The kinetics of nucleation and growth of sickle cell hemoglobin fibers

Polymerization of sickle cell hemoglobin (HbS) in deoxy state is one of the basic events in the pathophysiology of sickle cell anemia. For insight into the polymerization process, we monitor the kinetics of nucleation and growth of the HbS polymer fibers. We define a technique for the determination of the rates J and delay times theta of nucleation and the fiber growth rates R of deoxy-HbS fibers, based on photolysis of CO-HbS by laser illumination. We solve numerically time-dependent equations of heat conductance and CO transport, coupled with respective photo-chemical processes, during kinetics experiments under continuous illumination. After calibration with experimentally determined values, we define a regime of illumination ensuring uniform temperature and deoxy-HbS concentration, and fast (within <1 s) egress to steady conditions. With these procedures, data on the nucleation and growth kinetics have relative errors of <5% and are reproducible within 10% in independent experiments. The nucleation rates and delay times have steep, exponential dependencies on temperature. In contrast, the average fiber growth rates only weakly depend on temperature. The individual growth rates vary by up to 40% under identical conditions. These variations are attributed to instability of the coupled kinetics and diffusion towards the growing end of a fiber. The activation energy for incorporation of HbS molecules into a polymer is E(A)=50 kJ mol(-1), a low value indicating the significance of the hydrophobic contacts in the HbS polymer. More importantly, the contrast between the strong theta(T) and weak R(T) dependencies suggests that the homogenous nucleation of HbS polymers occurs within clusters of a precursor phase. This conclusion may have significant consequences for the understanding of the pathophysiology of sickle cell anemia and should be tested in further work.     

The neurotoxic effect of sickle cell hemoglobin

A growing body of experimental evidence suggests that the oxidative neurotoxicity of hemoglobin A may contribute to neuronal loss after CNS hemorrhage. Several hemoglobin variants, including hemoglobin S, are more potent oxidants in cell-free systems. However, despite the increased incidence of hemorrhagic stroke associated with sickle cell disease, little is known of the effect of hemoglobin S on cells of neural origin. In the present study, its toxicity was quantified and directly compared with that of hemoglobin A in murine cortical cell cultures. Reactive oxygen species production, as assessed by cellular fluorescence after treatment with dihydrorhodamine 123, was significantly increased by exposure to 10 μM hemoglobin S for 2-4 h. Neuronal death, as measured by propidium iodide staining and lactate dehydrogenase release, commenced at 4 h; for a 20-h exposure, the EC₅₀ was approximately 0.71 μm. Glial cells were not injured. Cell death was completely blocked by iron chelation with deferoxamine or phenanthroline. Direct comparison of sister cultures exposed to either hemoglobin A or hemoglobin S revealed a similar amount of cell injury in both groups. A significant difference was consistently observed only after treatment with 1 μM hemoglobin for 20 h, which resulted in death of approximately one third more neurons with hemoglobin S than with hemoglobin A. The results of this study suggest that sickle cell hemoglobin is neurotoxic at physiologically relevant concentrations. This toxicity is iron-dependent, oxidative, and quantitatively similar to that produced by hemoglobin A.     

Increased concentrations of IL-18 and uric acid in sickle cell anemia: contribution of hemolysis, endothelial activation and the inflammasome

Sickle cell anemia (SCA) is a common, severe monogenetic disorder characterized by chronic hemolysis, frequent infections, a chronic inflammatory state and recurrent occlusions of the microcirculation, resulting in painful crises, organ damage and premature death. This study evaluated associations between serum levels of IL-18, uric acid, hemolytic markers, and inflammatory molecules in SCA patients. A cross-sectional study was performed including 45 SCA patients (median age of 20.5 years) without general symptoms and who had not undergone blood transfusions. Inclusion criteria for the steady-state SCA patients were the absence of hospitalization and the absence of infections. Interleukin-18 and uric acid levels were correlated closely with markers of hemolysis, endothelial dysfunction and others cytokines levels. These findings suggest probable influences of IL-18 and uric acid in the pathophysiology of vascular occlusion in SCA. Additional studies should be performed to characterize similar prognosis markers and possible therapeutic targets.     

A Ca2+-refractory state of the Ca-sensitive K+ permeability mechanism in sickle cell anaemia red cells

Simultaneous measurements of Ca content and ⁴²K⁺ influx in sickle cell anaemia red cells confirm predictions from earlier data in the literature that the increased Ca content of sickle cell anaemia cells which are not metabolically depleted does not cause a quinine-sensitive increase in K⁺ permeability.It is shown that the ionophore, A23187, can cause the Ca contained inside sickle cell anaemia cells to activate the quinine-sensitive K⁺-permeability mechanism. This demonstrates the existence of a Ca²⁺-refractory state of the K⁺ channel in sickle cell anaemia cells and a direct stimulatory effect of the ionophore A23187 on its Ca sensitivity.     

