is supported by a second diagnostic test). The 75 g oral glucose tolerance test, as recommended by the WHO, is used for diagnosis in borderline cases or for academic studies. A plasma glucose concentration ≥11.1 mmol l−1 at 2 h following oral glucose confirms the diagnosis (Table 1). Table 1 Values for diagnosis of diabetes mellitus and other categories of hyperglycaemia.

Expression of functional soluble human alpha-globin chains of hemoglobin in bacteria.

Individual, soluble human alpha-globin chains were expressed in bacteria with exogenous heme and methionine aminopeptidase. The yields of soluble alpha chains in bacteria were comparable to those of recombinant non-alpha chains expressed under the same conditions. Molecular mass and gel-filtration properties of purified recombinant alpha chains were the same as those of authentic human alpha chains. Biochemical and biophysical properties of isolated alpha chains were identical to those of native human alpha chains as assessed by UV/vis, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy which contrasts with previous results of refolded precipitated alpha chains made in the presence of heme in vitro (M. T. Sanna et al., J. Biol. Chem. 272, 3478-3486, 1997). Mixtures of purified, soluble recombinant alpha-globin and native beta-globin chains formed heterotetramers in vitro, and oxygen- and CO-binding properties as well as the heme environment of the assembled tetramers were experimentally indistinguishable from those of native human Hb A. UV/vis, CD, and NMR spectra of assembled Hb A were also the same as those of human Hb A. These results indicate that individual expressed alpha chains are stable in bacteria and fold properly in vivo and that they then can assemble with free beta chains to form hemoglobin heterotetramers in vivo as well as in vitro.

The Effect of α-Thalassemia on the Level of Hybrid Hemoglobin Variants in Heterozygotes

The influence of a relative deficiency in alpha chain production on the amount of Hemoglobins Kenya, P-Nilotic, and Lepore was determined. The level of these hybrid hemoglobins in heterozygotes was correlated to various states of alpha chain deficiency by: 1) quantitation of the variants in blood samples and comparing these data with the number of alpha globin genes determined by gene mapping, 2) in vitro recombination experiments involving isolated non-alpha chains and normal alpha chains, and 3) in vitro heat stability analyses of the isolated hemoglobins. Hb Kenya, composed of normal alpha and gamma-beta hybrid chains, is heat labile, has a decreased ability to combine with alpha chains, and its level in heterozygotes is greatly decreased when a concomitant alpha-chain deficiency (alpha-thalassemia) is present. Such a posttranslational control mechanism was not observed for Hb Lepore, with normal alpha chains and delta-beta hybrid chains, and Hb P-Nilotic, with normal alpha chains and beta-delta hybrid chains. The latter two variants are heat stable, and their hybrid chains combine equally well as normal beta chains with normal alpha chains. Hb P-Nilotic is more heat stable than Hb A and its in vitro formation is increased over that of Hb S, and perhaps even Hb A, in conditions of severe alpha chain deficiency.

Expression of embryonic hemoglobin genes in mice heterozygous for α-thalassemia or β-duplication traits and in mice heterozygous for both traits

Hemoglobins of mouse embryos at 11.5 through 16.5 days of gestation were separated by electrophoresis on cellulose acetate and quantitated by a scanning densitometer to study the effects of two radiation-induced mutations on the expression of embryonic hemoglobin genes in mice. Normal mice produce three kinds of embryonic hemoglobins. In heterozygous α-thalassemic embryos, expression of EI (x 2y 2) and EII ( α 2y 2) is deficient because the x- and α-globin genes of one of the allelic pairs of Hba on chromosome 11 was deleted or otherwise inactivated by X irradiation. Simultaneous inactivation of the x- and α-globin genes indicates that these genes must be closely linked. Reduced x- and α-chain synthesis results in an excess of y chains that associate as homotetramers. This unique y 4 hemoglobin also appears in β-duplication embryos where excess y chains are produced by the presence of three rather than two functional alleles of y- and β-globin genes. In double heterozygotes, which have a single functional allele of x- and α-globin genes and three functional alleles of y- and β-globin genes, synthesis of α and non-α chains is severely imbalanced and half of the total hemoglobin is y 4. Mouse y 4 has a high affinity for oxygen, P 50 of less than 10 mm Hg, but it lacks cooperativity so is inefficient for oxygen transport. The death of double heterozygotes in late fetal or neonatal life may be due in large part to oxygen deprivation to the tissues.

