Hemoglobin Tetramer Research Articles

Cation-Induced Stabilization of Protein Complexes in the Gas Phase: Mechanistic Insights From Hemoglobin Dissociation Studies

Collision-induced dissociation (CID) of electrosprayed protein complexes usually involves asymmetric charge partitioning, where a single unfolded chain gets ejected that carries a disproportionately large fraction of charge. Using hemoglobin (Hb) tetramers as model system, we confirm earlier reports that bound metal ions can stabilize protein complexes under CID conditions. We examine the mechanism underlying this effect. Nonvolatile salts cause extensive adduct formation. Significant stabilization was observed for Mg(2+) and Ca(2+), whereas K(+), Rb(+), and Cs(+) had no effect. Precursor ion selection was used to examine Hb subpopulations with well-defined metal binding levels. K(+), Rb(+), and Cs(+)-adducted tetramers eject monomers that carry roughly one-quarter of the metal ions that were bound to the precursor. This demonstrates that charge migration during CID is exclusively due to proton transfer, not metal ion transfer. Also, replacement of highly mobile charge carriers (protons) with less mobile species (metal ions) does not exert a stabilizing influence under the conditions used here. Interestingly, Hb carrying stabilizing ions (Mg(2+) and Ca(2+)) generates monomeric CID products that are metal depleted. This effect is attributed to a combination of two factors: (1) Me(2+) binding stabilizes Hb via formation of chelation bridges (e.g., R-COO(-) Me(2+) (-)OOC-R); the more Me(2+) a subunit contains the more stable it is. (2) More than ~90% of the tetramers contain at least one subunit with a below-average number of Me(2+). The prevalence of monomeric CID products with depleted Me(2+) levels is caused by the tendency of these low metal-containing subunits to undergo preferential unfolding/ejection.

Paper Spray Ionization of Noncovalent Protein Complexes

Paper spray (PS) ionization, an ambient ionization method, has previously been explored as a direct and fast method for mass spectrometric analysis of complex mixtures. It has been applied to the analysis of a wide variety of compounds, mostly small molecules. The work reported here extends the application of PS ionization to noncovalent protein complexes on an ion mobility tandem mass spectrometer. Similar mass spectra for protein complexes were obtained by PS ionization and nanoflow electrospray ionization (nESI), indicating that intact protein complexes can be preserved in PS ionization. In addition, collisional cross sections measured by ion mobility provide evidence that the protein assemblies may remain compact by PS ionization. With PS, it is possible to detect hemoglobin tetramer from a blood sample with minimal sample preparation. This is the first report to show that PS ionization is a promising ionization method for nonconvalent protein complexes.

Structural modifications induced by the switch from an endogenous bis-histidyl to an exogenous cyanomet hexa-coordination in a tetrameric haemoglobin

Two EPR- and structurally-distinct bis-histidyl conformers of the ferric haemoglobin fromTrematomus bernacchiireact differently with CN−

Functional Modulation in a Typical Allosteric Protein Revisited - Beyond “T” and “R”

The classical tenet of allostery in proteins involves the existence of two alternative but inter-convertible molecular conformations, called T and R, each one exhibiting distinct functional characteristics. Customarily, the T conformation is characterized by a low activity, whereas the R conformation shows high activity. In the textbook case of tetrameric hemoglobin (Hb), ligation at the hemes triggers the structural transition from T to R, with the concomitant functional change from low to high affinity for heme ligands. These two conformations correspond to two distinct structures that differ in the way the two αβ dimers assemble to form an α2β2 tetramer.We have investigated the effects of chemical modification on an allegedly inert interface in Hb, i.e., the α1β1 interface, facing the central cavity, by specific alkylation of residues α104Cys and/or β112Cys, and found that substantial functional changes on Hb do indeed take place. In an extreme case, in the absence of strong heterotropic effectors, one of the derivatives showed a ∼200-fold drop in oxygen affinity and almost abolished cooperativity when compared against its native form. Dimerization degree measured by size exclusion chromatography and isothermal titration calorimetry, as well as 1H-NMR measurements revealed that the permanent-T characteristics for the fully ligated form of modified Hb were not due to the packing of the tetramer into a T conformation, but rather suggested that these characteristics originated from the dimer itself, since the dimerization degree for the fully ligated form was comparable, if not larger than that for the fully ligated native form.These results suggest that a control center for ligand affinity resides within the αβ dimer, and that functional modulation is not necessarily dictated by the tetrameric molecule adopting any of the typical T or R allosteric conformations.