Strategies for rare-event detection: an approach for automated fetal cell detection in maternal blood.

This article explores the feasibility of the use of automated microscopy and image analysis to detect the presence of rare fetal nucleated red blood cells (NRBCs) circulating in maternal blood. The rationales for enrichment and for automated image analysis for "rare-event" detection are reviewed. We also describe the application of automated image analysis to 42 maternal blood samples, using a protocol consisting of one-step enrichment followed by immunocytochemical staining for fetal hemoglobin (HbF) and FISH for X- and Y-chromosomal sequences. Automated image analysis consisted of multimode microscopy and subsequent visual evaluation of image memories containing the selected objects. The FISH results were compared with the results of conventional karyotyping of the chorionic villi. By use of manual screening, 43% of the slides were found to be positive (>=1 NRBC), with a mean number of 11 NRBCs (range 1-40). By automated microscopy, 52% were positive, with on average 17 NRBCs (range 1-111). There was a good correlation between both manual and automated screening, but the NRBC yield from automated image analysis was found to be superior to that from manual screening (P=.0443), particularly when the NRBC count was >15. Seven (64%) of 11 XY fetuses were correctly diagnosed by FISH analysis of automatically detected cells, and all discrepancies were restricted to the lower cell-count range. We believe that automated microscopy and image analysis reduce the screening workload, are more sensitive than manual evaluation, and can be used to detect rare HbF-containing NRBCs in maternal blood.     

Haplotype mapping of a major quantitative-trait locus for fetal hemoglobin production, on chromosome 6q23.

Fetal hemoglobin (Hb F) and fetal cell (FC) levels in adults show considerable variation and are influenced by several genetic variants; the major determinants appear to be unlinked to the beta-globin gene cluster. Recently, a trans-acting locus controlling Hb F and FC production has been mapped to chromosome 6q23 in an Asian Indian kindred that includes individuals with heterocellular hereditary persistence of Hb F (HPFH) associated with beta thalassemia. We have extended the kindred by 57 members, bringing the total studied to 210, and have saturated the region with 26 additional markers. Linkage analysis showed tight linkage of the quantitative-trait locus (QTL) to the anonymous markers D6S976 (LOD score 11.3; recombination fraction .00) and D6S270 (LOD score 7.4; recombination fraction .00). Key recombination events now place this QTL within a 1-2-cM interval spanning approximately 1.5 Mb between D6S270 and D6S1626. Furthermore, haplotype analysis has led to a reevaluation of the genealogy and to the identification of additional relationships in the kindred.     

Ligand binding and the gelation of sickle cell hemoglobin.

The solubility equilibrium between monomer and polymer which has been shown to exist in deoxyhemoglobin S solutions is examined in solutions partially saturated with carbon monoxide. The total solubility is found to increase monotonically with increasing fractional saturation. At low fractional saturations the increase is nearly linear, amounting roughly to an increase of 0.01 g cm^?3 in solubility for each 10% increase in fractional saturation. Linear dichroism measurements on the spontaneously aligned polymer phase are used to examine the composition of the polymer as a function of the fractional saturation of the corresponding solution phase. The dichroism experiments show that the polymer phase contains less than 5% of CO-liganded hemes even at supernatant fractional saturations in excess of 70%. The polymer selects against totally liganded hemoglobin molecules by a minimum factor of 65 and against singly liganded molecules by a factor of at least 2.5. Consequently, polymerized hemoglobin S has a ligand affinity which is significantly lower than that of monomeric hemoglobin S in the deoxy quaternary structure.The kinetics of the polymerization reaction in the presence of CO are similar to those observed in pure deoxyhemoglobin S solutions. The polymerization is preceded by a pronounced delay, the duration of which, t_d, is proportional roughly to the 30th power of the solubility. At low fractional saturations, this amounts to a tenfold increase in t_d for each 10% increase in the fractional saturation.These results show that the polymerization reaction is nearly specific for deoxyhemoglobin. Models for the dependence of the solubility and the polymer saturation on ligand partial pressure demonstrate the importance of solution phase non-ideality in determining the solubility of mixtures. The results require selection against partially liganded species which is significantly greater than is predicted by the two-state allosteric model. The data are compatible with either sequential or allosteric models in which the major polymerized component is the unliganded hemoglobin molecule.     