Cultured Erythroid Cells as a Model for Hb Regulation: Ability of Cultured Cells to Synthesize Hb Lepore and HbA2 and to Maintain Balanced Globin Synthesis

AbstractTo be useful in the study of mechanisms controlling hemoglobin synthesis, erythroid bursts cloned from peripheral blood burst-forming units (BFU-E) must be shown to perform functions characteristic of bone marrow erythroid cells. To this end, peripheral blood BFU-E were cloned from two carriers of Hb Lepore. This globin chain has the N-terminal sequence of the δ chain and C-terminal sequence of the β chain. Reticulocytes from carriers of Hb Lepore do not synthesize the δβ chain and also synthesize an excess of α chain. In contrast, nucleated erythroid cells from bone marrow of Hb Lepore carriers can both synthesize the δβ chain and maintain balanced synthesis of the α and non-α chains. Like bone marrow cells, colonies derived from BFU-E of the Hb Lepore carriers incorporated 3H-leucine into a peak that cochromatographed with the δβ chain and contained 9%–16% of the non-α chain counts. Colonies derived from peripheral blood BFU-E from normal persons incorporated little radioactivity into this position. The counts in the δβ position were unlikely to represent artifacts of the culture system, since little radioactivity was found in this position when 3H-isoleucine was used as a label. When hemoglobins A2, Lepore, and A + F were isolated before chain separation, both the δ and the δβ chains contained significant amounts of radioactivity. Neither of these chains was synthesized in reticulocytes from either subject. Thus, the cultured cells, like bone marrow cells, must synthesize and translate the mRNA for each of these globin chains. In addition, cultured cells also behaved like bone marrow cells in that an excess of radioactive α chains was not observed, suggesting that the cultured cells possess the balancing mechanisms found in bone marrow cells.

Cellular regulation of fetal hemoglobin synthesis in man: Investigation of γ and β mRNA accumulation in clonal erythroid cultures initiated from erythroid progenitors derived from fetuses, neonates, and adult individuals

The accumulation of globin mRNA in erythroid cells during human development was investigated in clonal cultures of fetal, newborn, and adult origin erythroid progenitors and in control uncultured erythroblasts and reticulocytes. The relative concentrations of α, β, and γ mRNA sequences were measured by hybridization with purified α, β, and γ[ 3H]cDNA probes. We found that the amount of γ mRNA is characteristic of the developmental stage from which the erythroid progenitor cells are obtained. In colonies from fetal liver cells, 89% of the non-α mRNA is γ mRNA. Colonies derived from neonatal (cord blood) cells produce an average of 55% γ mRNA, while colonies generated from either adult bone marrow or progenitors circulating in the peripheral blood of the adult individual produce γ mRNA ranging from 15 to 38% of the non-α sequences. When compared with erythroid cells (erythroblasts or reticulocytes) from the same tissues, the fetal liver colonies produce identical proportions of γ mRNA as liver cells in vivo. Colonies from circulating erythroid progenitors from the perinatal (switchover) period accumulate somewhat less γ mRNA (55%) than do circulating reticulocytes in vivo (64%). The concentration of γ mRNA (15 to 38%) in colonies of adult marrow or peripheral blood origin is substantially higher than the trace amounts (<1%) found in mature erythroid cells in vivo. The amounts of β and γ globin chain synthesis from fetal liver, cord blood, and adult peripheral blood are proportional to β and γ mRNA ( r = 0.97) and thus indicate that selective translation of β and γ mRNA is not a significant factor in the switch from Hb F to Hb A production. (However, both neonatal reticulocytes in vivo and erythroid colonies in vitro contain excess non-α mRNA but exhibit balanced globin chain synthesis; translational control mechanisms apparently prevent excess synthesis of total non-α chains.) The expression of stage-specific patterns of γ and β mRNA in erythroid colonies suggests that the erythroid progenitor cells in vivo possess the potential for unique programs of γ and β mRNA accumulation before they actually enter the pathway to terminal maturation.

Separation of hemoglobin chains.

Separation of Hb chains constitutes an important step in the procedures of (a) the studies on the primary structure of polypeptide chains of hemoglobins, (b) the detection of chain anomaly of the abnormal hemoglobins, and (c) the investigation on the biosyn-thetic ratio of the α chain to the non-α chain.

Haemoglobin Lepore trait: haematological and structural studies on the Italian population.