The conserved Phe GH5 of importance for hemoglobin intersubunit contact is mutated in gadoid fish

BackgroundFunctionality of the tetrameric hemoglobin molecule seems to be determined by a few amino acids located in key positions. Oxygen binding encompasses structural changes at the interfaces between the α1β2 and α2β1 dimers, but also subunit interactions are important for the oxygen binding affinity and stability. The latter packing contacts include the conserved Arg B12 interacting with Phe GH5, which is replaced by Leu and Tyr in the αA and αD chains, respectively, of birds and reptiles.ResultsSearching all known hemoglobins from a variety of gnathostome species (jawed vertebrates) revealed the almost invariant Arg B12 coded by the AGG triplet positioned at an exon-intron boundary. Rare substitutions of Arg B12 in the gnathostome β globins were found in pig, tree shrew and scaled reptiles. Phe GH5 is also highly conserved in the β globins, except for the Leu replacement in the β1 globin of five marine gadoid species, gilthead seabream and the Comoran coelacanth, while Cys and Ile were found in burbot and yellow croaker, respectively. Atlantic cod β1 globin showed a Leu/Met polymorphism at position GH5 dominated by the Met variant in northwest-Atlantic populations that was rarely found in northeast-Atlantic cod. Site-specific analyses identified six consensus codons under positive selection, including 122β(GH5), indicating that the amino acid changes identified at this position may offer an adaptive advantage. In fact, computational mutation analysis showed that the replacement of Phe GH5 with Leu or Cys decreased the number of van der Waals contacts essentially in the deoxy form that probably causes a slight increase in the oxygen binding affinity.ConclusionsThe almost invariant Arg B12 and the AGG codon seem to be important for the packing contacts and pre-mRNA processing, respectively, but the rare mutations identified might be beneficial. The Leu122β1(GH5)Met and Met55β1(D6)Val polymorphisms in Atlantic cod hemoglobin modify the intradimer contacts B12-GH5 and H2-D6, while amino acid replacements at these positions in avian hemoglobin seem to be evolutionary adaptive in air-breathing vertebrates. The results support the theory that adaptive changes in hemoglobin functions are caused by a few substitutions at key positions.

High Altitude Genetic Adaptation In Tibetans Does Not Include Increased Hemoglobin-Oxygen Affinity

