Human Fetal Hemoglobin Research Articles

Production of human normal adult and fetal hemoglobins in Escherichia coli.

A hemoglobin expression system in Escherichia coli is described. In order to produce authentic human hemoglobin, we need to co-express both methionine aminopeptidase and globin genes under the control of a strong promoter. We have constructed three plasmids, pHE2, pHE4 and pHE7, for the expression of human normal adult hemoglobin and a plasmid, pHE9, for the expression of human fetal hemoglobin, in high yields. The globin genes can be derived from either synthetic genes or human globin cDNAs. The extra amino-terminal methionine residues of the expressed globins can be removed by the co-expressed methionine aminopeptidase. The heme is inserted correctly into the expressed alpha-globin from our expression plasmids. A fraction (approximately 25%) of the heme is not inserted correctly into the expressed beta- or gamma-globin. However, the incorrectly inserted hemes can be converted into the correct conformation by carrying out a simple oxidation-reduction process on the purified hemoglobin molecule. We have investigated the functional properties of the expressed hemoglobins by measuring their oxygen-binding properties and their structural features by obtaining their 1H-NMR spectra. Our results show that authentic human normal adult and fetal hemoglobins can be produced from our expression plasmids in E. coli and in high yields. Our expression system allows us to design and to produce any recombinant hemoglobins needed for our research on the structure-function relationship in hemoglobin.

Significance of Oxygen Affinity of Fetal and Adult Human Hemoglobins

Abstract The oxygen equilibrium curves of human fetal and adult hemoglobins were reconstructed from the published Adair constants. The curves were then analyzed theoretically with respect to the amount of transferred oxygen, which is directly related to the saturation difference (▵S) of hemoglobin with oxygen in the artery and vein. In fetal blood, the oxygen affinity is optimized so as to provide the maximal ▵ S value in the fetus oxygen environment. In adult blood, on the other hand, the ▵ S value is far smaller than the theoretically obtained maximum, but it is most sensitive to the P50 changes around its physiological value of 27 torr. The present results imply that adult blood reserves an oxygen transport capacity for increased oxygen demands under resting conditions, and that the oxygen affinity is optimized so as to make the Bohr effect most effective to gaseous exchange.

A rapid combined immunocytochemical and fluorescence in situ hybridisation method for the identification of human fetal nucleated red blood cells

Fetal nucleated red blood cells are found in the maternal circulation during pregnancy. If a simple routine method of detection of these cells was developed, it could be used as the basis of non-invasive prenatal diagnosis of fetal genetic disorders. Fetal male and adult female blood were mixed to mimic maternal blood in pregnancy and used to establish a simple technique to unequivocally detect fetal nucleated red blood cells. These were identified by combined immunocytochemistry using a human fetal haemoglobin antibody and a rapid and simple-to-use fluorescence in situ hybridisation method using X and Y chromosome probes. Initial studies using the alkaline phosphatase anti-alkaline phosphatase technique as the first procedure showed that the stain was unstable and unsuitable for in situ hybridisation. An immunoperoxidase technique was found to produce a stable stain resistant to harsh fixation steps required in subsequent in situ hybridisation. This enabled the simultaneous visualisation of immunopositivity and in situ hybridisation signals on the same cell with neither procedure affecting the other's signal quality. We are currently using this procedure to detect a range of endoplasmic reticulum proteins in fetal nucleated red blood cells from maternal blood in an attempt to diagnose disorders of liver protein expression in early pregnancy.

Increases in human fetal hemoglobin oxygen saturation during late fetal heart rate decelerations as a response to intrauterine stress: [formula omitted], C. Harvey x, G. Anderson, T. Shailer x, L. Troyer. Dept. of OB/GYN, The University of Texas Medical Branch, Galveston, TX

Increases in human fetal hemoglobin oxygen saturation during late fetal heart rate decelerations as a response to intrauterine stress: [formula omitted], C. Harvey x, G. Anderson, T. Shailer x, L. Troyer. Dept. of OB/GYN, The University of Texas Medical Branch, Galveston, TX

The Mγ chain of human fetal hemoglobin is an Aγ chain with an in vitro modification of γ141 leucine to hydroxyleucine

