Education status among orphans and non-orphans in communities affected by AIDS in Tanzania and Burkina Faso.

The AIDS pandemic has created an estimated 15 million orphans who may face elevated risk of poor health and social outcomes. This paper compares orphans and non-orphans regarding educational status and delay using data collected in three low-income communities affected by AIDS in Tanzania and Burkina Faso. Orphans were significantly more likely not to attend school than were non-orphans and also to be delayed when in school, though, after controlling for confounders, the risk was borderline and non-significant. Multivariate analysis indicates that variables such as age, religion, family of origin, the relation between the child and the head of household and the dependency ratio of the household better explain differences in education than does orphan status. This study suggests, therefore, that orphans' educational status is relatively equivalent to non-orphans perhaps as a result of family based or community program safety nets.

Additive effects of beta chain mutations in low oxygen affinity hemoglobin betaF41Y,K66T.

In order to decrease significantly the oxygen affinity of human hemoglobin, we have associated the mutation betaF41Y with another point mutation also known to decrease the oxygen affinity of Hb. We have synthesized a recombinant Hb (rHb) with two mutations in the beta chains: rHb betaF41Y,K66T. In the absence of 2, 3-diphosphoglycerate, additive effects of the mutations are evident, since the doubly mutated Hb exhibits a larger decrease in oxygen affinity than for the individual single mutations. In the presence of 2,3-diphosphoglycerate, the second mutation did not significantly increase the P(50) value relative to the single mutations. However, the kinetics of CO binding still indicate combined effects on the allosteric equilibrium, as evidenced by more of the slow bimolecular phase characteristic of binding to the deoxy conformation. Dimer-tetramer equilibrium studies indicate an increase in stability of the mutants relative to rHb A; the double mutant rHb betaF41Y, K66T at pH 7.5 showed a K(4,2) value of 0.26 microM. Despite the lower oxygen affinity, the single mutant betaF41Y and double mutant betaF41Y,K66T show only a moderate increase of 20% in the autoxidation rate. These mutations are thus of interest in developing a Hb-based blood substitute.

Chimeric beta-EF3-alpha hemoglobin (Psi): energetics of subunit interaction and ligand binding.

Among the numerous strategies to design an oxygen carrier, we outline in this work the engineering of a stable homotetrameric hemoglobin, expressed in Escherichia coli. The chimeric globin (Psi) consists of the first 79 residues of human beta globin (corresponding to positions NA1 --> EF3) followed by the final 67 residues of human alpha globin (corresponding to positions EF3 --> HC3). The molecular mass for beta-EF3-alpha (Psi) globin was measured using mass spectrometry to be equal to its theoretical value: 15782 Da. Correct protein folding was assessed by UV/visible and fluorescence spectra. The subunit interaction free energies were estimated by HPLC gel filtration. In the cyanometHb species, the formation of the dimer-tetramer interface is 2 kcal/mol less favorable (Delta G = -7 kcal/mol) than that of Hb A (Delta G = -9 kcal/mol), whereas the dimer-monomer interface is tightly assembled (< -10 kcal/mol) as for the Hb A alpha 1 beta 1 interface. In contrast to Hb A, oxygen binding to Psi Hb is not cooperative. The free energy for binding four oxygen molecules to a Psi homotetramer is slightly increased compared to a Hb A heterotetramer (-28 and -27.5 kcal/4 mol of O2, respectively). The intrinsic O2 affinity of a Psi homodimer is 6-fold higher than that of a homotetramer. The linkage scheme between dimer-tetramer subunit assembly and the noncooperative oxygenation of Psi Hb predicts a stabilization of the tetramer after ligand release. This protein mechanism resembles that of Hb A for which the dimers exhibit a 100-fold higher O2 affinity relative to deoxy tetramers (which are 10(5) times more stable than oxy tetramers). A potent allosteric effector of Hb A, RSR4, binds to Psi Hb tetramers, inducing a decrease of the overall O2 affinity. Since RSR4 interacts specifically with two binding sites of deoxy Hb A, we propose that the chimeric tetramer folding is close to this native structure.