Increased hemoglobin-oxygen affinity has been shown to be an adaptive response to hypoxia in many high altitude animals such as the Andean goose, guinea pig and llamas (Reynafarje C. 1975; Hebbel R. 1978). It has been reported that people living in a high altitude, hypoxic environment have also developed a similar adaptation. Native Tibetans are known to have lived at an average of 3000-5000 meters on the Tibetan Plateau for more than 20,000 years, and have undergone genetic adaptations that have enabled them to thrive in this reduced oxygen environment. Most Tibetans are thus protected from polycythemia and other features of chronic mountain sickness. Several studies have reported higher arterial oxygen saturations among Tibetans as part of their genetic adaptation (Beall C. 1994; Moore L. 2001; Niermeyer S. 1995), thereby concluding that they have higher hemoglobin-oxygen affinity. Further, recent genomic studies have reported that beta-globin haplotypes (HBB and HBG2) have been selected in Tibetans, suggesting the presence of hemoglobin variants as a beneficial factor of Tibetan adaptation (Yi. 2010). However, some of the reports of increased hemoglobin-oxygen affinity are based on single readings of arterial oxygen measurements. Hemoglobin-oxygen affinity is more optimally measured by deriving the P50 value, which is the partial pressure of oxygen at which hemoglobin is 50% saturated with oxygen. A decreased P50 can be due to mutated globin genes resulting in high oxygen affinity hemoglobins, low 2,3 BPG, high pH or low temperature. The hemoglobin-oxygen dissociation is optimally derived by hemoximeter measurements of the percent saturation of hemoglobin at various partial pressures of oxygen. The resultant curve has a sigmoid shape due to the cooperative binding of oxygen to the four globins in the hemoglobin tetramer; this cooperative interaction can be enumerated as a Hill coefficient “n”. If a hemoximeter is not readily available, the P50 can be estimated from the venous blood gas using the measured pO2, hemoglobin oxygen percent saturation O2%, and pH (Lichtman M. 1976); however the Hill coefficient “n” cannot be derived by this method.To definitely establish whether the Tibetan adaptation to high altitude hypoxia involves increased hemoglobin-oxygen affinity, we conducted the following study of direct and indirect oxygen-hemoglobin affinity among Tibetans living at two different altitudes.We enrolled 14 healthy ethnic Tibetans and one closely related Nepalese Sherpa. There were 8 males and 7 females ages ranging 35-75 years. The first group consisted of 5 ethnic Tibetans living in Srinagar, India (1,600 meters), on whom venous blood gases were done and the P50 was derived using pH, PO2 and O2 saturation using the formula described by Lichtman and colleagues. Three were born in Tibet and two were offspring of Tibet-born parents. The second group consisted of 10 volunteers (9 Tibetans and one Nepalese Sherpa) residing in Salt Lake City, UT, (1,300 meters) whose peripheral blood was evaluated by Hemox Analyzer for obtaining P50 values and “n” Hill coefficients for hemoglobin oxygen binding. All the ethnic Tibetans in Salt Lake City were born in Tibet except for one, and the Nepalese Sherpa was born in Nepal.The results are depicted in Table. The P50 measured by venous blood gases on the Tibetan volunteers from Srinagar, India and those measured by Hemox Analyzer on the 10 volunteers from Salt Lake City, UT were normal, with values in the normal range (22-28 mmHg). No hemoglobin variants were detected by high pressure liquid chromatography in these 15 Tibetan volunteers.TableSubject IDP50 (mmHg)“n” Hill CoefficientS 0526.38n/aS 0825.95S 1326.55S 1523.68S 2722.72U 1926.962.97U 2025.162.83U 2124.202.89U 2225.462.84U 2322.502.89U 2424.062.87U 2524.282.83U 2622.352.82U 2723.292.79U 2825.992.75We report no evidence for the presence of high hemoglobin-oxygen affinity in Tibetans as a constituent of their genetic adaptation. Our data rule out the existence of hemoglobin variants and aberrant 2,3 BPG metabolism as possible features of Tibetan high-altitude adaptation; however acquired transient metabolic alterations at high altitudes, cannot be excluded to account for possible changes in hemoglobin-oxygen affinity but these are not evolved persistent features of Tibetan genetic adaptation. Studies of Tibetans living in these extreme hypoxic environment (>4,000m) are now planned. Disclosures:No relevant conflicts of interest to declare.

β-globin gene transfer to human bone marrow for sickle cell disease.

Autologous hematopoietic stem cell gene therapy is an approach to treating sickle cell disease (SCD) patients that may result in lower morbidity than allogeneic transplantation. We examined the potential of a lentiviral vector (LV) (CCL-βAS3-FB) encoding a human hemoglobin (HBB) gene engineered to impede sickle hemoglobin polymerization (HBBAS3) to transduce human BM CD34+ cells from SCD donors and prevent sickling of red blood cells produced by in vitro differentiation. The CCL-βAS3-FB LV transduced BM CD34+ cells from either healthy or SCD donors at similar levels, based on quantitative PCR and colony-forming unit progenitor analysis. Consistent expression of HBBAS3 mRNA and HbAS3 protein compromised a fourth of the total β-globin-like transcripts and hemoglobin (Hb) tetramers. Upon deoxygenation, a lower percentage of HBBAS3-transduced red blood cells exhibited sickling compared with mock-transduced cells from sickle donors. Transduced BM CD34+ cells were transplanted into immunodeficient mice, and the human cells recovered after 2-3 months were cultured for erythroid differentiation, which showed levels of HBBAS3 mRNA similar to those seen in the CD34+ cells that were directly differentiated in vitro. These results demonstrate that the CCL-βAS3-FB LV is capable of efficient transfer and consistent expression of an effective anti-sickling β-globin gene in human SCD BM CD34+ progenitor cells, improving physiologic parameters of the resulting red blood cells.

