Human Fetal Hemoglobin Research Articles

Neither DNA Hypomethylation or Changes in the Kinetics of Erythroid Differentiation Account for 5-Azacytidine's Ability To Induce Human Fetal Hemoglobin.

5-Azacytidine (5-Aza) is among the most potent inducers of fetal hemoglobin (HbF) in patients with β-thalassemia and sickle cell disease. Two models have been proposed to explain this activity. The first model is based on the drug's ability to inhibit DNA methyltransferase enzymes causing global DNA hypomethylation, including the promoters of the fetal globin genes, resulting in their expression during adult erythropoiesis. The second model is based on the drug's other well-know property - cytotoxicity. In this model, a cohort of differentiating erythroblasts either dies or undergoes growth arrest during drug exposure. The marrow then responds with a rapid proliferation of erythroblasts which express high levels of fetal globin mRNA and HbF. To determine which model best explains HbF induction by 5-Aza, we used an in vitro differentiation system in which CD34+ cells from normal donors are cultured with SCF, IL-3 and Flt-3 ligand for 7 days and then switched to Epo for an additional 14 days. This results in >90% erythroid cells, a 2,000-fold expansion in cell number and Hb expression similar to that seen in normal red cells (∼99% HbA, ∼1% HbF). To model common 5-Aza dosing schedules, we treated cultures daily with doses ranging from 0 to 1000 nM. Near-maximal induction of γ-globin mRNA (∼2-fold increase in γ/γ+β) and HbF (from 1% to 40%) occurred at the 300 nM dose level. While this dose was associated with hypomethylation of the 7 CpGs between −256 and +50 bp of the γ promoter (59% +/− 14% methylated CpGs vs. 92% +/− 8.1% in untreated controls, p < 0.001), no changes were found in cell expansion rate, differentiation kinetics (as judged by glycophorin A expression) or cell cycle distribution. These results argue against the cytotoxicity model. We also observed that global DNA methylation was not significantly changed by the 300 nM dose of 5-Aza. Because 5-Aza is incorporated into both RNA and DNA, it affects many cellular processes beyond DNA methylation. To determine if DNA hypomethylation is sufficient for γ-globin mRNA and HbF induction, we treated differentiating cells with DNMT1 siRNA. This resulted in transiently decreased DNMT1 mRNA and protein and lowered global DNA methylation. γ-globin promoter methylation decreased to levels equivalent to those seen with 300 nM 5-Aza (61% +/− 7% of CpGs methylated vs. 88% +/− 8% in controls, p < 0.001) but did not induce γ-globin mRNA or HbF. To further evaluate this unexpected finding we used a lentiviral vector expressing DNMT1 shRNA. This suppressed DNMT1 mRNA and protein throughout differentiation and decreased total γ-globin promoter methylation from 98% +/− 4% in control cells, to 20% +/− 11% (p<0.001) in treated cells. This level of γ promoter methylation is similar to that seen in fetal erythroid cells (13% +/− 7%). Global CpG methylation was also decreased vs. controls (30% +/− 10% vs. 83% +/− 11%, p<0.001). Again, despite significant γ promoter and global hypomethylation, no induction of γ-globin mRNA or HbF was observed. These results suggest that neither model explains 5-Aza's ability to induce fetal Hb and that alternative mechanisms await discovery. If verified, these findings have important implications for the future development of clinically useful HbF inducing agents.

Human embryonic, fetal, and adult hemoglobins have different subunit interface strengths. Correlation with lifespan in the red cell

The different types of naturally occurring, normal human hemoglobins vary in their tetramer-dimer subunit interface strengths (stabilities) by three orders of magnitude in the liganded (CO or oxy) state. The presence of embryonic zeta-subunits leads to an average 20-fold weakening of tetramer-dimer interfaces compared to corresponding hemoglobins containing adult alpha-subunits. The dimer-monomer interfaces of these hemoglobins differ by at least 500-fold in their strengths; such interfaces are weak if they contain zeta-subunits and exchange with added beta-subunits in the form of beta(4) (HbH) significantly faster than do those with alpha-subunits. Subunit exchange occurs at the level of the dimer, although tetramer formation reciprocally influences the amount of dimer available for exchange. Competition between subunit types occurs so that pairs of weak embryonic hemoglobins can exchange subunits to form the stronger fetal and adult hemoglobins. The dimer strengths increase in the order Hb Portland-2 (zeta(2)beta(2)) < Hb Portland-1 (zeta(2)gamma(2)) approximately equal Hb Gower-1 (zeta(2)epsilon(2)) < Hb Gower-2 (alpha(2)epsilon(2)) < HbF(1) < HbF (alpha(2)gamma(2)) < HbA(2) (alpha(2)delta(2)), i.e., from embryonic to fetal to adult types, representing maturation from weaker to stronger monomer-monomer subunit contacts. This increasing order recapitulates the developmental order in which globins are expressed (embryonic --> fetal --> adult), suggesting that the intrinsic binding properties of the subunits themselves regarding the strengths of interfaces they form with competing subunits play an important role in the dynamics of protein assemblies and networks.