Tetramer-dimer equilibrium of oxyhemoglobin mutants determined from auto-oxidation rates.

One of the main difficulties with blood substitutes based on hemoglobin (Hb) solutions is the auto-oxidation of the hemes, a problem aggravated by the dimerization of Hb tetramers. We have employed a method to study the oxyHb tetramer-dimer equilibrium based on the rate of auto-oxidation as a function of protein concentration. The 16-fold difference in dimer and tetramer auto-oxidation rates (in 20 mM phosphate buffer at pH 7.0, 37 degrees C) was exploited to determine the fraction dimer. The results show a transition of the auto-oxidation rate from low to high protein concentrations, allowing the determination of the tetramer-dimer dissociation coefficient K4,2 = [Dimer] 2/[Tetramer]. A 14-fold increase in K4,2 was observed for addition of 10 mM of the allosteric effector inositol hexaphosphate (IHP). Recombinant hemoglobins (rHb) were genetically engineered to obtain Hb with a lower oxygen affinity than native Hb (Hb A). The rHb alpha2beta2 [(C7) F41Y/(G4) N102Y] shows a fivefold increase in K4,2 at pH 7.0, 37 degrees C. An atmosphere of pure oxygen is necessary in this case to insure fully oxygenated Hb. When this condition is satisfied, this method provides an efficient technique to characterize both the tetramer-dimer equilibrium and the auto-oxidation rates of various oxyHb. For low oxygen affinity Hb equilibrated under air, the presence of deoxy subunits accelerates the auto-oxidation. Although a full analysis is complicated, the auto-oxidation studies for air equilibrated samples are more relevant to the development of a blood substitute based on Hb solutions. The double mutants, rHb alpha2beta2 [(C7) F41Y/(G4) N102A] and rHb alpha2beta2 [(C7) F41Y/(E10) K66T], show a lower oxygen affinity and a higher rate of oxidation than Hb A. Simulations of the auto-oxidation rate versus Hb concentration indicate that very high protein concentrations are required to observe the tetramer auto-oxidation rate. Because the dimers oxidize much more rapidly, even a small fraction dimer will influence the observed oxidation rate.

Influence of the A helix structure on the polymerization of hemoglobin S.

Hb S variants containing Lys-beta132 --> Ala or Asn substitutions were engineered to evaluate the consequences of the A helix destabilization in the polymerization process. Previous studies suggested that the loss of the Glu-beta7-Lys-beta132 salt bridge in the recombinant Hb betaE6V/E7A could be responsible for the destabilization of the A helix. The recombinant Hb (rHb) S/beta132 variants polymerized with an increased delay time as well as decreased maximum absorbance and Hb solubility values similar to that of Hb S. These data indicate that the strength of the donor-acceptor site interaction may be reduced due to an altered conformation of the A helix. The question arises whether this alteration leads to a true inhibition of the polymerization process or to qualitatively different polymers. The oxygen affinity of the beta132 mutated rHbs was similar to that of Hb A and S, whereas the cooperativity and effects of organic phosphates were reduced. This could be attributed to modifications in the central cavity due to loss of the positively charged lysine. Since Lys-beta132 is involved in the stabilization of the alpha1-beta1 interface, the loss of the beta132(H10)-beta128(H6) salt bridge may be responsible for the marked thermal instability of the beta132 mutated rHbs.

Recombinant [Phe(beta)63]hemoglobin shows rapid oxidation of the beta chains and low-affinity, non-cooperative oxygen binding to the alpha subunits.