Determination of the transition-state entropy for aggregation suggests how the growth of sickle cell hemoglobin polymers can be slowed

Sickle cell anemia is associated with the mutant hemoglobin HbS, which forms polymers in red blood cells of patients. The growth rate of the polymers is several micrometers per second, ensuring that a polymer fiber reaches the walls of an erythrocyte (which has a 7-μm diameter) within a few seconds after its nucleation. To understand the factors that determine this unusually fast rate, we analyze data on the growth rate of the polymer fibers. We show that the fiber growth follows a first-order Kramers-type kinetics model. The entropy of the transition state for incorporation into a fiber is 95 J mol^− 1 K^− 1, very close to the known entropy of polymerization. This agrees with a recent theoretical estimate for the hydrophobic interaction and suggests that the gain of entropy in the transition state is due to the release of the last layer of water molecules structured around contact sites on the surface of the HbS molecules. As a result of this entropy gain, the free-energy barrier for incorporation of HbS molecules into a fiber is negligible and fiber growth is unprecedentedly fast. This finding suggests that fiber growth can be slowed by components of the red cell cytosol, native or intentionally introduced, which restructure the hydration layer around the HbS molecules and thus lower the transition state entropy for incorporation of an incoming molecule into the growing fiber.     

Mechanisms of homogeneous nucleation of polymers of sickle cell anemia hemoglobin in deoxy state

The primary pathogenic event of sickle cell anemia is the polymerization of the mutant hemoglobin (Hb) S within the red blood cells, occurring when HbS is in deoxy state in the venous circulation. Polymerization is known to start with nucleation of individual polymer fibers, followed by growth and branching via secondary nucleation, yet the mechanisms of nucleation of the primary fibers have never been subjected to dedicated tests. We implement a technique for direct determination of rates and induction times of primary nucleation of HbS fibers, based on detection of emerging HbS polymers using optical differential interference contrast microscopy after laser photolysis of CO-HbS. We show that: (i). nucleation throughout these determinations occurs homogeneously and not on foreign substrates; (ii). individual nucleation events are independent of each other; (iii). the nucleation rates are of the order of 10(6)-10(8)cm(-3)s(-1); (iv). nucleation induction times agree with an a priori prediction based on Zeldovich's theory; (v). in the probed parameter space, the nucleus contains 11 or 12 molecules. The nucleation rate values are comparable to those leading to erythrocyte sickling in vivo and suggest that the mechanisms deduced from in vitro experiments might provide physiologically relevant insights. While the statistics and dynamics of nucleation suggest mechanisms akin to those for small-molecule and protein crystals, the nucleation rate values are nine to ten orders of magnitude higher than those known for protein crystals. These high values cannot be rationalized within the current understanding of the nucleation processes.     

Membrane components in the red cells of patients with sickle cell anemia Relationship to cell aging and to irreversibility of sickling

Cholesterol, phospholipid and sialic acid were measured in red cells from patients with sickle cell anemia to determine whether the cells had abnormal concentrations of these components and whether the amounts of these compounds differed in irreversibly sickled cells as compared to non-irreversibly sickled cells. Sickle cells had significantly higher levels of both lipids than similar populations of normal cells, however, comparisons to populations of young control cells showed that the differences were generally not significant. Sialic acid levels in sickle cells were not significantly different from normal cells. Irreversibly sickled cells had lower lipid and sialic acid concentrations than those not irreversibly sickled, but the differences were either not significant or did not occur when compared to young control cells. The studies show that the increased lipid concentrations in the membrane of sickle cells are not abnormal but are related to cell age and that the decrease in membrane components in irreversibly sickled cells is no greater than would be predicted for similarly aged populations of cells.     

An in silico approach to map the binding site of doxorubicin on hemoglobin

Binding modalities of doxorubicin (DOX), a widely used antineoplastic anthracyline antibiotic with hemoglobin (Hb) have been studied. The protein and the ligand were prepared using CORINA and protonated with insight II. The best conformation was sought by employing GOLDV. Molecular modeling calculations showed that DOX binds Hb to a non-classical drug binding site. The alpha subunit of Hb has been assigned to posses the binding site for DOX with a binding affinity (Ka) = 16.98 x10(3) mol(-1). The interaction was found to be thermodynamically favorable (DeltaG degrees = -66.23 KJmol(-1)). The analysis of DOX binding site to Hb suggested that the types of interactions that contribute in this binding are hydrophobic contacts, hydrogen bonding and electrostatic interactions.     