Haematological data on 59 heterozygotes for haemoglobin (Hb) Lepore and 10 double heterozygotes for Hb Lepore and beta thalassaemia from 36 Italian families are reported. The red cell indices are defined and compared with those of groups of non-thalassaemic and beta thalassaemic subjects of comparable number, age and sex distribution. The relative level of each haemoglobin fraction and the absolute production of single polypeptide chains are calculated in order to compare the expression of the non-alpha chain genes in Hb Lepore trait and beta thalassaemia. Structural studies demonstrate that the haemoglobin Lepore is of the Boston type (delta 87 beta 116) in all subjects, confirming that this type of fusion variant is probably the only one which occurs in Mediterranean populations. The distribution and incidence of the Lepore haemoglobinopathy are discussed.

Oxygen affinity of hemoglobins F and A partially oxidized to methemoglobin: influence of 2,3-diphosphoglycerate.

Summary: Oxygen equilibrium curves of partially autoxidized adult (HbA) and fetal hemoglobins (HbF) were determined at 2–75% methemoglobin (met-Hb). Hemoglobin concentration of 5 mM, pH 7.15 (Tris 0.05 M), and temperature of 37° corresponded to intraerythrocytic in vivo conditions. With increasing met-Hb fractions the curves became shifted to the left and approached a hyperbolic form: P50 of both HbA and HbF decreased in absence of 2, 3-diphosphoglycerate (2, 3-DPG) from about 13 to 7 Torr, and Hill's n from 2.7 to 1.25. Independent of the met-Hb fraction the alkaline Bohr effect was increased by 2, 3-DPG. The allosteric effect of 2, 3-DPG on P50 decreased with increasing met-Hb. At all oxidation levels this effect was smaller on HbF than on HbA: At 50% met-Hb P50 of HbF is increased by 0.7 Torr/mM 2, 3-DPG increase, P50 of HbA by 1.3 Torr, respectively. There was no evidence of preferential autoxidation of α or non-α chains of the hemoglobin molecule. Oxidized hemoglobin does not take part in oxygen transport. In addition a left-shifted, more hyperbolic curve means restricted oxygen unloading from the remaining active hemoglobin. Thus, from application of our results to clinical conditions it must be concluded that sustained methemoglobinemia, despite partial compensation by 2, 3-DPG, may interfere with oxygen supply to tissue, particularly in the perinatal period, when HbF prevails. Speculation: Tissue oxygen supply, as calculated from the present data on O2 capacity, P50, 2, 3-DPG reactivity, Hill's n, and Bohr factors, appears to be markedly lowered in methemoglobinemia, particularly in presence of fetal hemoglobin. Reduced tissue oxygen supply in the perinatal period, in addition to a generalized cytochrome b5 reductase deficiency (22), might contribute to irreversable cerebral dysfunction in hereditary methemoglobinemia.

Rat haemoglobin heterogeneity. Two structurally distinct α chains and functional behaviour of selected components

Six haemoglobins were separated analytically from haemolysates of adult Wistar rats (Rattus norvegicus) by cellulose acetate electrophoresis and preparatively by DEAE-cellulose chromatography. The globin chains were separated from unfractionated haemolysates by CM-cellulose chromatography by using a non-linear formic acid-pyridine gradient followed by CM-cellulose chromatography in 8M-urea by using a gradient of increasing Na+ concentration in phosphate buffer, pH 6.7. Two alpha chains and three non-alpha chains were identified. Chains isolated from purified haemoglobins were correlated with chains isolated from unfractionated haemolysates by electrophoresis on urea-starch gels to make presumptive assignments of the subunit composition of the six haemoglobin tetramers. Partial amino acid sequences were determined for the major and minor alpha chains. The oxygen equilibria of two of the major haemoglobin components and of the unfractionated haemolysate were examined at pH 7.5 and 8.0. The two purified haemoglobins exhibited similar oxygen affinities; the haemolysate, however, had a lower oxygen affinity than either of the two purified haemoglobins. Both the haemolysate and the two haemoglobins showed an alkaline Bohr effect larger than that of human haemoglobin A.

Synthesis in vitro of anti-Lepore haemoglobin.

HUMAN erythrocytes contain two haemoglobins, a major component Hb A (α2β2) comprising 97 to 98% of the total Hb, which is actively synthesized in both nucleated red cells and reticulocytes and a minor component Hb A2 (α2δ2), which is produced chiefly in nucleated cells. This is because there is little or no δ chain synthesis by the reticulocyte stage of red cell maturation1,2. It has been shown recently that this early cessation of synthesis is not confined to the δ chain of Hb A2 but is observed also in the δβ fusion chain of Hb Lepore2–4. As the latter has the same N-terminal amino acid sequence as the δ chain of Hb A2 and presumably their mRNAs have the same 5′ nucleotide sequence, these observations suggest that the early decline of δ and δβ chain synthesis may somehow be connected with this region of mRNA. We have tested this possibility by examining the in vitro synthesis of the anti-Lepore haemoglobin, Hb Miyada, which has a non-α chain comprising the N-terminal residues of the β chain fused to the C-terminal residues of the δ chain5.

