Human Fetal Hemoglobin Research Articles

The synthesis of the G gamma and A gamma chains of human fetal hemoglobin in erythroid colonies cultured from peripheral blood BFUe's of normal adults and newborn and of subjects with an A gamma or a G gamma chain abnormal fetal hemoglobin.

AbstractPeripheral blood mononuclear cells from normal adults, normal newborn infants, and from newborn and adult subjects with one of three γ chain variants (GγF‐Malta I, GγF‐Port Royal, AγF‐Hull) were cultured in vitro with erythropoietin. The 35S‐methionine‐labelled hemoglobin from 13‐ to 15‐day‐old BFUe‐derived colonies was studied by chromatography on columns of DEAE‐cellulose and the quantities of Hbs A2, Fx, and F0 determined. The percentages of Gγ and Aγ chains in isolated Hb Fx and Hb F0 were determined using high‐performance liquid chromatography (HPLC) of the tryptic peptides of these proteins. Calculation of these percentages was based on the total activities of the GγT‐15 and AγT‐15 peptides which contain one (35S‐labelled) methionyl residue each and can be separated by the HPLC procedure.The data show an increased synthesis of Hb F in the „adult”︁ colonies and a decreased synthesis in the „newborn”︁ colonies. The Gγ to Aγ ratio of the Hb F from adult colonies varied greatly. The percentages of Gγ and Aγ chains in the Hb F from adult colonies correlated with the percentages in the Hb F isolated from the Hb F of circulating red blood cells. The Gγ to Aγ ratio in the Hb F from newborn colonies was high as in the Hb F from cord blood samples.Gγ and Aγ chain abnormal Hb F variants were readily detectable in colonies from both adults and newborn. The Gγ to Aγ ratio in the Hb F of colonies of adult Hb F‐Malta I and Hb F‐Hull heterozygotes approached 1, but that of adult Hb F‐Port Royal heterozygotes remained about as high as in colonies from newborn heterozygotes. The percent Hb F‐Port Royal in the Hb F of adult colonies was twice that in the Hb F of newborn colonies.These results are discussed in the light of information from recent detailed studies of genomic DNA assuming controlling functions for the segments of DNA that are interspersed between the various structural genes.

Polymorphism of human fetal haemoglobin studied by isoelectric focusing

Human fetal haemoglobin (Hb F, o?ys) is a mixture of different molecular species. Two of them differ in position 136 of the 7 chain which is occupied either by glycine (G-r chain) or by alanine (*r chain) [l]. These chains are the products of two closely linked genes present in all normal individuals [ 1,2]. A variant of Hb F, Hb F Sardinia (ars$s 75 Ile + Thr) [3] is of unusual high frequency in various populations at birth [4,5]. Very recent results of peptide analysis suggested that the 775 Thr chain is a variant produced by the A7 locus [6,7] and corresponds to a genetic polymorphism. The study of this polymorphism is made difficult by the complexity of the methodology involved in peptide analysis [ 1,4,5]. In contrast, we report that isoelectric focusing (IEF) on acrylamide slabs using a shallow gradient of laboratory made ampholytes (pH 6.5-7.8) allows to separate Hb F 775 Thr from Hb F 775 Ile. It is a reliable and a simple method for the study of such polymorphism. We provide a direct evidence that the 775 Thr chain variant is produced by the A7 locus.

Globin chain electrophoresis: a new approach to the determination of the G gamma/A gamma ratio in fetal haemoglobin and to studies of globin synthesis.

Separation of globin chains by electrophoresis provides a simple and rapid method for the determination of the G gamma/A gamma ratio in human fetal haemoglobin, and of biosynthetic rates of the globin chains. Whole haemolysates were analysed by electrophoresis on polyacrylamide gels in urea, acetic acid and Triton X-100. Electrophoresis of haemolysates from newborn infants led to four bands: A gamma, G gamma, beta and alpha. The identity of these bands was indicated by examination of haemoglobins of known globin chain composition. In 15 samples, the % G gamma was similar by Triton gels and by amino acid analysis of the gamma CB-3 peptide. Some mutant globin chains were also separable with the electrophoretic technique. Triton gel electrophoresis provides rapid analysis of very small amounts of haemoglobin, and permits examination of globin chain composition as well as globin synthetic ratios.