We have engineered alpha2beta2 [Phe63]hemoglobin by changing the highly conserved distal histidine of the beta chains to a phenylalanine. The mutant tetramer binds four high-affinity ligands, such as CO or NO, to the ferrous form, or CN to the oxidized iron; however, it binds only two low-affinity ligands, oxygen and azide. The absorption spectrum of the ferrous deoxy or ferric forms are not normal, displaying an enhanced absorption of the visible band near 560 nm. Half of the autooxidation process, attributed to the mutated beta subunits, is over 1000-fold faster than for Hb A. The mutant Hb exhibits non-cooperative binding of two oxygens with an affinity about fivefold lower than those of HbA valency hybrids (alpha met beta)2. Functional properties of this mutant Hb resemble those of Hb Saskatoon ([Tyr63]Hb) [Suzuki, T., Hayashi, A., Shimizu, A. & Yamamura, Y. (1966) Biochim. Biophys. Acta 127, 280-282]. Flash-photolysis experiments also indicate non-cooperative behaviour: the CO-recombination kinetics were independent of the fraction dissociated. Furthermore, the amplitude of the CO bimolecular phase was the same for the (alpha(CO)metbeta)2 valency hybrid or the (alphaCO betaCO)2 form, suggesting mainly geminate CO-recombination kinetics to the beta chains. EPR and Resonance Raman spectra did not show evidence for a hemichrome, normally considered as a six-coordinated iron with low-spin character. The EPR and resonance Raman spectra for the mutated beta subunits demonstrate the presence of a high-spin compound in the ferric and deoxy ferrous forms. In particular, the ferrous mutated beta subunits are penta-coordinated. The abnormal absorption spectra are possibly due to an interaction between the porphyrin and the phenyl ring in the distal position rather than to direct binding to the iron.

Functional studies and polymerization of recombinant hemoglobin Glu-alpha2beta26(A3) --> Val/Glu-7(A4) --> Ala.

In hemoglobin (Hb) S the hydrophobic mutated residue Val-beta6(A3) (donor site) closely interacts with the hydrophobic side groups of Phe-beta85(F1) and Leu-beta88(F4) (EF pocket, acceptor site) of a neighboring tetramer, resulting in decreased solubility and polymerization of the deoxy-Hb. The beta6(A3) residue is followed by two charged residues Glu-beta7(A4) and Lys-beta8(A5). This cluster has no attraction for the hydrophobic EF pocket. We have modified the beta7(A4) residue next to the donor site Val-beta6(A3), replacing the charged Glu by a hydrophobic Ala-(rHb betaE6V/E7A). The single mutant Glu-beta7 --> Ala-(rHb betaE7A) was also engineered. Both rHbs exhibit a heat instability and an increased oxygen affinity compared to Hb A and Hb S. There was a concentration dependence of the ligand binding properties (1-300 microM in heme) indicating an increased amount of dimers relative to Hb A. The deoxy form of rHb betaE6V/E7A polymerizes in vitro, with a decreased rate of polymer formation relative to Hb S, while the single mutant betaE7A does not polymerize in the same experimental conditions. The Glu-beta7(A4) --> Ala substitution does not increase the hydrophobic interaction between donor and acceptor site. We speculate that the loss of the normal saline bridge between Glu-beta7(A4) and Lys-beta132(H10) leads to an increased flexibility of the A helix and may account for the difference of the polymerization for this Hb S mutant.

The role of proline beta 5(A2) in the functional properties of human adult hemoglobin.

The replacement of beta 5(A2)Pro by Arg in Hb Warwickshire appears to be without an effect on the functional properties of human Hb A, despite adding two external positive charges close to the central cavity of the hemoglobin tetramer, along the dyad axis. To clarify the role of this portion of the molecule involved in oxygen-linked anion binding, we have engineered the recombinant hemoglobin alpha 2 beta (2)5(A2)Pro-->Ala[rHb beta 5(A2)Pro-->Ala]. The rHb beta 5(A2)Pro-->Ala exhibits an increased oxygen affinity compared to Hb A, with normal heterotropic effects in standard conditions. The increased oxygen affinity may be attributed to the absence of proline, which would render the A helix more flexible, thus destabilizing the T structure. The normal functional properties of Hb Warwickshire may be due to the regulation of oxygen affinity by electrostatic effects involving diffusible anions not bound to any specific site.

Two mutations in recombinant Hb beta F41(C7)Y, K82 (EF6)D show additive effects in decreasing oxygen affinity.