Haemoglobin P-Nilotic containing a - chain.

HAEMOGLOBIN P has been found amongst Nilotic populations of the then Belgian Congo, now called Zaire1,2 (Hb P Congo), and in the USA (Hb P Galveston)3. The abnormality in Hb P Galveston has been identified as β117 G19 His→Arg3, but the abnormality in Hb P Congo seems to be more complex and it has been suggested by Lehmann and Charlesworth4 that it is a “reversed Lepore” haemoglobin with a hybrid β-δ chain. In an extensive family study Dherte, Lehmann and Vandepitte1 found one man who had inherited Hbs A, S and P. His wife was a normal A/A homozygote and their children were all either A/S or A/P heterozygotes. Hybridization experiments demonstrated that the abnormality in the Hb P Congo was not in the α chain5. As the genes for the βA and βS chains are alleles at a single locus it is impossible for one individual to inherit Hbs A, S and P unless the gene for the non-α chain of Hb P is at a separate locus. A new genetic locus for a hybrid β-δ chain might be generated as shown in Fig. 1, by unequal but homologous crossing over of the genes for the δ and β chains6. One of the products of a crossing over would be a hybrid δ-β gene which would give rise to Hb Lepore. The other product would contain the genes for the δ and β chain and for a hybrid β-δ chain. The existence of homozygotes for Hb Lepore7 who make no Hb A or A2 suggests that the δ and β genes are in the order shown in Fig. 1. If a homozygote for the β-δ gene existed he would be expected to make Hb A and A2 as well.

The structure of Mus musculus bactrianus hemoglobin. I. Isolation and preliminary characterization of the globin chains.

The minor component of Japanese dancing mice hemoglobin, which runs more slowly on electrophoresis, differs from its major component in the non-α chains. The alkaline denaturation results show that the x chains are more alkali labile than α and β chains. Globin electrophoresis provides evidence that the charge difference between the major and minor components is located in non-α chains.

Gene selection in hemoglobin and in antibody-synthesizing cells.

Close linkage of mutually exclusive genes occurs in the non-alpha chain hemoglobin genes and in the immunoglobulin genes of man and other mammals. The expression of one gene in the cluster precludes the expression of any other linked gene. A simple, testable theory of gene selection called "looping-out excision"which was designed only to explain this mutual exclusivity in the hemoglobin system is described. The theory is closely concordant with a wide range of previously unexplained findings concerning hematopoiesis- including the developmental changes of hemoglobins, the increases in immature or fetal forms of hemoglobin that accompany anemia, and with the distribution of adult and fetal hemoglobins among erythrocytes during normal embryogenesis and in various pathological conditions. One corollary of this theory is that erythroid tissue in the normal adult bone marrow is constantly recapitulating the developmental stages of its embryogenesis. Another corollary is that the selection from among the linked globin genes occurs independently on the two chromosomes of the diploid organism. Both of these corollaries are supported by the available data. The same theory of gene selection is also remarkably consistent with known data for immunoglobulin synthesis; it could explain not only the mutually exclusive activation of linked variable genes but also the splicing which occurs between genetically linked variable and constant region genes for the immunoglobulin polypeptide chains. The agreement between these two different tissues is considered to be strong evidence that the proposed mechanism is correct at least in broad outline. Evidence from the genetics of maize and of drosophila also supports this theory of somatic tissue variegation. On the basis of these comparisons, I suggest that looping-out excision probably occurs also in other tissues and may be one means of gene selection and activation in differentiating cells.

Multiple hemoglobins in the golden hamster

1. 1. Seven distinct benzidine-positive bands were found on starch-gel electrophoresis at pH 8.6 of the hemolysates from fetal, newborn and adult golden hamsters. Four of the seven benzidine-positive bands were analyzed and named Hemoglobins I, II, III and IV according to their increasing electrophoretic mobility. Hemoglobins I and II were found as major components in the fetal stage. Hemoglobin I gradually decreased and disappeared by the 11th day after birth. Hemoglobin II, however, gradually decreased but remained as a minor component in the newborn and adult. Hemoglobins III and IV were found as minor components in the fetal stage, the former as a major component and the latter as a minor one in the newborn and adult stages. 2. 2. The amino acid compositions of the polypeptide chains from these hemoglobins were determined. Few differences among the α I, α III and α IV chains were found in the amino acid compositions. There were apparent differences between the α II and other α chains involving a minimum of eleven amino acid residues. Among the non-α chains, the β II chain was very similar to the β III chain and there were distinct differences among the γ I, β III and β IV chains. 3. 3. The tryptic peptide patterns of the aminoethylated (AE) α I, AE α II and AE α IV chains were closely similar to one another. In the patterns of the AE α III and AE α II chains, one peak of the AE α III was replaced by one acidic peak and two basic peaks of the AE α II chain. On the other hand the AE γ I, AE β III and AE β IV chains were quite different from one another in the tryptic pattern, while the pattern of the AE β II chain was similar to that of the AE β III chain.