Nature of the negative ellipticity of human fetal hemoglobin in the 280 nm region

The uv circular dichroism (CD) spectra of aquomet hemoglobins A and F were followed to monitor their R→T conformational change. Titration studies with inositol hexaphosphate (IHP) for both adult and fetal hemoglobin showed identical total ellipticity changes although HbF was found to possess an inherently negative ultraviolet CD spectrum. By monitoring changes in the protein portion of the molecule, a dissociation constant for IHP of 16 μM was obtained for HbF. Chemical modification of HbF was found to leave the negative ellipticity unperturbed relative to native HbF. The results suggest that the negative ellipticity seen for stripped aquomet HbF is not due to a T conformation, but rather to an amino acid substitution in the γ chain of HbF.

Phosphate binding by llama and camel hemoglobin

1. We have estimated the binding constant of 2,3-diphosphoglycerate (DPG) to llama and camel hemoglobin at 37°C and pH 6.7, 7.2, and 7.7 from the DPG induced change in the half saturation pressure (P50). The values obtained were compared with the ones derived from similar measurements on human fetal and adult hemoglobin. At pH 7.2 the binding constant of DPG to llama deoxy-hemoglobin is smaller by a factor of 3 in comparison to camel hemoglobin whilst human fetal hemoglobin binds DPG six times less firmly than the human adult pigment. Camel and human adult hemoglobin have about the same affinity for DPG. 2. It is concluded that in llama hemoglobin an amino acid replacement at the DPG binding site explains the reduced phosphate affinity as compared to camel hemoglobin. In human fetal hemoglobin, in addition to an amino acid exchange, there are alterations of the anatomy of the DPG binding site which result in a poorer fit of the phosphate molecule. 3. Our data show that llama hemoglobin has a lower intrinsic oxygen affinity than camel hemoglobin so that despite the reduced DPG binding constant the oxygen affinity of llama and camel blood is very similar at equal phosphate concentration. Possibly, the amino acid exchange at the DPG binding site has compensated for the low oxygen affinity of llama hemoglobin in the course of phylogeny

The determination of the percentages of G gamma and A gamma chains in human fetal hemoglobin by HPLC.

A newly developed HPLC procedure (1) was used to study the percentages of G gamma and A gamma chains in the Hb F from newborn and patients with sickle cell anemia. The method gives data comparable to those obtained with a more laborious chemical procedure. However, the presence of the A gamma T chain, i.e., the A gamma chain with an Ile replaced by Thr substitution at position 75, interferes with the determination of the G gamma to A gamma ratio because the A gamma T and G gamma chains have the same chromatographic mobility.

Antigens present on human myeloid leukemia cell lines

Two human acute myelogenous leukemia cell lines have recently been established: KG-1 and HL-60. Both lines retain the morphology and cytochemistry of myeloid cells, are not infected with EB virus, and respond to colony-stimulating activity (CSA) with increased colony formation in soft-gel culture. The K 562 cell line is composed of undifferentiated blast cells which are rich in glycophorin and may be induced to produce human fetal and embryonic hemoglobin. We used a variety of antisera to characterize the antigen composition of the leukemic colony-forming cells (CFC) and the general population of leukemic cells. Cells were incubated with antisera and complement, washed, plated in agar, and the colonies counted after 12 days' incubation. Culture data were correlated with the results obtained using direct and indirect immunofluorescence and dye exclusion microcytotoxicity. The majority of KG-1 cells, including CFC, expressed the ‘Ia-like’ antigen. The HL-60 and K562 cells did not express the Ia antigen, although indirect evidence using cytogenetic and isoenzyme studies suggests that the HL-60 gene locus for the antigen was probably intact. We found that the leukemia CFC had the HLA-A and -B antigens as did the general leukemia cell population. The leukemia CFC did not express a T-lymphocyte antigen, certain erythrocyte antigens, nor antigens associated with acute lymphocytic leukemia. HL-60 cells underwent morphological and functional maturation in the presence of DMSO, but no change in surface antigenic composition was noted. All antigens expressed on the leukemia CFC were also present on the general cell population.

The synthesis and chemical heterogeneity of human fetal hemoglobin: overview and present concepts.

The synthesis and chemical heterogeneity of human fetal hemoglobin: overview and present concepts.