Stabilities of hemoglobins A and S in dried blood spots stored under controlled conditions

ObjectiveWe aimed to measure separately the contributions of heat and humidity to changes in levels of hemoglobins A and S in dried-blood-spot (DBS) samples. Design and methodsWe stored paired sets of DBSs at 37°C for predetermined intervals in low-humidity and high-humidity environments. Hemoglobin A and S levels of all samples in each complete set were measured in a single high performance liquid chromatography run. ResultsDuring the one-month storage intervals, both hemoglobin species lost about 35% of initial levels in low-humidity storage and almost all of initial levels in high-humidity storage. ConclusionsMinimizing both humidity and temperature in the transportation and storage environments of DBS samples is essential to maintaining the integrity of the hemoglobin tetramer molecules.

Haptoglobin Preferentially Binds β but Not α Subunits Cross-Linked Hemoglobin Tetramers with Minimal Effects on Ligand and Redox Reactions

Human hemoglobin (Hb) and haptoglobin (Hp) exhibit an extremely high affinity for each other, and the dissociation of Hb tetramers into dimers is generally believed to be a prerequisite for complex formation. We have investigated Hp interactions with native Hb, αα, and ββ cross-linked Hb (ααXLHb and ββXLHb, respectively), and rapid kinetics of Hb ligand binding as well as the redox reactivity in the presence of and absence of Hp. The quaternary conformation of ββ subunit cross-linking results in a higher binding affinity than that of αα subunit cross-linked Hb. However, ββ cross-linked Hb exhibits a four fold slower association rate constant than the reaction rate of unmodified Hb with Hp. The Hp contact regions in the Hb dimer interfaces appear to be more readily exposed in ββXLHb than ααXLHb. In addition, apart from the functional changes caused by chemical modifications, Hp binding does not induce appreciable effects on the ligand binding and redox reactions of ββXLHb. Our findings may therefore be relevant to the design of safer Hb-based oxygen therapeutics by utilizing this preferential binding of ββXLHb to Hp. This may ultimately provide a safe oxidative inactivation and clearance pathway for chemically modified Hbs in circulation.

Tertiary and Quaternary Allostery in Tetrameric Hemoglobin from Scapharca inaequivalvis

The clam Scapharca inaequivalvis possesses two cooperative oxygen binding hemoglobins in its red cells: a homodimeric HbI and a heterotetrameric A2B2 HbII. Each AB dimeric half of HbII is assembled in a manner very similar to that of the well-studied HbI. This study presents crystal structures of HbII along with oxygen binding data both in the crystalline state and in wet nanoporous silica gels. Despite very similar ligand-linked structural transitions observed in HbI and HbII crystals, HbII in the crystal or encapsulated in silica gels apparently exhibits minimal cooperativity in oxygen binding, in contrast with the full cooperativity exhibited by HbI crystals. However, oxygen binding curves in the crystal indicate the presence of a significant functional inequivalence of A and B chains. When this inequivalence is taken into account, both crystal and R state gel functional data are consistent with the conservation of a tertiary contribution to cooperative oxygen binding, quantitatively similar to that measured for HbI, and are in keeping with the structural information. Furthermore, our results indicate that to fully express cooperative ligand binding, HbII requires quaternary transitions hampered by crystal lattice and gel encapsulation, revealing greater complexity in cooperative function than the direct communication across a dimeric interface observed in HbI.