Based on the properties of two low oxygen affinity mutated hemoglobins (Hb), we have engineered a double mutant Hb (rHb beta YD) in which the beta F41Y substitution is associated with K82D. Functional studies have shown that the Hb alpha 2 beta 2(C7)F41Y exhibits a decreased oxygen affinity relative to Hb A, without a significantly increased autooxidation rate. The oxygen affinity of the natural mutant beta K82D (Hb Providence-Asp) is decreased due to the replacement of two positive charges by two negative ones at the main DPG-binding site. The functional properties of both single mutants are interesting in the view of obtaining an Hb-based blood substitute, which requires: (1) cooperative oxygen binding with an overall affinity near 30 mm Hg at half saturation, at 37 degrees C, and in the absence of 2,3 diphosphoglycerate (DPG), and (2) a slow rate of autooxidation in order to limit metHb formation. It was expected that the two mutations were at a sufficient distance (20 A) that their respective effects could combine to form low oxygen affinity tetramers. The double mutant does display additive effects resulting in a fourfold decrease in oxygen affinity; it can insure, in the absence of DPG, an oxygen delivery to the tissues similar to that of a red cell suspension in vivo at 37 degrees C. Nevertheless, the rate of autooxidation, 3.5-fold larger than that of Hb A, remains a problem.

Loss of allosteric behaviour in recombinant hemoglobin alpha 2 beta 2(92)(F8) His-->Ala: restoration upon addition of strong effectors.

In the stereochemical model proposed by Perutz [1], the Fe-His(F8) bond plays a significant role in the allosteric transition in hemoglobin and the resulting cooperativity in ligand binding. When this bond is ruptured, there is a loss in the transmission of the information concerning ligand binding; examples are Hb(NO)4 in the presence of inositol hexakisphosphate (IHP), or nickel substituted Hb hybrids which, despite being liganded, exhibit deoxy-like properties. To study the effects of the loss of the iron proximal histidine bond, we have engineered the alpha 2 beta 2(F8)H92A recombinant Hb. The replacement of the highly conserved proximal histidine F8 residue by an alanine results in a low affinity for the heme group and a loss of the allosteric properties; kinetics of CO recombination after photodissociation show only the rapid bimolecular phase, characteristic of the high affinity R-state. However, a significant amount of deoxy (T-state) kinetics are observed after addition of external effectors such as IHP. The iron-histidine bond is apparently crucial for the heme-heme interaction, but the allosteric equilibrium may still be influenced by external constraints.

[Expression of recombinant human hemoglobin in plants]. / Expression d'hémoglobine humaine recombinante dans les plantes.

Human utilization of recombinant proteins of therapeutical interest, as hemoglobin, implies that the transgenic host allows a low cost production of the active proteins with minimal risks of pathogen contamination. In this regard, the use of transgenic plants could be of great interest. In particular, the systems based on plants could be one of the most economical transgenic system, compared with the others, because biomass obtention in fields is not expensive.

[Recombinant human hemoglobin with low oxygen affinity: additional effects of two mutations]. / Hémoglobine humaine recombinante de faible affinité pour l'oxygène: effets additionnels de deux mutations.

The search for human Hb variants exhibiting a low oxygen affinity without requiring 2,3-diphosphoglycerate, together with a low oxygation rate, is of an increased interest in the view of producing an artificial oxygen carrier. We have synthesized the recombinant Hb beta 41Phe-->Tyr (rHb beta F41Y) which exhibits a low oxygen affinity due to the stabilization of the deoxy state of tetrameric Hb [1]. Interestingly, the autooxydation rate for this mutant is similar to that for Hb A. We have associated the mutation beta F41Y with the naturally occurring beta 82Lys-->Asp substitution (Hb Providence) known to be responsible for a low oxygen affinity [2]. The second-site mutation further decreases the oxygen affinity of the rHb beta F41Y. The effects of the beta F41Y and K82D mutations are additive, resulting in a four fold decrease in oxygen affinity of the artificial mutant Hb beta F41Y-K82D, compared to Hb A. In spite of the marked decrease in oxygen affinity, the autooxydation rate is 2- to 3 fold larger than that of Hb A. These data show that it is possible to adjust the oxygen binding properties of human Hb by using protein engineering methods. Because of the low oxygen affinity coexisting with a moderately increased autooxydation rate, this variant is a good candidate for the development of a Hb-based oxygen carrier.