The initiation of fetal hemoglobin biosynthesis

Biosynthesis of the α and β chains of rabbit and human adult hemoglobin is initiated with a methionyl residue, which is removed during elongation of the peptide chain. To study the initiation of biosynthesis of the γ chain of human fetal hemoglobin, fresh placental blood was used for labeling experiments with radioactive amino acids. Labeled nascent peptide chains were purified from the polysomal fraction of placental blood reticulocytes. The number of amino acid residues in nascent γ chain at the time of removal of its N-terminal methionine was estimated to be 40–60 from the relative yields of labeled tryptic peptides.

Separation of the Aγ and Gγ cyanogen bromide peptides of human fetal hemoglobin by high-pressure liquid chromatography

The G γ and A γ variants of human fetal hemoglobin are defined on the basis of a single glycine/alanine substitution at position 136 of the γ chain which is located in the carboxyterminal cyanogen bromide fragment (γCB-3). This communication describes the rapid separation of these two tridecapeptides by high-pressure liquid chromatography. Application of this new technique will facilitate the study of the biosynthesis and genetics of human fetal hemoglobin.

G γ and A γ globin chains separation and quantitation by isoelectric focusing

Isoelectric focusing in the presence of Nonidet P-40 splits human chromatographically pure γ globin chains into two bands of isoelectric points 6.95 and 6.85, respectively. The comparison of the relative proportions of the two bands with the ratios between the G γ and A γ non allelic chains of human fetal hemoglobin suggests that the band at pI 6.95 corresponds to G γ and the band at pI 6.85 corresponds to the A γ chain; the latter is the only band present in a patient with Greek type hereditary persistence of fetal hemoglobin, producing only A γ chains. Fluorography of electrofocusing-separated radioactive γ globin chains synthesized by thalassemic reticulocytes indicates that the relative G γ A γ synthetic ratios are similar to the relative amounts of G γ and A γ chains accumulated in the erythrocytes, suggesting that the activities for the G γ and A γ mRNAs decay at roughly similar rates.

Hemoglobin synthesis in cultures of hepatic erythroid cells from the human fetus

A recent theory of the control of human fetal hemoglobin synthesis, based on studies in cultured adult marrow, proposes that the phenotypic expression of fetal hemoglobin is largely dependent on the level of differentiation of the parental stem cells; that is, the earlier the progenitor, the greater the ability of its progeny to express fetal hemoglobin [Papayannopoulou, Th., Brice, M. & Stamatoyannopoulos, G. (1977) Proc. Natl. Acad. Sci. USA 74, 2923-2927]. To test this relationship with fetal tissue, we have studied hemoglobin synthesis in cultured human fetal liver, comparing gamma chain synthesis in the descendants of the early progenitors ("bursts") with that in the descendants of the later progenitors ("colonies"). Cells from the livers of midtrimester fetuses were cultured in methylcellulose with erythropoietin. The beta/(beta + gamma) globin synthetic ratio on days 5 to 7, when colonies predominated, was 0.09-0.11, a value characteristic of fetal reticulocytes, and on days 11 and 12, when bursts predominated, was 0.15-0.17. Thus, in fetal liver, the descendants of the earlier progenitor, the burst-forming unit, may be making more beta chains rather than more gamma chains, compared to descendants of the later progenitor, the colonyforming unit. Our data on fetal liver, taken together with the data on adult marrow by others, suggest that the erythroid colonies express the gene characteristic of the age of the organism to a greater degree than bursts, which express beta and gamma genes less specifically. Thus, the capacity for highly selective gene expression characteristic of differentiated cells appears to be less well developed in the burst-forming unit than in the colony-forming unit.

Further studies of the frequency and significance of the Tgamma-chain of human fetal hemoglobin.