Impact of Interfacial Chemical Modifications on the Assembly of an Allosteric Protein - Isothermal Calorimetry and Oxygenation Measurements

Most allosteric proteins -if not all- present in living organisms tend to associate naturally to form macromolecular assemblies. The classical tenet of allostery invokes that structural changes at quaternary level are responsible for triggering functional changes in the assembly. That is to say, functional changes are not expected to occur in macromolecular systems that lack quaternary structure (with a few exceptions).In the present work, we have investigated the role of the α1β1 interface in α2β2 tetrameric hemoglobin (Hb), long considered inert, and studied the effects of chemical modifications on this interface on the dissociation of tetramers into dimers, as well as oxygenation properties of tetrameric Hb.Chemical modifications of sulfhydryl groups of α104Cys and β112Cys present in the α1β1 interface allow the study of intradimeric communication. Reactions of these Cys residues were carried out with dithiopyridine. Disulfide bonded thiopyridyl groups thus produced faced the central cavity of the tetramer.Tetramers were reassembled and dimer association equilibrium constants and oxygenation properties were measured. We found that any modification on the α1β1 interface produced a pronounced decrease in oxygen affinity. Surprisingly, the dimerization of the ligated derivatives was enhanced rather than impaired. These results suggest that the chemically modified α1β1 interface of the dimer produced such a striking effect. In other words, the quaternary effect originated from the dimer rather than the tetramer.

Case study: haemoglobin rothschild in a case presenting with cyanosis

Introduction Here we describe a case presenting with episodic cyanosis in the absence of any evidence of cardio-pulmonary disease. Case The patient was a 27-year-old female who sought medical attention for a productive cough. On examination she was cya-nosed but not in distress. Systematic examination, notably cardio-respiratory system, revealed no abnormalities. A positive family history of episodic cyanosis in one of the siblings, but not the parents, was obtained. Results The full blood count, renal and liver functions were essentially normal. A normal haptoglobin level effectively excluded haemolysis. The oxygen saturation at the time of examination was 87%. An abnormal band was noted on Hb electrophoresis confirm-ing the presence of an Hb variant. Gene sequencing revealed a cod 37 (Tryp>Arg) mutation in exon 2 of the beta-globin gene, identi-fied as Hb Rothschild. Discussion Tryptophan at β37 is recognised as an important α1β2 contact point of the haemoglobin molecule. Disruption of the β37 contact point favours a T-quaternary (low oxygen affinity) state of the haemoglobin tetramer. Premature oxygen off-loading decreases the percentage oxygen saturation and may lead to cyanosis. Causes of cyanosis include cardio-respiratory disease, meth-aemoglobinaemia, sulfhaemoglobinaemia and low oxygen affinity haemoglobin variants.

Signal Enhancement in Disperse Solutions for the Analysis of Biomedical Samples by Photothermal Spectroscopy

Photothermal lens spectrometry not only shows high sensitivity of colored heme protein determination, but also provides a change in the sensitivity compared to the theoretical values due to changes in the heat transfer in dispersed media. This can be used for estimating the size of disperse particles exemplified by hemoglobin cyanide, photothermal examination of the state of existence of hemoglobin in highly saline solutions by changes in photothermal properties upon dissociation of hemoglobin tetramers into dimers and monomers. The example of determination of contrast agents (dyes) in blood as the versification of the platform of photoacoustic/photothermal measurement of circulating blood volume is shown.

The globins: classic structures and new insights

The oxygen-binding proteins myoglobin and haemoglobin have a special place in the history of structural biology, and, by implication, of molecular biology and molecular medicine. When the first structures of these proteins were published, in 19581 and 19602, they were the only protein structures known. And even these structures bore very little resemblance to what we now think of as ‘protein structures’. The structures were far too crude – low resolution, as structural biologists say – to reveal the positions of any of the atoms. The beautiful and meticulous hand-drawn diagrams of the myoglobin monomer and the haemoglobin tetramer in these proteins show fat sausage-like shapes representing the eight -helices of the now well-known globin fold, with the haem group nestling in between. It took the genius of the principal scientists involved in that work, Max Perutz and John Kendrew, to imagine the potential of these rather unpromising looking structures. Perutz and Kendrew were jointly awarded the Nobel Prize for Chemistry in 1962, which was a particularly good year for structural biology; Watson, Crick and Wilkins were awarded the Physiology or Medicine Nobel for the DNA structure then too.