Coordinated changes of histone modifications, transcription factors and p38/MAPK signaling pathway contribute to apicidin inducement of human fetal hemoglobin expression in K562 cells

Coordinated changes of histone modifications, transcription factors and p38/MAPK signaling pathway contribute to apicidin inducement of human fetal hemoglobin expression in K562 cells

Histone deacetylase inhibitor FK228 is a potent inducer of human fetal hemoglobin

We investigated the induction of the human fetal globin gene using five potent histone deacetylase (HDAC) inhibitors: FK-228, HC-Toxin, Trichostatin, MS-275, and Apicidin, using in vitro assays and cultures of primary human erythroblasts. The results showed that FK228 is the most potent inducer of fetal hemoglobin and exhibits its effects in picomolar concentrations. FK228 should be considered as a potential therapeutic for induction of fetal hemoglobin in patients with beta chain hemoglobinopathies.

Regulation of human fetal hemoglobin: new players, new complexities

AbstractThe human globin genes are among the most extensively characterized in the human genome, yet the details of the molecular events regulating normal human hemoglobin switching and the potential reactivation of fetal hemoglobin in adult hematopoietic cells remain elusive. Recent discoveries demonstrate physical interactions between the β locus control region and the downstream structural γ- and β-globin genes, and with transcription factors and chromatin remodeling complexes. These interactions all play roles in globin gene expression and globin switching at the human β-globin locus. If the molecular events in hemoglobin switching were better understood and fetal hemoglobin could be more fully reactivated in adult cells, the insights obtained might lead to new approaches to the therapy of sickle cell disease and β thalassemia by identifying specific new targets for molecular therapies.

The Course of Plasmodium.yoelii Infection in Hereditary Persistence of Fetal Hemoglobin.

Malaria, caused by plasmodium species has great impact on human generations. Each year 300–500 million of people are diagnosed and 1 million die of the disease. Malaria probably become endemic about 10,000 years ago and it has affected human genome by strong selective pressure. Several human genetic diseases are thought to be the outcome of this pressure. Our current research is focused on understanding natural host defense mechanism of this disease, especially effect to human fetal hemoglobin expression to plasmodium species.Other investigator indicated growth of Plasmodium falciparum is retarded in the cell containing human fetal hemoglobin in vivo (Nature, 270:171), and transgenic mouse (transgenic of human fetal hemoglobin and knockout of mouse beta-major gene) infected with Plasmodium has longer survival compare to control.(Blood , 92:2520). Here we use transgenic mouse express human fetal hemoglobin (human Gamma globin) which simulate human hereditary persistence of fetal hemoglobin (PNAS 86:7033) to investigate fetal hemoglobin itself has a protective effect on Plasmodium infection in vivo.We injected 1,000 Plasmodium.yoelii infected RBCs to transgenic mouse expressing human fetal hemoglobin (n=5) and C57BL6J mouse (n=8). Blood smears obtained daily and smears were stained with Giemsa. Parasitemia is calculated from about 1000 RBCs. 5 days after injection, infected RBCs was 26.4 ±4.7% for C57BL6J and 32.6± 17.3% for human fetal hemoglobin transgenic mouse. We couldn't show significant difference between these groups. And 60% of C57BL6J mice (3 of 5) were recovered from illness. In contrast, 20% of transgenic mouse (1of 5) was recovered from illness.These results demonstrates only human fetal hemoglobin expression is not enough to contribute defense mechanism against plasmodium species.

Generation of a Novel Mouse Model of Cooley's Anemia.