We have reanalyzed the structure of the γT-15 peptide from the minor Mγ chain of human hemoglobin (Hb) F. Amino acid analysis confirmed that the Leu 141 residue was missing from position 9 of this peptide, and liquid secondary ion mass spectrometry indicated that it was replaced, not by methionine (residue mass 131) as previously believed, but by an amino acid of mass 129. By analogy with the recently reported oxidation of the corresponding leucine at position γ141 of the unstable Hb Atlanta, it appears that the Mγ chain also results from the oxidation of γ141 to hydroxyleucine (residue mass 129). The finding that the proportion of the Mγ chain increased when red cell lysates were prepared with carbon tetrachloride prompted us to reinvestigate the oxidation mechanism involved in the formation of β141 hydroxyleucine in Hb Atlanta. Oxidation of the β141 residue could be detected when carbon tetrachloride was used in the lysis protocol, while conversion of oxyhemoglobin to carbon monoxyhemoglobin prior to carbon tetrachloride treatment prevented oxidation. It therefore appears that the hydroxylation of Leu 141 is not an in vivo process in the circulating red cell. Perhaps leucine at position 141 of the β, γ, and δ chains (and at position 136 of the α chain), which forms a contact with heme and is located directly across the heme plate from the E helix, is oxidized to hydroxyleucine at a very low rate forming minute amounts of modified chains; this process is accelerated by treatment with agents such as carbon tetrachloride and prolonged exposure to air.

Physiological Relevance of the Overall ΔH of Oxygen Binding to Fetal Human Hemoglobin

Human fetal hemoglobin is known to display, at 20°C, a lower affinity than human adult hemoglobin for oxygen when both proteins are in the absence of organic phosphates. The physiologically important reverse situation is achieved at 37°C upon addition of 2,3-bisphosphoglycerate (DPG), whose lower effect on fetal hemoglobin is related to some amino acid substitutions present in γ-chains. However, the difference in oxygen affinity observed at 37°C is not solely due to the different modulation power of DPG with respect to adult and fetal hemoglobins. In fact, the results presented here reveal new aspects linked to the interplay of temperature and organic phosphates.In particular, the lower effect of DPG on fetal hemoglobin renders almost identical the oxygen affinity of the two hemoglobins at 20°C, abolishing the difference observed in the absence of the effector. Successively on going from 20°C to 37°C, by virtue of the lower overall heat of oxygenation (ΔH) displayed by fetal hemoglobin when in the presence of DPG, adult hemoglobin shows a lower oxygen affinity, as it should if oxygen has to be transferred from maternal to fetal blood.

Changes is human fetal cerebral hemoglobin concentration and oxygenation during labor measured by near-infrared spectroscopy

Changes is human fetal cerebral hemoglobin concentration and oxygenation during labor measured by near-infrared spectroscopy

Determination of Fetal Hemoglobin by Time-Resolved Immunofluorometric Assay

We have developed a "sandwich"-type time-resolved immunofluorometric assay (IFMA) for fetal hemoglobin (HbF) in hemolysates from adults and newborns, amniotic fluid, and plasma, based on a polyclonal and a monoclonal antibody against human fetal hemoglobin. Microtiter wells are coated with polyclonal capture antibody, and the gamma-chain-specific monoclonal tracer antibody is labeled with a europium chelate. In a simple and fast assay procedure, prediluted hemolysates are incubated in the microtiter wells first with capture antibody for 1 h and, after washing, for 1 h with tracer antibody. The wells are washed and the fluorescence of europium is measured. The mean analytical recovery is 102% and results by IFMA agreed well with values obtained by high-performance liquid chromatography. The analytical range of IFMA is large and well suited for clinical purposes. The detection limit of the assay is 0.2 microgram/L and the measuring range extends to 500 micrograms/L.

Capillary electrophoresis of hemoglobins and globin chains

Capillary isoelectric focusing (cIEF) and free zone capillary electrophoresis were evaluated for separation of native hemoglobins and globin chains. High-resolution separations of adult human hemoglobin A, fetal human hemoglobin F, and hemoglobin variants S and C were obtained using cIEF with cathodic mobilization. Absorbance detection in the UV and visible regions were compared, and on-line fast UV or visible-wavelength scanning detection was used to obtain spectral information on separated components. Globin chain analysis was performed on the same hemoglobin species by free zone capillary electrophoresis following precipitation of the protein with acidic acetone. Free zone separations were carried out at low pH in the presence of 7 M urea.