A further study of the Tgamma-chain in a variety of conditions has revealed its presence in the cord bloods of ethnic groups previously unstudied. Heterozygous newborn average 17-19% Tgamma-chain while the mean value in four presumed homozygotes was 31%. The Tgamma-chain is readily detectable in beta-thalassemia of various ethnic groups (although infrequent in Blacks) as well as in deltabeta-thalassemia. Studies of a few families have provided an opportunity to determine whether or not certain individuals are heterozygous or homozygous for the Tgamma-gene. The Tgamma-chain has not been detected in the human fetal hemoglobin that is synthesized in increased amounts in persons with the hereditary persistence of fetal hemoglobin. Although the Tgamma-chain is detectable in sickle cell anemia, its frequency appears to be lower than in normal individuals. By focusing upon the relationship of the percentage of Tgamma-chain to the sources of human fetal globulin from determinants in cis and in trans, the conclusion has been reached that the Tgamma-chain is the product of a mutant Agamma-locus which should be named the TAgamma-chain.

The synthesis of fetal hemoglobin types in red blood cells and in BFU-E derived colonies from peripheral blood of patients with sickle cell anemia, beta+ - and delta beta-thalassemia, various forms of hereditary persistence of fetal hemoglobin, normal adults and newborn.

The biosynthesis of two types of human fetal hemoglobin (Hb F), namely Hb F with G gamma chains having glycine in position 136 and Hb F with A gamma chains having alanine in position 136, was studied in blood samples and in cultures of erythroid precursors from blood of patients with different hemoglobinopathies. High pressure liquid chromatography (HPLC) was adapted to allow the separation of the methionyl-containing tryptic peptides G gamma T-15 and A gamma T-15 (which include the Gly leads to Ala polymorphism at position 136) from a digest of microquantitites of 35S-methionyl labelled Hb F. This method was sensitive enough to quantitate the relative production of the G ygamma and A gamma chains by erythroid colonies derived from cloned Burst Forming Units (bfu-e) which were cultured for 16 days on methylcellulose. The production of Hb F in these colonies was generally higher than the level of Hb F in blood except for subjects with the G gamma A gamma-HPFH heterozygosity. The G gamma to A gamma ratio in the Nb F produced in cultures of cells from G gamma delta beta-thalassemia or G gamma-HPFH heterozygotes was lower and that from A gamma-HPFH heterosygotes was higher than the ratios in the Hb F of the corresponding peripheral blood cells. Mixtures of G gamma and A gamma chains were present in cell cultures of SS patients, beta+-thalassemia homozygotes and G gamma A gamma-HPFH heterozygotes in a ratio similar to that in the Hb F of mature red cells. These data suggest that erythroblasts in BFU-E derived colonies reactivate all available gamma chain structural genes, both in cis and in trans to the abnormal determinant. Hb F biosynthesis by adult blood samples concerns primarily the G gamma chains. This was particularly striking for blood samples in which erythroblasts were absent and the biosynthesis took place in fetal reticulocytes. Thus, the F-retuculocytes in blood of A gamma-HPFH heterozygotes with about 5% Hb F of the A gamma type produced primarily Hb F with G gamma chains. Similar differences were observed for G gamma A gamma-HPFH heterozygotes and, less strinkingly, for SS patients. A satisfactory explanation for this observation has not yet been obtained.

Composition of the <i>γ</i>-Chains of Human Fetal Hemoglobin at Birth and during Intrauterine Life

The composition of the <i>&#947;</i>-chains has been examined in 21 fetuses and premature babies and in 18 full-term newborns. G<i>&#947;</i>/A<i>&#947;</i>ratio was slightly higher in fetuses (0.81 ± 0.04 expressed as glycine residue at position 136) than in full-term newborns (0.73 ± 0.06), while the incidence and the percentage of ^T<i>&#947;</i>-chains were clearly higher in the first group and overlap the values found in the Hb F from <i>β</i>-thalassemia homozygotes. On the basis of these data the authors hypothesize that ^T<i>&#947;</i>/-chains may be codified by a gene more active during intrauterine life than at birth. Possibly, their synthesis may be reactivated when high levels of Hb F are present during adult life, as in <i>β</i>-thalassemia.