A novel mouse model of Cooley's Anemia (CA) has been generated by targeted gene replacement of the adult murine α-globin genes with human α-globin and the adult mouse β-globin genes with a human γ- to β-globin gene switching cassette containing a β0 thalassemic allele. A positive-negative gene replacement construct was designed to simultaneously delete both of the adult mouse α-globin genes by inserting a 3.8kb human α1-globin gene and a hygromycin marker gene flanked by loxP sites in murine embryonic stem (ES) cells. Both adult murine β-globin genes were deleted by insertion of an Hprt marker gene that was later replaced by a 5.6kb human Aγ-globin gene, 4.1kb human β0-globin gene, and a loxP flanked hygromycin marker gene by a “tag and exchange” strategy. The human β0-globin knock-in allele contains a single G to A nucleotide mutation in the first base of intervening sequence 1 [β0-IVS1(GtoA)-globin]. This single base change destroys the splice donor site of IVS-1 resulting in the recruitment of several cryptic splice sites. Use of these cryptic splice sites produces a frameshift in the mRNA that results in no functional β-globin polypeptide synthesis from this allele. This β0-IVS1(GtoA)-globin gene mutation is a naturally occurring β0 thalassemia allele found in Mediterranean populations. Chimeric mice were generated from both the α- and β-globin targeted cells lines. After germline transmission the α- and β-globin targeted mice were bred to cre recombinase transgenic mice to remove the marker genes. Heterozygous CA mice exhibit β thalassemia intermedia. The α- and β-globin targeted mice were interbred to produce animals homozygous for the human α1- and γβ0-globin knock-in alleles. Instead of dying early in fetal life as all current homozygous β0 thalassemia mouse models, these novel homozygous CA mice survive solely on high levels of human fetal hemoglobin (α2γ2) throughout fetal development. Newborn homozygous CA mice are blood transfusion dependent similar to β thalassemia major infants. This novel model of CA has multiple improvements over existing models of β thalassemia. Namely, CA mice express 100% human hemoglobin in their RBCs, mimic the human γ- to β-globin gene switch, synthesize no functional β-globin chains after birth, have a single mutant human β0-globin knock-in allele at each β-globin locus, and are blood transfusion dependent for life after birth.

Enhanced Inhibition of Polymerization of Sickle Cell Hemoglobin in the Presence of Recombinant Mutants of Human Fetal Hemoglobin with Substitutions at Position 43 in the γ-Chain

Four recombinant mutants of human fetal hemoglobin [Hb F (alpha2gamma2)] with amino acid substitutions at the position 43 of the gamma-chain, rHb (gammaD43L), rHb (gammaD43E), rHb (gammaD43W), and rHb (gammaD43R), have been expressed in our Escherichia coli expression system and used to investigate their inhibitory effect on the polymerization of deoxygenated sickle cell hemoglobin (Hb S). Oxygen-binding studies show that rHb (gammaD43E), rHb (gammaD43W), and rHb (gammaD43R) exhibit higher oxygen affinity than human normal adult hemoglobin (Hb A), Hb F, or rHb (gammaD43L), and all four rHbs are cooperative in binding O2. Proton nuclear magnetic resonance (NMR) studies of these four rHbs indicate that the quaternary and tertiary structures around the heme pockets are similar to those of Hb F in both deoxy (T) and liganded (R) states. Solution light-scattering experiments indicate that these mutants remain mostly tetrameric in the liganded (R) state. In equimolar mixtures of Hb S and each of the four rHb mutants (gammaD43L, gammaD43E, gammaD43R, and gammaD43W), the solubility (Csat) of each of the pairs of Hbs is higher than that of a similar mixture of Hb S and Hb A, as measured by dextran-Csat experiments. Furthermore, the Csat values for Hb S/rHb (gammaD43L), Hb S/rHb (gammaD43E), and Hb S/rHb (gammaD43R) mixtures are substantially higher than that for Hb S/Hb F. The results suggest that these three mutants of Hb F are more effective than Hb F in inhibiting the polymerization of deoxy-Hb S in equimolar mixtures.

Analysis of interaction of cloned human and/or sheep fetal hemoglobin γ-chain and LPS in augmenting induction of inflammatory cytokine production in vivo and in vitro

We have reported earlier that purified preparations of sheep fetal hemoglobin, but not adult hemoglobin, in concert with non-stimulatory doses of lipopolysaccharide (LPS) (lipid A), act cooperatively to regulate in vitro production of a number of cytokines, including TNFα, TGFβ and IL-6 from murine and human leukocytes. Following in vivo treatment of mice with the same combination of hemoglobin and LPS, harvested spleen or peritoneal cells showed a similar augmented capacity to release these cytokines into culture supernatants. We report below that genetically cloned γ-chain of human or sheep fetal hemoglobin, but not cloned α- or β-chains, can produce this cooperative effect, as indeed can HPLC purified, heme-free, γ-chains derived from cord blood fetal hemoglobin, and that purified haptoglobin completely abolishes the cooperative interaction.

Dissecting a Multi-Step Mechanism of Beta-Globin Transcriptional Activation.