Structure of the haptoglobin–haemoglobin complex

Red cell haemoglobin is the fundamental oxygen-transporting molecule in blood, but also a potentially tissue-damaging compound owing to its highly reactive haem groups. During intravascular haemolysis, such as in malaria and haemoglobinopathies, haemoglobin is released into the plasma, where it is captured by the protective acute-phase protein haptoglobin. This leads to formation of the haptoglobin-haemoglobin complex, which represents a virtually irreversible non-covalent protein-protein interaction. Here we present the crystal structure of the dimeric porcine haptoglobin-haemoglobin complex determined at 2.9 Å resolution. This structure reveals that haptoglobin molecules dimerize through an unexpected β-strand swap between two complement control protein (CCP) domains, defining a new fusion CCP domain structure. The haptoglobin serine protease domain forms extensive interactions with both the α- and β-subunits of haemoglobin, explaining the tight binding between haptoglobin and haemoglobin. The haemoglobin-interacting region in the αβ dimer is highly overlapping with the interface between the two αβ dimers that constitute the native haemoglobin tetramer. Several haemoglobin residues prone to oxidative modification after exposure to haem-induced reactive oxygen species are buried in the haptoglobin-haemoglobin interface, thus showing a direct protective role of haptoglobin. The haptoglobin loop previously shown to be essential for binding of haptoglobin-haemoglobin to the macrophage scavenger receptor CD163 (ref. 3) protrudes from the surface of the distal end of the complex, adjacent to the associated haemoglobin α-subunit. Small-angle X-ray scattering measurements of human haptoglobin-haemoglobin bound to the ligand-binding fragment of CD163 confirm receptor binding in this area, and show that the rigid dimeric complex can bind two receptors. Such receptor cross-linkage may facilitate scavenging and explain the increased functional affinity of multimeric haptoglobin-haemoglobin for CD163 (ref. 4).

A model of the NO producing nitrite reductase activity of hemoglobin

The production of nitric oxide by the nitrite reductase activity of deoxyhemoglobin has been proposed to play a major role in hypoxic vasodilation. A feature of this mechanism is that deoxyhemoglobin sites in the R state of tetrameric hemoglobin favoured at high pO2 have enhanced activity compared to deoxyhemoglobin sites in the T state tetramer favoured at low pO2. The nitrite reductase activity versus the pO2 curve is a complex function of the internal properties of the allosteric transition. We therefore performed a full analysis of the nitrite reductase activity using the Monod–Wyman–Changeux allosteric model. We derived an equation for the deoxyhemoglobin heme sites over the total heme sites as a function of oxygen partial pressure. This enabled us to calculate an expression for the normalized bimolecular reaction rate of nitrite and deoxyhemoglobin. We then used these expressions to explore the factors controlling the shape of the pO2 versus nitrite reductase activity curve. Our model has predictive and explanatory powers. For example it is known that fetal hemoglobin has an enhanced maximum nitrite reductase activity compared to adult hemoglobin. However, the mechanism for this effect is unknown. Our model is able to show that enhanced NO production in the fetal case is a simple consequence of the differences in p50 between the fetal and adult forms, rather than any specific change in the nitrite reductase chemistry itself.

Modeling sickle hemoglobin fibers as one chain of coarse-grained particles

Sickle cell disease (SCD) is caused by a single point mutation in the beta-chain hemoglobin gene, resulting in the presence of abnormal hemoglobin S (HbS) in the patients' red blood cells (RBCs). In the deoxygenated state, the defective hemoglobin tetramers polymerize forming stiff fibers which distort the cell and contribute to changes in its biomechanical properties. Because the HbS fibers are essential in the formation of the sickle RBC, their material properties draw significant research interests. Here, a solvent-free coarse-grain molecular dynamics (CGMD) model is introduced to simulate single HbS fibers as a chain of particles. First, we show that the proposed model is able to efficiently simulate the mechanical behavior of single HbS fibers. Then, the zippering process between two HbS fibers is studied and the effect of depletion forces is investigated. Simulation results illustrate that depletion forces play a role comparable to direct fiber-fiber interaction via Van der Waals forces. This proposed model can greatly facilitate studies on HbS polymerization, fiber bundle and gel formation as well as interaction between HbS fiber bundles and the RBC membrane.