A model for the sickle hemoglobin fiber using both mutation sites

The standard molecular model of the fiber of the sickle hemoglobin (HbS: beta6 Glu-->Val) has been revised to allow both beta6 mutation sites to participate in intermolecular contacts, rather than only one beta6 site as previously thought, for four molecules per 14-molecule fiber cross section. This structure accurately predicts the copolymerization of hybridized mixtures of HbS with HbA or HbC (beta6 Glu-->Lys), which could not be reconciled with prior models in which only half the beta6 sites were required for assembly. This model suggests new contacts within the fiber and raises the question of whether these cross-linked double strands could possess added stability important in such processes as nucleation.     

Effect of nitric oxide on the transport and release of oxygen in fetal blood

It is well known that nitric oxide (NO), the most important vasodilator agent, plays an important role in lowering vascular resistance in the human umbilical-placental circulation and that its deficiency is related to the pathogenesis of pre-eclamptic disorder. Besides it has recently been demonstrated that human hemoglobin (HbA) is able to transport nitric oxide, as S-nitrosohemoglobin (SNO-Hb), from the arterial to the venous blood. In the present study we examine the functional properties of the adult and fetal nitrosated hemoglobins to see if the double transport of oxygen and NO may influence the fetal oxygenation and the relation between maternal and fetal blood. Our results show that S-nitrosation significantly increases the oxygen affinity of the adult Hb (HbA) with respect to native protein (no-nitrosated) while the functional properties of HbF are less influenced. The oxygen affinity modification, found for SNO-HbA, was ascribed to the nitrosation of cysteine beta 93: really, the same residue is also present in the gamma chains of fetal hemoglobin, while the increase of affinity is less evidenced; hence, it is probable that the 39 aminoacidic substitutions between beta and gamma chains allay the effects of S-nitrosation. As regards the physiological modulators (protons, chloride ions, 2,3-diphosphoglyceric acid, and temperature), they influence the oxygen affinity of the two hemoglobins S-nitrosated, in equal mode with respect to the native forms determining the same variation on the oxygen affinity. Hence, our results evidence the fact that the NO release by SNO-HbA "in vivo" would be limited to regions of extremely low oxygen tension (such as hypoxic regions), while in fetus, SNO-HbF would unload nitric oxide and oxygen at pressure values close to normal.     

Challenges for red blood cell biomarker discovery through proteomics

Red blood cells are rather unique body cells, since they have lost all organelles when mature, which results in lack of potential to replace proteins that have lost their function. They maintain only a few pathways for obtaining energy and reducing power for the key functions they need to fulfill. This makes RBCs highly sensitive to any aberration. If so, these RBCs are quickly removed from circulation, but if the RBC levels reduce extremely fast, this results in hemolytic anemia. Several causes of HA exist, and proteome analysis is the most straightforward way to obtain deeper insight into RBC functioning under the stress of disease. This should result in discovery of biomarkers, typical for each source of anemia. In this review, several challenges to generate in-depth RBC proteomes are described, like to obtain pure RBCs, to overcome the wide dynamic range in protein expression, and to establish which of the identified/quantified proteins are active in RBCs. The final challenge is to acquire and validate suited biomarkers unique for the changes that occur for each of the clinical questions; in red blood cell aging (also important for transfusion medicine), for thalassemias or sickle cell disease. Biomarkers for other hemolytic anemias that are caused by dysfunction of RBC membrane proteins (the RBC membrane defects) or RBC cytosolic proteins (the enzymopathies) are sometimes even harder to discover, in particular for the patients with RBC rare diseases with unknown cause. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.     

Proliferation and morphology of chick embryo cells cultured in the presence of horse serum and hemoglobin

Summary We have shown previously that hemoglobin greatly stimulates chick embryo cell proliferation in Eagle's minimal essential medium supplemented with horse serum. In the present study we compared the effects of horse serum plus 10 μM hemoglobin to those of fetal bovine serum on subcultures of chick embryo cells serially propagated at high cell densities. The cells became elongated in the presence of fetal bovine serum and their rate of proliferation progressively decreased, whereas they became polygonal in the presence of horse serum plus hemoglobin and proliferated well in successive cell passages. The polygonal cell obtained in the presence of horse serum plus hemoglobin rapidly elongated if cultured at low cell densities in the presence of fetal bovine serum, but, in contrast, elongated cells did not yield polygonal cells if cultured at low densities in the presence of horse serum plus hemoglobin. It is possible that the polygonal and elongated cells are undifferentiated cells and differentiating myogenic cells, respectively.     