Defining factors and signals that establish and maintain the native nucleoprotein structure of endogenous chromatin domains represents a powerful approach for elucidating transcriptional mechanisms. In adult erythroid cells, the locus control region (LCR) and the adult beta-globin genes of the murine beta-globin locus are highly enriched in acetylated histones H3 and H4 (acH3, acH4) and H3 methylated at lysine 4 (H3-meK4). By contrast, the embryonic beta-globin genes reside in a broad region of reduced acetylation. Histone H3 methylated at lysine 79 (H3-meK79) is highly enriched at the adult beta-globin genes, but not at the LCR. To elucidate the molecular steps in beta-globin transcriptional activation, genetic complementation experiments were conducted in GATA-1-null G1E cells containing an estrogen receptor hormone binding domain-GATA-1 fusion protein (ER-GATA-1). Kinetic analysis of ER-GATA-1 occupancy of chromatin and establishment of the histone modification pattern by chromatin immunoprecipitation (ChIP) revealed that GATA-1 occupies multiple regions within the LCR prior to the beta-major promoter. Chromatin accessibility at the promoter was low until ER-GATA-1 assembled into regulatory complexes at the LCR. Subsequently, ER-GATA-1 accessed the beta-major promoter, induced acH3, RNA polymerase II (Pol II) recruitment, and elevated H3-meK79. Acquisition of transcriptional competence appears to require establishment of H3-meK4, which is GATA-1-independent. Blocking transcriptional elongation did not erase H3-meK79, indicating that maintenance of H3-meK79 does not require ongoing elongation. Analysis of N-terminal GATA-1 deletion mutants that retain Friend of GATA-1 (FOG-1) binding and DNA binding activities revealed that FOG-1 binding and DNA binding activities are insufficient for Pol II recruitment and chromatin modification at the promoter. These results support a model in which ER-GATA-1 binding to the LCR increases acH3 at the promoter as an early event in transcriptional activation, which is tightly coupled to ER-GATA-1 access to the promoter, increased promoter accessibility, and Pol II recruitment. Increased promoter accessibility, which likely permits ER-GATA-1 access to the promoter, precedes maximal induction of H3-meK79, a late event in activation. Given the dynamic regulation of H3-meK79 by GATA-1 and NF-E2 and the modulation of H3-meK79 levels during erythropoiesis, we propose that H3-meK79 is a crucial signal that controls the rate of beta-globin transcription. Studies are underway to test this hypothesis and to dissect mechanisms underlying the requirement of N-terminal sequences of GATA-1 for Pol II recruitment and chromatin modification. Furthermore, having identified individual steps in transcriptional activation of the endogenous beta-globin genes, we are testing whether inducers of human fetal hemoglobin affect these specific steps. GATA-1 has been reported by the Crispino group to be expressed as an N-terminally truncated species in megakaryoblastic leukemia. Defining how the N-terminus functions should therefore lead to a molecular understanding of this disorder. As the N-termini of GATA factors differ considerably, one might expect these divergent sequences to establish GATA factor-specific functions, and this prediction is being tested via detailed analysis of the activities of chimeric GATA factors.

The cooperativity of human fetal and adult hemoglobins is optimized: a consideration based on the effectiveness of the Bohr shift.

The physiological significance of the cooperativity of human hemoglobin (Hb) is considered from the viewpoint of the effectiveness of the Bohr shift at the sites of O(2) release and uptake across the placental membrane. The effects of the Bohr shift was examined by changing the O(2) saturation of Hb (S(pO2)) per unit change in P(50), -dS(PO2)/d P(50), where P(50) is partial pressure of O(2) at half saturation. The Bohr shift at the sites of O(2) uptake and release was found to be highly effective in both fetal and maternal bloods at physiological degree of cooperativity (Hill's coefficient, n=2.65). From the results obtained in this paper, it is concluded that the positions of OECs of fetal and maternal Hbs are regulated to receive a maximal benefit from the Bohr shift, and that a relatively low n value of human tetrameric Hb is adequate for the O(2) and CO(2) exchange across the placental membrane.

Probing the importance of the amino-terminal sequence of the beta- and gamma-chains to the properties of normal adult and fetal hemoglobins.