[Transfer of functional properties from bovine hemoglobin to human hemoglobin]. / Transfert des propriétés fonctionnelles de l'hémoglobine bovine à l'hémoglobine humaine.

Bovine hemoglobin (Hb) has been proposed as a potential blood substitute because of its low intrinsic oxygen affinity in the absence of chloride anions and 2,3-diphosphoglycerate. The use of bovine blood as a source of Hb does not eliminate the risks of viral infections. Biotechnology techniques allow to produce modified recombinant Hbs. We have engineered human Hb mutants with the aim of mimicking the functional properties of bovine Hb. The argument for this work resides in the crystallographic studies and in the comparison of human and bovine beta globin sequences. The mutant recombinant Hbs exhibit the heterotropic effects of bovine Hb do not exhibit the low intrinsic oxygen affinity of bovine Hb.

Chimeric hemoglobin subunits: functional properties of a recombinant beta/alpha hemoglobin.

Our goal was to design a single hemoglobin subunit able to assemble into a stable tetrameric structure with cooperative O2 binding and low oxygen affinity. We have synthesized in E. coli a chimeric beta/alpha globin subunit composed of the first 73 residues of the beta chain and the last 73 residues of the alpha chain. Molecular building indicated that this construction could result in Hb homotetramers possessing the alpha 1 beta 2 interface, responsible for the heme-heme interaction in Hb heterotetramers. The results show that the chimeric subunits assemble into tetramers which bind oxygen reversibly without cooperativity but with an oxygen affinity slightly lower than observed for human Hb. The strong effector RSR 4 lowers the oxygen affinity. Kinetics of CO recombination in the presence of RSR 4 reveal a biphasic bimolecular rebinding. Functional studies suggest that the quaternary structure of the oligomer is intermediary between R-and T-state.

Functional properties of beta(NA1)Val-deleted,(NA2)His-->Met hemoglobin synthesized in Escherichia coli.

Bovine Hb (hemoglobin) has a low oxygen affinity in the absence of chloride ions and DPG. Because of the increasing interest of this Hb as a potential blood substitute we have engineered a human Hb mutant with the aim of mimicking the functional properties of bovine Hb. This was achieved by deleting residue beta NA1 Val and substituting a methionine for histidine at the beta NA2 position as previously suggested by Perutz and Imai in 1980. Our results show that the artificial mutant exhibits some of the characteristics of bovine Hb but does not show the low oxygen affinity which is measured in bovine blood.

A novel allosteric mechanism in haemoglobin. Structure of bovine deoxyhaemoglobin, absence of specific chloride-binding sites and origin of the chloride-linked Bohr effect in bovine and human haemoglobin.