Changes in human fetal cerebral hemoglobin concentration and oxygenation during labor measured by near-infrared spectroscopy

The purpose of this study was to measure by near-infrared spectroscopy changes in human fetal cerebral oxyhemoglobin, deoxyhemoglobin, and cerebral blood volume during labor and to calculate mean cerebral hemoglobin oxygen saturation. The effects of uterine contractions with and without fetal heart rate decelerations were compared in eight singleton term fetuses. Results were analyzed by analysis of variance. In six of eight fetuses normal uterine contractions were associated with proportional decreases in both oxyhemoglobin and deoxyhemoglobin and a fall in cerebral blood volume without desaturation of cerebral hemoglobin. Contractions with fetal heart rate decelerations produced different results in that oxyhemoglobin fell but deoxyhemoglobin rose, indicating cerebral desaturation. In two of the eight fetuses normal contractions were associated with increases in oxyhemoglobin, deoxyhemoglobin, and cerebral blood volume; no decelerations were seen in either fetus. Mean cerebral hemoglobin oxygen saturation calculated during normal contractions was 43% +/- 10% (SD). Uterine contractions were associated with detectable changes from baseline in cerebral oxyhemoglobin, deoxyhemoglobin, and cerebral blood volume.

Postnatal transition of γ-globin gene expression in normal Japanese population

Temporal course of postnatal changes in the gamma isoform composition of human fetal haemoglobin (HbF) was studied in 259 cord, 272 infantile and 216 adult Japanese blood samples. Reversed phase high-performance liquid chromatography was used for determination of the three isoforms of the gamma chain (A gamma T, A gamma I and G gamma), and the adult samples, usually with less than 1% of Hb F, were enriched for Hb F by an alkali denaturation-salting out procedure. The results show that the G gamma gene expression, kept at a higher and strictly-controlled level in the neonatal period, undergoes a gradual change during 1 year, beginning 3-4 months after birth, to the adult stage which is characterized by a generally lower and loosely-controlled expression of the gene. Further change seems to occur after 1 year, settling to the final adult level. The A gamma T gene frequency is estimated as 0.139 in the present Japanese adult population, and is essentially identical to the value for the newborns in the same population (0.141).

Measurement of changes in human fetal cerebral haemoglobin concentration and oxygenation by near infrared spectroscopy during labour : D.M. Peebles, A.D. Edwards, J.S. Wyatt, A.P. Bishop, M. Cope, D.T. Delpy and E.O.R. Reynolds, Departments of Paediatrics, Obstetrics, and Medical Physics and Bioengineering, University College and Middlesex School of Medicine, London, U.K.

Measurement of changes in human fetal cerebral haemoglobin concentration and oxygenation by near infrared spectroscopy during labour : D.M. Peebles, A.D. Edwards, J.S. Wyatt, A.P. Bishop, M. Cope, D.T. Delpy and E.O.R. Reynolds, Departments of Paediatrics, Obstetrics, and Medical Physics and Bioengineering, University College and Middlesex School of Medicine, London, U.K.

The effect of organic cosolvents on the oxygen affinity of fetal hemoglobin

We have studied the effects of organic cosolvents (monohydric alcohols and formamide) on the oxygen affinity of human fetal hemoglobin stripped of phosphates and have compared them with the effects of the same cosolvents on the oxygen affinity of human adult hemoglobin under the same experimental conditions. Our results confirm that, in fetal hemoglobin, the T in equilibrium R conformational equilibrium is more displaced toward the T conformation than in the adult form and indicate that increased electrostatic and hydrophobic protein-solvent interactions contribute to this effect. The data reported are discussed in terms of the known amino acid substitutions between the beta- and gamma-chains and an attempt is made to rationalize the results with a molecular mechanism based on the crystallographic structure of fetal deoxyhemoglobin.

Monoclonal antibody to the gamma chain of human fetal hemoglobin used to develop an enzyme immunoassay.

A monoclonal antibody (mAb) that recognizes the gamma chain of human fetal hemoglobin (Hb F) has been produced by cell hybridization techniques. The mAb reacts with Hb F (alpha 2 gamma 2), Hb Bart's (gamma 4), and Hb Kenya (gamma-beta hybrid), but does not cross-react with Hb A (alpha 2 beta 2) or Hb A2 (alpha 2 delta 2). We describe a direct enzyme-linked immunoassay (ELISA) for measurement of Hb F, in which hemoglobins from standards or from unknown hemolysates are covalently bound to the wells of microtiter plates. The antigen is quantified by addition of the gamma-specific mAb, followed by anti-mouse IgG conjugated with horseradish peroxidase, and incubation with the substrate, tetramethylbenzidine. Absorbances at 630 nm are directly proportional to the amount of Hb F present in the standards or samples. Results for Hb F in 53 hemolysates agreed well with values obtained by "high-performance" liquid chromatography, RIA, alkali denaturation, and magnetic affinity immunoassay. This ELISA can detect a 0.5% proportion of Hb F in 1 h and offers distinct advantages over other techniques currently in use.