α-Thalassemia Impairs the Cytoadherence of Plasmodium falciparum-Infected Erythrocytes

Backgroundα-thalassemia results from decreased production of α-globin chains that make up part of hemoglobin tetramers (Hb; α2β2) and affects up to 50% of individuals in some regions of sub-Saharan Africa. Heterozygous (−α/αα) and homozygous (−α/−α) genotypes are associated with reduced risk of severe Plasmodium falciparum malaria, but the mechanism of this protection remains obscure. We hypothesized that α-thalassemia impairs the adherence of parasitized red blood cells (RBCs) to microvascular endothelial cells (MVECs) and monocytes – two interactions that are centrally involved in the pathogenesis of severe disease.Methods and FindingsWe obtained P. falciparum isolates directly from Malian children with malaria and used them to infect αα/αα (normal), −α/αα and −α/−α RBCs. We also used laboratory-adapted P. falciparum clones to infect −/−α RBCs obtained from patients with HbH disease. Following a single cycle of parasite invasion and maturation to the trophozoite stage, we tested the ability of parasitized RBCs to bind MVECs and monocytes. Compared to parasitized αα/αα RBCs, we found that parasitized −α/αα, −α/−α and −/−α RBCs showed, respectively, 22%, 43% and 63% reductions in binding to MVECs and 13%, 33% and 63% reductions in binding to monocytes. α-thalassemia was associated with abnormal display of P. falciparum erythrocyte membrane protein 1 (PfEMP1), the parasite’s main cytoadherence ligand and virulence factor, on the surface of parasitized RBCs.ConclusionsParasitized α-thalassemic RBCs show PfEMP1 display abnormalities that are reminiscent of those on the surface of parasitized sickle HbS and HbC RBCs. Our data suggest a model of malaria protection in which α-thalassemia ameliorates the pro-inflammatory effects of cytoadherence. Our findings also raise the possibility that other unstable hemoglobins such as HbE and unpaired α-globin chains (in the case of β-thalassemia) protect against life-threatening malaria by a similar mechanism.

Low NO Concentration Dependence of Reductive Nitrosylation Reaction of Hemoglobin

The reductive nitrosylation of ferric (met)hemoglobin is of considerable interest and remains incompletely explained. We have previously observed that at low NO concentrations the reaction with tetrameric hemoglobin occurs with an observed rate constant that is at least 5 times faster than that observed at higher concentrations. This was ascribed to a faster reaction of NO with a methemoglobin-nitrite complex. We now report detailed studies of this reaction of low NO with methemoglobin. Nitric oxide paradoxically reacts with ferric hemoglobin with faster observed rate constants at the lower NO concentration in a manner that is not affected by changes in nitrite concentration, suggesting that it is not a competition between NO and nitrite, as we previously hypothesized. By evaluation of the fast reaction in the presence of allosteric effectors and isolated β- and α-chains of hemoglobin, it appears that NO reacts with a subpopulation of β-subunit ferric hemes whose population is influenced by quaternary state, redox potential, and hemoglobin dimerization. To further characterize the role of nitrite, we developed a system that oxidizes nitrite to nitrate to eliminate nitrite contamination. Removal of nitrite does not alter reaction kinetics, but modulates reaction products, with a decrease in the formation of S-nitrosothiols. These results are consistent with the formation of NO(2)/N(2)O(3) in the presence of nitrite. The observed fast reductive nitrosylation observed at low NO concentrations may function to preserve NO bioactivity via primary oxidation of NO to form nitrite or in the presence of nitrite to form N(2)O(3) and S-nitrosothiols.

Gestación en paciente con anemia de Cooley

Thalassemia syndromes are inherited disorders of alpha- or beta-globin biosynthesis. The reduced availability of globin decreases the production of hemoglobin tetramers, giving rise to hypochromia and microcytosis. There is unbalanced accumulation of alpha or beta subunits because the synthesis of unaffected globins continues at a normal speed. This unbalanced accumulation dominates the clinical phenotype. The clinical severity varies widely, depending on the degree of disorder of the synthesis of the affected globin, the altered synthesis of other globin chains and simultaneous inheritance of other abnormal globin alleles. The incidence of these syndromes during pregnancy in all races is one in 300 to 500.