The structure of bovine deoxyhaemoglobin has been determined at 2.2 A resolution and refined to an R-factor of 0.193 for all 32,583 reflections, and a free R-factor of 0.249 for 1527 reflections excluded from the refinement. The structure shows no significant differences between the alpha-carbon positions of bovine and human haemoglobin, except at the N-terminal segment and the first helix (A) which are closer to the dyad symmetry axis and pushed more tightly against the rest of the beta-subunits in the bovine form. In a search for the predicted chloride-binding sites, three-dimensional data were collected from crystals suspended in 50% polyethylene glycol buffered either with 50 mM Na phosphate (pH 7.3) +/- 0.1 M NaCl or with 0.1 M Hepes (pH 7.3) +/- 0.1 M NaBr. Difference electron density maps with and without NaCl or NaBr showed no evidence of specific halide ion-binding sites. Oxygen equilibria were measured in 10 mM Hepes buffer without added NaCl, with 0.1 mM NaCl, 0.1 M NaCl + 1 mM 2,3-diphosphoglycerate, and 0.1 M NaCl + 1 mM inositol hexaphosphate. Without added chloride, P50 of stripped bovine haemoglobin was similar to that of human haemoglobin with 0.1 M NaCl. With 0.1 M NaCl it was similar to that of human haemoglobin saturated with 2,3-diphosphoglycerate. In 0.1 M NaCl neither organic phosphate significantly affected the oxygen affinity. Titration of P50 with NaCl showed delta log P50/delta log[Cl-] of bovine and human haemoglobin to be identical. Analysis of the oxygen equilibrium curves showed the low intrinsic oxygen affinity of bovine haemoglobin to be due to a larger oxygen dissociation constant from the T-structure. The influence of chloride on P50 and on the alkaline Bohr effect is the same in bovine and human haemoglobins. It is proposed that this is due to the excess positive charges in the central cavity and its widening in the transition from the R to the T-structure. The widening would allow more chloride ions to enter and neutralize the positive charges, but these ions would remain mobile and therefore do not show up as peaks of high electron density. Repulsion between excess positive charges in the central cavity raises the free energy of the T-structure relative to the R-structure, thereby raising the oxygen affinity. Conversely, entry of chloride ions on widening of the cavity reduces the free energy of the T-structure and therefore lowers the oxygen affinity.(ABSTRACT TRUNCATED AT 400 WORDS)

Functional consequences of mutations at the allosteric interface in hetero- and homo-hemoglobin tetramers.

A seminal difference exists between the two types of chains that constitute the tetrameric hemoglobin in vertebrates. While alpha chains associate weakly into dimers, beta chains self-associate into tightly assembled tetramers. While heterotetramers bind ligands cooperatively with moderate affinity, homotetramers bind ligands with high affinity and without cooperativity. These characteristics lead to the conclusion that the beta 4 tetramer is frozen in a quaternary R-state resembling that of liganded HbA. X-ray diffraction studies of the liganded beta 4 tetramers and molecular modeling calculations revealed several differences relative to the native heterotetramer at the "allosteric" interface (alpha 1 beta 2 in HbA) and possibly at the origin of a large instability of the hypothetical deoxy T-state of the beta 4 tetramer. We have studied natural and artificial Hb mutants at different sites in the beta chains responsible for the T-state conformation in deoxy HbA with the view of restoring a low ligand affinity with heme-heme interaction in homotetramers. Functional studies have been performed for oxygen equilibrium binding and kinetics after flash photolysis of CO for both hetero- and homotetramers. Our conclusion is that the "allosteric" interface is so precisely tailored for maintaining the assembly between alpha beta dimers that any change in the side chains of beta 40 (C6), beta 99 (G1), and beta 101 (G3) involved in the interface results in increased R-state behavior. In the homotetramer, the mutations at these sites lead to the destabilization of the beta 4 hemoglobin and the formation of lower affinity noncooperative monomers.

[Polymerization and solubility of recombinant hemoglobins alpha 2 beta 2 6 Glu-->Ala (Hb Makassar) and alpha 2 beta 2 6 Glu-->Ala, 23 Val-->Ile]. / Polymérisation et solubilité des hémoglobines recombinantes alpha 2 beta 2 6 Glu-->Ala (Hb makassar) et alpha 2 beta 2 6 Glu-->Ala, 23 Val-->Ile.

The Hb S (beta 6 Glu-->Val) fiber is formed by the packing of double strands which constitute the basic unit of the deoxyHb S polymer. The specific interaction responsible for the stabilization of the double strand involves the mutated beta 6 Val side chain (lateral contact). Recombinant Hb beta 6 Glu-->Ala and the double mutant beta 6 Glu-->Ala, 23 Val-->Ile exhibit a decreased solubility compared to Hb A. While the Hb beta 6 Ala does not polymerize, the association of the beta 23 Val-->Ile mutation at the axial contact allows the double mutant to polymerize. These results show that: (1) the hydrophobic interactions between donor and acceptor sites depend on both the hydrophobicity and the stereospecificity of the amino acid side chain at the beta 6 position; (2) increasing the hydrophobic interactions at the axial contact (connecting molecules along the same strand) result in an increased stability of the lateral contact between filaments.