Heterogeneity of the isolated subunits of the fetal and adult human hemoglobin in solution, detected by XANES spectroscopy

Differences in the local structure of the heme in the isolated α-, β- and γ-chains of the adult and fetal human hemoglobin are detected by XANES (X-ray absorption near-edge structure) spectroscopy. The ligand bonding angle to the iron ion in the ligated forms and the displacement of the Fe respect to the porphyrin plane in the deoxy forms are found to be different for each chain.

Temperature- and pH-dependence of the oxygen-binding reaction of human fetal haemoglobin

O2 binding to human haemoglobin F0 was studied at high haem concentrations (3 mM) in the temperature range 15-35 degrees C and in the pH range 6.8-8.7 at 25 degrees C. Comparison with O2 binding to human adult haemoglobin A0 under identical solution conditions reveals striking similarities in the Bohr effect and the enthalpy of oxygenation between the two haemoglobins.

Regulation of human fetal hemoglobin gene expression.

An understanding of the mechanism involved in the regulated expression of the human gamma and beta globin genes requires the detailed definition of the cis-acting DNA sequences and trans-acting protein factors responsible for their developmental stage specific expression. To determine the critical cis-acting elements, hybrid genes containing elements of the gamma and beta globin genes were transfected into K562 cells, a human erythroleukemia line. The regulated expression of the gamma and beta genes was also studied by transferring hybrid genes containing the gamma or beta promoters linked to the neomycin resistance gene (neoR) into erythroid (K562) cells and nonerythroid (Hela) cells. DNA sequences found to be important to the expression of the gamma gene were assayed for the presence of transacting factors by studying the binding of protein factors using the gel mobility shift assay. The results suggest that there are multiple cis-acting elements 5' and 3' to the gamma and beta genes, and perhaps within these genes contributing to their regulation. In addition, there are multiple trans-acting protein factors interacting with these regions which may determine their transcriptional regulation in erythroid cells.

Absorption characteristics of human fetal hemoglobin at wavelengths used in pulse oximetry

The absorption characteristics of fetal and adult human hemoglobin samples were determined for the range of 600 to 1,050 nm. Over this range, fetal hemoglobin absorption is nearly identical to that of adult hemoglobin. Since currently available two-wavelength pulse oximeters base their calculations of arterial oxyhemoglobin saturation on absorption at the wavelengths of 660 and 920 nm, we conclude that the accuracy of two-wavelength pulse oximetry previously demonstrated in adults can be extrapolated to infants with high concentrations of fetal hemoglobin.

A physiological delay in human fetal hemoglobin switching is associated with specific globin DNA hypomethylation

The human fetal-to-adult globin switch normally occurs on a fixed schedule, beginning at 32–34 weeks gestation, and recent studies have suggested an association between this developmental inactivation of the fetal (γ) globin genes and the appearance of methylation within and around these genes. We have studied a population of infants in whom this switch does not occur before birth (infants of diabetic mothers, IDM) and examined the patterns of methylation surrounding their active γ-globin genes, in comparison to the γ-globin genes of age-matched controls who have switched their pattern of globin gene expression on schedule. All genomic DNA samples from infants with delays in the globin switch demonstrated extensive hypomethylation in the region of the γ-globin genes, comparable to that found in the genomes of fetuses of less than 21 weeks gestation. DNA from the erythroid cells of infants of 32–40 weeks gestation had no detectable hypomethylation in the γ-globin region. These findings support the concept that hypomethylation is an accurate developmental marker of globin gene switching, and suggest that globin gene expression in IDM may be arrested at an early preswitch stage.

The Mγ chain of human fetal hemoglobin; its identification and occurrence

High-performance liquid chromatographic procedures have been used in the detection and identification of a new γ chain of human fetal hemoglobin (Hb). This Mγ chain is characterized by a Leu → Met replacement at position γ141; no other structural variations have been observed. The Mγ chain has been detected in red cell lysates of subjects with a heterozygosity for one of many types of so-called hereditary persistence of fetal hemoglobin conditions, which are characterized by an increased level of HB F in adult life, in sickle cell anemia, and in a few cord blood samples. At present it is not possible to definitely identify the genetic cause of this newly discovered heterogeneity; an infidelity in translation or the existence of an unrecognized γ globin gene should be considered.