High yield photoreagents for protein crosslinking and affinity labeling

4-Nitrophenyl ethers are proposed as new high-yield photoreagents for protein crosslinking and affinity labeling. These are totally unreactive in the dark under biological conditions, but react quantitatively with amines at pH 8 upon irradiation with 366-nm light. The reaction of monoalkoxy-p-nitrobenzenes with an amine yields the corresponding free alcohol and substituted nitrophenylamine. In essence, the nitrophenyl group is transferred from the alcohol to the amine. Bifunctional affinity reagents of this type could be especially useful for placing the p-nitrophenyl chromophore adjacent to a binding site without blocking it. The corresponding 2-methoxy-4-nitrophenyl ethers react with amines by displacement of the methoxyl group. Thus, bifunctional reagents of this class could be photocrosslinkers. A maleimide-containing 2-methoxy-4-nitrophenyl ether was attached to human fetal hemoglobin at gamma-cysteine F9 stoichiometrically. Subsequent ultraviolet irradiation yielded a gamma-gamma crosslinked hemoglobin in 80% yield. The oxygenation properties of the derivative indicate that it is locked in a high affinity conformation and that all cooperativity is lost.

Analogous effect of protons and inositol hexaphosphate on the alteration of structure of nitrosyl fetal human hemoglobin.

We have determined the low temperature EPR spectra and room temperature ligand dissociation rate constants of human NO-hemoglobins F and A as a function of pH and inositol hexaphosphate levels in order to assess the contribution of a quaternary structural equilibrium in the two proteins to their spectral and functional properties. Our results are consistent with an increased stability of a ligated low affinity structure in the fetal protein; the functional properties of this structure appear to be essentially the same in the two hemoglobins, even though its stability relative to a high affinity conformation is different. The pH dependence of the NO dissociation constant in both adult and fetal hemoglobin can be assigned primarily to the pH-dependent equilibria of high and low affinity forms as monitored by EPR.

High pK value of the N-terminal amino group of the γ-chain causes low CO 2 binding of human fetal hemoglobin

The N-terminal amino acid residue of the γ-chains of human fetal hemoglobin (Hb F II) is glycine rather than valine like in many other hemoglobins including the human adult pigment (Hb A). In the course of an evaluation of functional implications associated with this replacement we have studied the CO 2 binding properties of Hb F II in comparison with Hb F Ic where the N-termini of the γ-chains are blocked. By comparing Hb F II and Hb F Ic it is possible to specifically estimate carbamate formation at the N-termini of the γ-chains in Hb F II. These data were used to calculate the carbamate equilibrium and ionization constant of these groups. At 37 °C, −log 10 of the ionization constant (pK z) was found to be 8.1 and is thus significantly higher than pK z of the N-terminal valines of the β-chains of Hb A which has been reported to be 6.6 at 37 °C. The high pK z value of the γ-chain α-amino group explains the much lower carbamate formation in Hb F II compared to Hb A.

The T?? Chain of Human Fetal Hemoglobin at Birth and in Several Abnormal Hematologic Conditions

The T gamma chain of human fetal hemoglobin has a threonyl in stead of an isoleucyl residue in position 75. When the cord bloods from infants from varied ethnic backgrouds and geographic areas were tested for the presence of the T gamma chain, it was present in 28 or 98 samples. In some groups as many as 40% had the T gamma chain whereas none was detected in other. When the T gamma chain was present, its quantity was about 20% of the total gamma chains, but one case had 35%. Among beta-thalassemia homozygotes of the Mediterranean region, 70% and the T gamma chain in the amount of 20-50% of the total gamma chains, but seven Black beta-thalassemia homozygotes were negative for the T gamma chain. The fetal hemoglobin of 16 adult patients with sickle cell anemia had no T gamma chains, but 2 of 9 newborn children with sickle cell anemia had the T gamma chain. The frequency of the T gamma gene (16), the relationship of the T gamma gene to the G gamma and A gamma genes, and the significance of the T gamma gene are discussed.

Anomaly in the gamma chain heterogeneity of the newborn.

THE two types of γ chain in human foetal haemoglobin (HbF) are the products of nonallelic structural genes and have either glycine (Gγ type) or alanine (Aγ type) in position 136 (ref. 1). Although the examination of 108 cord blood samples from worldwide sources showed 102 to have a Gγ : Aγ ratio within a relatively narrow range, one had a low ratio and five a high ratio2. It seemed possible that analytical error was responsible for these extreme values. But as we studied cord blood from normal black babies of residents from Burke County, Georgia, where an abnormal HbF (designated FX (ref. 3)) has been detected repeatedly, we observed high ratios in further samples. The present study of almost 700 cord bloods has detected the high Gγ : Aγ ratio in about 7.5% of samples from black newborns as compared with perhaps 0.5% from Caucasian infants.