A recombinant mutant of human fetal hemoglobin (Hb F), named rHb Oscar, has been constructed to explore the importance of the sequence of the amino-terminal region of the gamma-chain to the structural and functional properties of Hb F as compared to human normal adult hemoglobin (Hb A). Substitutions in the N-terminal region of Hb A have shown this region to be important to its structural and functional properties. Recent studies of recombinant mutants of Hb A with gamma-chain mutations have been used to probe the significance of the N-terminal sequence to the properties of Hb F. One of these mutants of Hb A, called rHb Felix, contains eight substitutions in the N-terminal region of the beta-chain corresponding to the sequence of the gamma-chain in that region [Dumoulin et al. (1998) J. Biol. Chem. 273, 35032-35038]. rHb Felix exhibits a 2,3-bisphosphoglycerate (2,3-BPG) response like that of Hb A, but its tetramer-dimer dissociation constant is similar to that of Hb F. In contrast, rHb Oscar contains a gamma-chain with eight mutations at the N-terminal end corresponding to the sequence of the beta-chain of Hb A in that region. (1)H NMR studies of rHb Oscar indicate a global structure like that of Hb F. rHb Oscar is not as stable against alkaline denaturation as Hb F but is more stable than Hb A, and it exhibits a stronger response to 2,3-BPG and inositol hexaphosphate as compared to Hb F. The 2,3-BPG effect in rHb Oscar also appears to be slightly enhanced compared to that in Hb A. Subzero isoelectric focusing experiments suggest that rHb Oscar does not have dissociation properties like those of Hb A. These results along with those of rHb Felix illustrate that the effects of the N-terminal region on structure and function of the Hb molecule are complicated by interactions with the rest of the molecule that are not yet well defined. However, studies of complementary mutations of Hb A and Hb F may eventually help to define such interactions and lead to a better understanding of the relationship between the amino acid sequence and the properties of the Hb molecule.

The acetylation state of human fetal hemoglobin modulates the strength of its subunit interactions: long-range effects and implications for histone interactions in the nucleosome.

The source of the 70-fold increased tetramer strength of liganded fetal hemoglobin relative to that of adult hemoglobin between pH 6.0 and 7.5 reported earlier [Dumoulin et al. (1997) J. Biol. Chem. 272, 31326] has been identified as the N-terminal Gly residue of the gamma-chain, which is replaced by Val in adult hemoglobin. This was revealed by extending the study of the pH dependence of the tetramer-dimer equilibrium of these hemoglobins into the alkaline range as far as pH 9. From pH 7.5 to 9.0, the 70-fold difference in the association equilibrium constant between hemoglobins F and A lessened progressively. This behavior was attributed to the difference in the pK(a) 8.1 of Gly-1(gamma) compared to the pK(a) 7.1 value of Val-1(beta) of hemoglobins F and A, respectively. Evidence for this conclusion was obtained by demonstrating that natural hemoglobin F(1), which is specifically acetylated at Gly-1(gamma) and hence unable to be protonated, behaves like HbA and not HbF in its tetramer-dimer association properties over the pH range studied. An increased degree of protonation of the gamma-chain N-terminus of hemoglobin F from pH 9.0 to 8.0 is therefore suggested as responsible for its increased tetramer strength representing an example of transmission of a signal from its positively charged N-terminal tail to the distant subunit allosteric interface where the equilibrium constant is measured. An analogy is made between the effects of acetylation of the fetal hemoglobin tetramer on the strength of its subunit interactions and acetylation of some internal Lys residues within the N-terminal segments of the histone octamer around which DNA is wrapped in the nucleosome.

Assembly of γ- with α-Globin Chains to Form Human Fetal Hemoglobin in Vitro and in Vivo

Soluble gamma-globin chains were expressed in bacteria and purified to assess the mechanism of gamma- and alpha-chain assembly to form Hb F. Formation of Hb F in vitro following incubation of equimolar mixtures of gamma and alpha chains was about 4 x 10(5)-fold slower than assembly of alpha and beta chains to form Hb A in vitro. Results of assembly for gamma(116Ile-->His) and gamma(112Thr-->Asp) chains with alpha chains were similar to that of beta chains, whereas assembly of gamma(112Thr-->Cys) and alpha chains was similar to wild type gamma chains, indicating that amino acid differences at alpha1beta1 and alpha1gamma1 interaction sites between gamma116 Ile and beta116 His are responsible for the different assembly rates in vitro in the formation of Hb F and Hb A. Homoassembly in vitro of individual gamma chains as assessed by size-exclusion chromatography shows that gamma and gamma(112Thr-->Cys) chains form stable dimers like alphabeta and alphagamma that do not dissociate readily into monomers like beta chains. In contrast, gamma(116Ile-->His) chains form monomers and dimers upon dilution. These results are consistent with the slower assembly rate in vitro of gamma and gamma(112Thr-->Cys) with alpha chains, whereas the faster rate of assembly of gamma(116Ile-->His) and gamma(112Thr-->Asp) chains with alpha chains, like beta chains, may be caused by dissociation to monomers. These results suggest that dissociation of gamma(2) dimers to monomers limits formation of Hb F in vitro. However, yields of soluble Hb F expressed in bacteria were similar to Hb A, and no unassembled alpha and gamma chains were detected. These results indicate that gamma chains assemble in vivo with alpha chains prior to forming stable gamma(2) dimers, possibly binding to alpha chains as partially folded nascent gamma-globin chains prior to release from polyribosomes.