Exploring the binding dynamics of etoricoxib with human hemoglobin: A spectroscopic,calorimetric, and molecular modeling approach

Etoricoxib, widely used for the treatment of osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, and related conditions has ample affinity to bind with globular proteins. Here, the molecular interaction between purified human hemoglobin (HHb), a major heme protein and etoricoxib, a cyclooxygenase-2 inhibitor was studied by various spectroscopic, calorimetric, and molecular modeling techniques. The binding affected hypochromic changes in the Soret band of hemoglobin (Hb) and induced remarkable quenching of the intrinsic fluorescence property of protein molecules. Synchronous fluorescence studies revealed alterations in tryptophan (Trp) and tyrosine (Tyr) microenvironments of HHb molecule in presence of etoricoxib. Flouremetric and isothermal titration calorimetric studies suggested two binding sites in HHb for etoricoxib at three different temperatures (298.15, 303.15, and 310.15 K). Negative values of Gibbs energy change (ΔG⁰) and enthalpy change (ΔH⁰) strongly suggest that it is spontaneous and exothermic reaction, mainly stabilized by hydrogen bonding as evidenced by sucrose binding assay. These findings support our in silico molecular docking study, which specified the binding site and the non-covalent interactions involved in the association. Moreover, the interaction impacts on structural integrity and functional aspects of HHb as confirmed by decreased α helicity, increased free iron release, increased rate of co-oxidation, and decreased rate of esterase activity. Overall, these studies conclude that etoricoxib leads to a remarkable alteration in the conformational aspects of binding to HHb.

Communicated by Ramaswamy H. Sarma.     

Studies of the fiber to crystal transition of sickle cell hemoglobin in acidic polyethylene glycol

The crystallization of deoxygenated sickle cell hemoglobin in acidic (pH 5.2) polyethylene glycol (10%) has been studied in order to determine if the mechanism of crystal formation under such conditions has features in common with the mechanism of crystal formation at higher pH values in the absence of polyethylene glycol. The existence of a common mechanism of crystallization under different conditions is relevant in validating the use of the known high resolution crystal structure to interpret the fiber structure. Our findings indicate that deoxygenated sickle cell hemoglobin crystallization in acidic polyethylene glycol is initiated by fiber formation. Fibers, in turn, convert to larger structures called macrofibers within several hours (Wellems et al., 1981). Fibers and macrofibers (and their respective optical transforms) formed in acidic polyethylene glycol appear to have the same structure as their counterparts formed at higher pH values in the absence of polyethylene glycol. Early in the transition one can observe macrofibers in the process of alignment and fusion. The structural characterization of the intermediates leaves little doubt that crystallization in acidic polyethylene glycol is mediated by the same mechanism as that occurring under more physiological conditions, and that fibers are a metastable intermediate whose ultimate fate is to crystallize.     

The hemoglobins of Artemia salina. IV. A model for genetic control of hemoglobin 1, hemoglobin 2, and hemoglobin X

Two loci account for all genetic variation resulting in difference in electrophoretic mobility in three hemoglobins (Hb1, Hb2, and HbX) in the hemolymph of the brine shrimp. Four alleles and nine alleles have been studied. In shrimps of all genotypes and in electrophoresis in media with varying degrees of molecular sieving, Hb2 is approximately equidistant from Hb1 and HbX. A shrimp heterozygous at both loci has a three-banded Hb1, a four-banded Hb2, and a three-banded HbX. We conclude that Hb2 contains n -polypeptides and n -polypeptides. Hb1 contains 2n -polypeptides. HbX contains 2n -polypeptides. During electrophoresis, the three native hemoglobins undergo reversible dissociation to n subunits. Subunits with the same charge reassemble to migrate as molecules of the same size as the native molecules. Although there is no evidence for an additional polypeptide in the three hemoglobins, we cannot exclude such a possibility. If it exists, it is under three constraints: (1) it must be present in equal amounts in each of the three hemoglobins; (2) it must have the same molecular weight as the - and -polypeptides; and (3) it must be free of genetic variation (detectable by electrophoresis).Supported by National Institutes of Health Grant HE-11445.     

Computerized scheme for the reaction of hemoglobin with ligands

A scheme for the reaction of hemoglobin with ligands is described, which postulates the functional heterogeneity of the chains, considers all possible combinations of the distribution of the ligand on the four chains of hemoglobin, and does not require simplifying assumptions about the hemoglobin reactivity. Ten tetrameric species are considered, together with 16 reactions between these species, each with an on and an off rate constant. The dissociation of hemoglobin tetramers into dimers is also considered, with four on and four off rate constants for the reactions between dimers, and ten equilibrium constants for the reactions between tetramers and dimers. Moreover, some side reactions, such as the trapping of ligands by a hemoglobin competitor, are included. A FORTRAN program, suitable for microcomputers, is described for handling this scheme, with some examples showing its advantages.