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.

Allosteric properties of haemoglobin beta 41 (C7) Phe-->Tyr: a stable, low-oxygen-affinity variant synthesized in Escherichia coli.

In human deoxy haemoglobin, the alpha 42(C7)Tyr-residue is hydrogen-bonded to beta 99(G1)Asp which stabilizes the low-oxygen-affinity deoxy conformation. We engineered a haemoglobin with Tyr for Phe at the homologous C7 position in beta-chains. The oxygen affinity of the variant is decreased about two-fold relative to Hb A while keeping similar KR and KT values. This mutant may be a candidate for the development of an artificial oxygen carrier, as it would not require an external effector for significant oxygen unloading in vivo.

Functional role of the distal valine (E11) residue of alpha subunits in human haemoglobin.

We have expressed human alpha-globin to a high level in Escherichia coli as a fusion protein, purified it and removed the N-terminal leader sequence by site-specific proteolysis with blood coagulation factor Xa. The apo globin has been refolded and reconstituted with haem and native beta-globin to form fully functional haemoglobin (Hb) with properties identical to those of native human Hb. By site-directed mutagenesis we have altered the distal residues of the alpha subunits and compared the functional properties of these mutant proteins. The rates of various ligands binding to these proteins in the R-state have been reported by Mathews et al. Here, we present the oxygen equilibrium curves of three E11 alpha mutants and the crystal structures of two of these mutants in the deoxy form. Replacing the distal valine residue of alpha-globin with alanine, leucine or isoleucine has no effect on the oxygen affinity of the protein in either quaternary state, in contrast to the equivalent mutations of beta subunits. The crystal structure of the valine E11 alpha----isoleucine mutant shows that the larger E11 residue excludes water from the haem pocket, but causes no significant movement of other amino acid residues. We conclude that the distal valine residue of alpha-globin does not control the oxygen affinity of the protein by sterically hindering ligand binding.

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)

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.

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.

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.

Abnormal hemoglobin synthesis in some leukemic patients.

Hemoglobin chain synthesis during leukemic processes has been studied on patients having fetal hemoglobin. All cases showed the following abnormalities : (1) a relatively increased synthesis of the beta chain ; (2) an important increase of the free dimeric precursors pool, with, most of the time, a predominance of alpha chain. If the first point suggests an alpha-thalassemia feature, the presence of free alpha chains shows evidence for a more complex mechanism not only due to a decrease of messenger RNA. The hypothesis of a clonal disorder could neither be demonstrated nor ruled out. The observed abnormalities could be due to a defect in a alpha chain depending regulation mechanism.

NMR characterization of a diamagnetic model of unliganded alpha chains from human hemoglobin.

This paper reports the reconstitution and spectroscopic characterization of a complex between alpha globin from human adult hemoglobin and protoporphyrin IX-Zn(II). Optical and proton one-dimensional (1-D) NMR spectra indicate that the prosthetic group binds in a 1:1 stoichiometry to the apoglobin in a single conformation. Using 2-D proton NMR techniques we assigned resonances corresponding to the majority of porphyrin substituents and to several side chains of amino acids in contact with the porphyrin. Analysis of nuclear Overhauser enhancement interactions between identified protons indicated that the complex contains only one rotation isomer of the prosthetic group. The diamagnetic Zn(II) ion is coordinated to the proximal histidine (His87) and does not bind O2 or CO as a sixth ligand. The ring current effects on protons from the distal valine (Val62) are considerably higher than in the liganded form providing strong evidence for a more compact ligand binding pocket relative to the carbon monoxy state. Therefore, protoporphyrin-Zn(II)/alpha globin complex is a suitable diamagnetic model for unliganded alpha chains and will be used for structure determination by NMR and modeling methods.

[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.

[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.

[Expression of recombinant human hemoglobin]. / Expression d'hémoglobine humaine recombinante.

The well recognized prevalence of infectious agents in products derived from human whole blood and the increasing number of transfusion-transmitted diseases has made urgent the search for a safe alternative to conventional blood transfusion. Sources of hemoglobin (Hb) different from outdated human bank blood are under active scrutiny in several laboratories. Different approaches have been proposed to produce recombinant human Hb in bacteria (E. coli), yeast (S. cerevisiae) and transgenic mammals. These efforts have lead to the synthesis of recombinant human Hb with functional properties similar to those of native human Hb A. Site directed mutagenesis enables one to modify the structure of the recombinant globin chains with the view of lowering the oxygen affinity and increasing the stability of the tetramers. Progress is still necessary to ensure scaling-up and safe purification procedures, and to prolong shelf life of these solutions.

[Molecular engineering of hemoglobin for transfusion]. / Ingéniérie moléculaire de l'hémoglobine à visée transfusionnelle.

The search for a safe alternative to conventional blood transfusion has been directed towards either the use of synthetic perfluorochemicals or the biochemical manipulation of highly purified stroma-free Hb solutions prepared from outdated bank blood. However when using human blood, one does not eliminate the risks of viral infections. A novel source of Hb appeared with recent biotechnology techniques enabling one to synthesize recombinant Hb from microorganisms (E coli or S. cerevisiae) whose genome has been modified to contain globin genes. Normal human Hb A in solution, i.e. outside the red cells, is not suited for direct usage as a blood substitute because i) its high oxygen affinity, due to the absence of 2,3 DPG in the plasma, precludes sufficient O2 unloading to the tissues; ii) at low concentration, relative to that in the red cells, tetrameric Hb dissociates into dimers which escape the circulatory system by renal filtration or rapidly oxidize to the non functional metHb form. Expression of alpha- and beta-globins in Escherichia coli and in Saccharomyces cerevisiae enables one to introduce appropriate mutation(s) in the globin genes resulting in the expression of a synthetic Hb with low oxygen affinity, resembling that of normal whole blood; functional Hb has also been produced in a soluble form either in E. coli or in yeast. The coexpression of beta globin chains and alpha globin subunits linked by a peptide bond results in the direct synthesis of stabilized and fully functional Hb tetramers. Lastly, dilute haemoglobin solutions are prone to autooxidize and the rate of oxidation appears to be inversely proportional with the oxygen affinity of the heme groups.(ABSTRACT TRUNCATED AT 250 WORDS)