A biochemical and biophysical characterization of recombinant mutants of fetal hemoglobin and their interaction with sickle cell hemoglobin.

Three recombinant mutants of human fetal hemoglobin (Hb F) have been constructed to determine what effects specific amino acid residues in the gamma chain have on the biophysical and biochemical properties of the native protein molecule. Target residues in these recombinant fetal hemoglobins were replaced with the corresponding amino acids in the beta chain of human normal adult hemoglobin (Hb A). The recombinant mutants of Hb F included rHb F (gamma 112Thr --> Cys), rHb F (gamma 130Trp --> Tyr), and rHb F (gamma 112Thr --> Cys/gamma 130Trp --> Tyr). Specifically, the importance of gamma 112Thr and gamma 130Trp to the stability of Hb F against alkaline denaturation and in the interaction with sickle cell hemoglobin (Hb S) was investigated. Contrary to expectations, these rHbs were found to be as stable against alkaline denaturation as Hb F, suggesting that the amino acid residues mentioned above are not responsible for the stability of Hb F against the alkaline denaturation as compared to that of Hb A. Sub-zero isoelectric focusing (IEF) was employed to investigate the extent of hybrid formation in equilibrium mixtures of Hb S with these hemoglobins and with several other hemoglobins in the carbon monoxy form. Equimolar mixtures of Hb A and Hb S and of Hb A(2) and Hb S indicate that 48-49% of the Hb exists as the hybrid tetramer, which is in agreement with the expected binomial distribution. Similar mixtures of Hb F and Hb S contain only 44% hybrid tetramer. The results for two of our recombinant mutants of Hb F were identical to the results for mixtures of Hb F and Hb S, while the other mutant, rHb F (gamma 130Trp --> Tyr), produced 42% hybrid tetramer. The sub-zero IEF technique discussed here is more convenient than room-temperature IEF techniques, which require Hb mixtures in the deoxy state. These recombinant mutants of Hb F were further characterized by equilibrium oxygen binding studies, which indicated no significant differences from Hb F. While these mutants of Hb F did not have tetramer-dimer dissociation properties significantly altered from those of Hb F, future mutants of Hb F may yet prove useful to the development of a gene therapy for the treatment of patients with sickle cell anemia.

Metal Complexes as Allosteric Effectors of Human Hemoglobin: An NMR Study of the Interaction of the Gadolinium(III) Bis( m-boroxyphenylamide)diethylenetriaminepentaacetic Acid Complex with Human Oxygenated and Deoxygenated Hemoglobin

The boronic functionalities on the outer surface of the Gd(III) bis( m-boroxyphenylamide)DTPA complex (Gd(III)L) enable it to bind to fructosamine residues of oxygenated glycated human adult hemoglobin. The formation of the macromolecular adduct can be assessed by NMR spectroscopy via observation of the enhancement of the solvent water proton relaxation rate. Unexpectedly, a strong binding interaction was also observed for the oxygenated unglycated human adult hemoglobin, eventually displaying a much higher relaxation enhancement. From relaxation rate measurements it was found that two Gd(III)L complexes interact with one hemoglobin tetramer ( K D = 1.0 × 10 −5 M and 4.6 × 10 −4 M, respectively), whereas no interaction has been observed with monomeric hemoproteins. A markedly higher affinity of the Gd(III)L complex has been observed for oxygenated and aquo-met human adult hemoglobin derivatives with respect to the corresponding deoxy derivative. Upon binding, a net change in the quaternary structure of hemoglobin has been assessed by monitoring the changes in the high-resolution 1H-NMR spectrum of the protein as well as in the Soret absorption band. On the basis of these observations and the 11B NMR results obtained with the diamagnetic La(III)L complex, we suggest that the interaction between the lanthanide complex and deoxygenated, oxygenated, and aquo-met derivatives of human adult hemoglobin takes place at the 2,3-diphosphoglycerate (DPG) binding site, through the formation of N→B coordinative bonds at His 143 β and His 2 β residues of different β-chains. The stronger binding to the oxygenated form is then responsible for a shift of the allosteric equilibrium toward the high-affinity R-state. Accordingly, Gd(III)L affinity for oxygenated human fetal hemoglobin (lacking His 143 β ) is significantly lower than that observed for the unglycated human adult tetramer.