Unequal Synthesis of Complementary Globin Chains of Human Fetal Hemoglobin by the Effect of L-O-Methylthreonine

Abstract Incorporation of radioactive amino acids into the α, β, and γ chains of human hemoglobin was studied with erythroid cells from newborn infants. When the cells were incubated in isoleucine-free medium together with l-O-methylthreonine, which is an antagonist of l-isoleucine, inhibition of γ chain synthesis occurred. The concomitant synthesis of α and β chains, which do not contain isoleucine, was not significantly affected. Inhibition of γ chain synthesis by as much as 90% had virtually no effect on the synthesis of complementary α chains, suggesting that the synthesis or release of α chains does not require prior synthesis of these non-α chains. As a result of the selective inhibition of synthesis of γ chains, cells incubated with the isoleucine antagonist synthesized a 2- to 3-fold excess of α chains over the total non-α chains. A major portion of the excess α chains was present in the cell lysates uncombined with hemoglobin, and had Sephadex gel filtration properties consistent with their being present as α dimers. When βa subunits were added to the lysates prior to Sephadex gel filtration, the excess α chain radioactivity appeared in the hemoglobin fraction, suggesting that the free α chains existed as completed globin-heme subunits which retained the capacity to combine with complementary subunits to form hemoglobin. When cells were incubated with l-O-methylthreonine and a radioactive amino acid in label-chase experiments, followed by 24-hour incubations, the excess radioactive α chains remained in the soluble cell fraction. This was in contrast with reported studies with β-thalassemia cells in which α chain precipitation was demonstrated during cell incubations in vitro. This difference appeared to be the result of intracellular exchange of the radioactive α chains with α chains of preexisting hemoglobin in the cells incubated with l-O-methylthreonine, a reaction which may be undetectable in β-thalassemia cells because of the presence of a preexisting pool of nonradioactive α chains in these cells.

Exchange of Heme among Hemoglobins and between Hemoglobin and Albumin

Intact heme groups undergo exchange between molecules of human hemoglobin under physiological conditions. This phenomenon requires the presence of ferrihemoglobin and is blocked by heme ligands or the prior binding of hemoglobin to haptoglobin. The kinetics of heme exchange between hemoglobins A and F allows a calculation of the rate constants for the dissociation of heme and globin. The rate of exchange of hemes between the non-α chains of ferrihemoglobins A and F is approximately 8 times that between the α chains. Mixtures of ferrihemoglobins and oxyhemoglobins exchange hemes at a rate greater than predicted from the rates of exchange observed in mixtures containing only oxyhemoglobin or ferrihemoglobin. Ferriheme groups also undergo exchange between hemoglobin not bound to haptoglobin and methemalbumin, although at equilibrium the affinity of human albumin for ferriheme is only about one-fifteenth that of globin. Only primate albumins possess appreciable affinity for ferriheme; albumins from the nonprimate species examined appeared to be incapable of forming methemalbumin. Depletion of hemes from ferrihemoglobin by excess human albumin leads to the accumulation of free, precipitable globin.

The synthesis of alpha, beta, and delta peptide chains by reticulocytes from subjects with thalassemia or hemoglobin Lepore.

Abstract The incorporation of radioactive amimo acids into the α and β chains of hemoglobin A, the α and δ chains of hemoglobin A 2 , and the α and non-α chains of hemoglobin Lepore by reticulocytes from nonhemoglobinopathic, Lepore trait, and thalassemic patients has been compared. Significant imbalance of α and β chain production was detected only in thalassemic subjects. This was characteristic of thalassemia minor as well as thalassemia major. The relative rates of δ chain production were greater than those of β thalassemia chains. There was little depression of the synthesis of the non-α chains of hemoglobin Lepore relative to α chain formation in vitro by reticulocytes from patients heterozygous for hemoglobin Lepore when compared with control subjects.