Bibliography of resources by and about André Hellegers.

Bibliography of Resources by and about André E. Hellegers* Compiled by Doris Mueller Goldstein (bio) This bibliography is derived from the holdings of the National Reference Center for Bioethics Literature and the BIOETHICSLINE© database (both of which are at the Kennedy Institute of Ethics and supported by the National Library of Medicine); the archives of Lauinger Library, Georgetown University; the Medline databases of the National Library of Medicine; the WorldCat database of OCLC, Dublin, Ohio, and selected Internet sites. Primarily comprised of writings by Dr. Hellegers, the list also includes interviews and media programs in which he participated, as well as writings about him that were written following his death. The arrangement within each section is by date. The 231 authored pieces reflect the transition from Dr. Hellegers’s early career as a researcher in fetal physiology at Johns Hopkins Medical School, where he published his first research report at the age of 29, to the early years of the Kennedy Institute, where he produced so many provocative columns on bioethical issues that influenced the growth of the field. Due to the diversity of sources and the 40-year time span covered, there are some variations in bibliographic style. Time and availability constraints precluded verifying all of the citations with the original documents. Any additions or corrections should be submitted to the compiler. Works Authored by André E. Hellegers No Date Ethical Issues in Sterilization of the Retarded. Unpublished paper. 1956 Vitamin B12 Serum Level and Pregnancy. American Journal of Clinical Nutrition 4: 440+, 1956. [With Bacon F. Chow and Kunio Okuda] [End Page 89] 1957 Vitamin B12 Absorption in Pregnancy and in the Newborn. American Journal of Clinical Nutrition 5: 327+, 1957. [With Bacon F. Chow, Robert E. L. Nesbitt, Kunio Okuda, and David W. Smith] 1958 The Effects of Acute Hypoxia on the Osmotic Pressure of the Plasma. Quarterly Journal of Experimental Physiology 43: 197+, 1958. [With Donald H. Barron, Frederick C. Battaglia, and Giacomo Meschia] Studies on the Absorption of Vitamin B12 in Human Pregnancy with Especial Reference to the Effect of D-Sorbitol. American Journal of Obstetrics and Gynecology 76: 91+, 1958. [With Bacon F. Chow, Harry Prystowsky, and Vernon Wong] 1959 The Alveolar PCO2 and PO2 in Pregnant and Nonpregnant Women at Altitude. Proceedings of the Society for Clinical Investigation 38: 1010+, 1959. [With Donald Barron, William Huckabee, Giacomo Meschia, James Metcalfe, and Harry Prystowsky] A Comparison of the Oxygen Dissociation Curves of the Bloods of Maternal and Fetal Goats at Various pHs. Quarterly Journal of Experimental Physiology 44: 215–21, July 1959. [With Donald Barron, Giacomo Meschia, Harry Prystowsky, and A. Stark Wolkoff] Fetal Blood Studies. XII. On the Relationship Between the Position of the Oxygen Dissociation Curve of Human Fetal Blood and Adult Fetal Hemoglobin. American Journal of Obstetrics and Gynecology 77: 585+, 1959. [With Paul D. Bruns, Julian Cotter, and Harry Prystowsky] Fetal Blood Studies. XIV. A Comparative Study of the Oxygen Dissociation Curve of Nonpregnant, Pregnant and Fetal Human Blood. American Journal of Obstetrics and Gynecology 78: 489–93, September 1959. [With Paul D. Bruns and Harry Prystowsky] Fetal Blood Studies. XVI. On the Changes in Total Osmotic Pressure and Sodium and Potassium Concentration of Amniotic Fluid During the Course of Human Gestation. Surgery, Gynecology and Obstetrics 109: 509–12, October 1959. [With Frederick Battaglia, Paul D. Bruns, Harry Prystowsky, and Clayton Smisson] Further Observations on the Metabolism of Vitamin B12 in Human Pregnancy. American Journal of Obstetrics and Gynecology 77: 1+, 1959. [With Bacon F. Chow, Harry Prystowsky, Bruno Ranke, and Elma Ranke] The Oxygen Dissociation Curves of the Bloods of Adult and Fetal Llamas. Federation Proceedings 18, 1959. [With Donald Barron, William Huckabee, Giacomo Meschia, James Metcalfe, and Harry Prystowsky] [End Page 90] Transfer of Oxygen Across the Sheep Placenta at High Altitude. Federation Proceedings 18, 1959. [With Donald Barron, William Huckabee, Giacomo Meschia, and Harry Prystowsky] Uterine Blood Flow and Metabolism in Pregnant Sheep at High Altitude. Federation Proceedings 18, 1959. [With Donald Barron, William Huckabee, Giacomo Meschia, James Metcalfe, and Harry Prystowsky] Uterine Blood Flow and Metabolism in Unanesthetized Pregnant Sheep. Federation Proceedings 18, 1959. [With Donald Barron, William Huckabee, Giacomo...

Modulating the oxygen affinity of human fetal haemoglobin with synthetic allosteric modulators.

Improving the delivery of oxygen to the tissues by decreasing the oxygen affinity of haemoglobin has been a major aim of several laboratories over recent years because this may reduce the consequences of anaemia and/or improve tissue oxygenation in cases of decreased blood perfusion. Within the same context, lowering the oxygen affinity may prove valuable in the application of native or recombinant haemoglobin solutions as a blood substitute. The shift of the oxygen equilibrium curve to the right is obtained by various modulators. Among them, the bezafibrate derivatives are considered as a most interesting group. These principles are of the utmost importance in thalassaemia and other haemoglobinopathies where the beneficial effects of the compensatory synthesis of fetal haemoglobin are diminished by the increased oxygen affinity of this pigment. In this paper we present the results of a study initiated to determine whether a potent oxygen affinity modifier, RSR-4, could satisfactorily decrease the oxygen affinity of fetal haemoglobin, thus improving tissue oxygenation. The experiments were carried out on whole blood and on purified haemoglobin solutions and showed that the effector markedly decreased the oxygen affinity of HbF (from 18.7 to 3.73 mmHg in whole blood). At the same time the cooperativity index (n50) and the oxygen saturation levels remained within normal limits under the conditions of the main experiment. These observations have important implications for the potential application of oxygen affinity modifiers in vivo.

Stabilization of the T-state of ferrous human adult and fetal hemoglobin by Ln(III) complexes: A thermodynamic study

The effect of the lanthanide(III) complexes [Gd(1,4,7,10-tetraazacyclododecane- N, N′, N″, N′′′-tetrakis(methylenephosphonate))] 5− (Gd-DOTP) and La-DOTP on the oxygen binding and spectroscopic properties of human adult and fetal hemoglobin (HbA and HbF, respectively) has been investigated. The affinity of Gd-DOTP and La-DOTP for oxygenated HbA (HbAO 2; K HbAO 2 =2.6 × 10 −3 M) is closely similar to that observed for Ln(III) complexes association to nitrosylated HbA (HbANO K HbANO=1.8 × 10 −3 M) and to aquo-met HbA (met-HbA; K met-HbA=1.9 × 10 −3 M), being lower than that determined for Gd-DOTP and La-DOTP binding to the deoxygenated form of the tetramer (HbAd; K HbAd=3.0 × 10 −4 M). The affinity of Gd-DOTP for deoxygenated HbF (HbFd; K HbFd=9.5 × 10 −4 M) and oxygenated HbF (HbFO 2; K HbFO 2 =3.7 × 10 −3 M) is lower than that observed for Ln(III) complexes association to HbAd and HbAO 2, respectively. Gd-DOTP and La-DOTP bind to HbA and HbF with a 1:1 stoichiometry per tetramer. Increasing Gd-DOTP and La-DOTP concentration, oxygen affinity for HbA decreases (i.e. P 50 increases), this effect being minor for HbF. Upon binding of Ln(III) complexes to HbANO, the X-band EPR spectrum and the absorption spectrum in the Soret region display the characteristics which have been attributed to the T-state of the ligated tetramer. These results represent a clear cut evidence for the specific binding of Gd-DOTP and La-DOTP to the 2,3- D-glycerate bisphosphate (BPG) pocket (i.e. at the dyad axis, in between the β-chains) of HbA and HbF. The effect of Ln(III) complexes on the ligand binding and spectroscopic properties of HbA and HbF is reminiscent that of BPG, the physiological modulator of human Hb action.

A 1H NMR comparative study of human adult and fetal hemoglobins

The affinities of the individual subunits in human adult and fetal hemoglobins to azide ion have been determined from the combined analysis of NMR and optical titration data. Structural and functional non-equivalence of the constituent subunits, i.e. α and β subunits in human adult hemoglobin and α and γ subunits in human fetal hemoglobin, has been confirmed. The function of the α subunits, which are common to both hemoglobins, is essentially identical in these hemoglobins and, in spite of the substitutions of 39 amino acid residues between β and γ subunits, they exhibit similar azide ion affinities. The present study also demonstrates that the NMR spectral comparison between the two proteins provides signal assignments to the individual subunits in intact tetramer.