Metastable mesoscopic clusters in solutions of sickle-cell hemoglobin

Sickle cell hemoglobin (HbS) is a mutant, whose polymerization while in deoxy state in the venous circulation underlies the debilitating sickle cell anemia. It has been suggested that the nucleation of the HbS polymers occurs within clusters of dense liquid, existing in HbS solutions. We use dynamic light scattering with solutions of deoxy-HbS, and, for comparison, of oxy-HbS and oxy-normal adult hemoglobin, HbA. We show that solutions of all three Hb variants contain clusters of dense liquid, several hundred nanometers in size, which are metastable with respect to the Hb solutions. The clusters form within a few seconds after solution preparation and their sizes and numbers remain relatively steady for up to 3 h. The lower bound of the cluster lifetime is 15 ms. The clusters exist in broad temperature and Hb concentration ranges, and occupy 10(-5)-10(-2) of the solution volume. The results on the cluster properties can serve as test data for a potential future microscopic theory of cluster stability and kinetics. More importantly, if the clusters are a part of the nucleation mechanism of HbS polymers, the rate of HbS polymerization can be controlled by varying the cluster properties.     

Free Heme and the Polymerization of Sickle Cell Hemoglobin

In search of novel control parameters for the polymerization of sickle cell hemoglobin (HbS), the primary pathogenic event of sickle cell anemia, we explore the role of free heme, which may be excessively released in sickle erythrocytes. We show that the concentration of free heme in HbS solutions typically used in the laboratory is 0.02-0.04 mole heme/mole HbS. We show that dialysis of small molecules out of HbS solutions arrests HbS polymerization. The addition of 100-260 μM of free heme to dialyzed HbS solutions leads to rates of nucleation and polymer fiber growth faster by two orders of magnitude than before dialysis. Toward an understanding of the mechanism of nucleation enhancement by heme, we show that free heme at a concentration of 66 μM increases by two orders of magnitude the volume of the metastable clusters of dense HbS liquid, the locations where HbS polymer nuclei form. These results suggest that spikes of the free heme concentration in the erythrocytes of sickle cell anemia patients may be a significant factor in the complexity of the clinical manifestations of sickle cell anemia. The prevention of free heme accumulation in the erythrocyte cytosol may be a novel avenue to sickle cell therapy.     

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.     

Heterogeneous nucleation in sickle hemoglobin: experimental validation of a structural mechanism

Sickle hemoglobin polymerizes by two types of nucleation: homogeneous nucleation of aggregates in solution, and heterogeneous nucleation on preexisting polymers. It has been proposed that the same contact that is made in the interior of the polymer between the mutant site beta6 and its receptor pocket on an adjacent molecule is the primary contact site for the heterogeneous nucleus. We have constructed cross-linked hybrid molecules in which one beta-subunit is from HbA with Glu at beta6, and the other is from HbS with a Val at beta6. We measured solubility (using sedimentation) and polymerization kinetics (using laser photolysis) on cross-linked hybrids, and cross-linked HbS as controls. We find approximately 4000 times less heterogeneous nucleation in the cross-linked AS molecules than in cross-linked HbS, in strong confirmation of the proposal. In addition, changes in stability of the nucleus support a further proposal that more than one beta6 contact is involved in the homogeneous nucleus.     

The role of pH on instability and aggregation of sickle hemoglobin solutions

Understanding the physical basis of protein aggregation covers strong physical and biomedical interests. Sickle hemoglobin (HbS) is a point-mutant form of normal human adult hemoglobin (HbA). It is responsible for the first identified "molecular disease," as its propensity to aggregation is responsible for sickle cell disease. At moderately higher than physiological pH value, this propensity is inhibited: The rate of aggregate nucleation becomes exceedingly small and solubility after polymerization increases. These order-of-magnitude effects on polymer nucleation rates and concurrent relatively modest changes of solubility after polymerization are here shown to be related to both pH-induced changes of location and shape of the liquid-liquid demixing (LLD) region. This allows establishment of a self-consistent contact between the thermodynamics of the solution as such (i.e., the LLD region), the kinetics of fiber nucleation, the theory of percolation, and the thermodynamics of gelation. The observed pH-induced changes are largely attributable to strong perturbations of hydrophobic hydration configurations and related free energy by electric charges. Similar mechanisms of effective control of aggregate nucleation rates by means of agents such as cosolutes, pH, salts, and additives, shifting the LLD and associated regions of anomalous fluctuations, promise to be relevant to the whole field of protein aggregation pathologies.     

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.     

Potential of isoquercitrin as antisickling agent: a multi-spectroscopic,thermophoresis and molecular modeling approach

Abstract

Sickle cell disease is an inherited disease caused by point mutation in hemoglobin (β-globin gene). Under oxygen saturation, sickle hemoglobin form polymers, leading to rigid erythrocytes. The transition of the blood vessels is altered and initiated by the adhesion of erythrocytes, neutrophils and endothelial cells. Sickle Hemoglobin (HbS) polymerization is a major cause in red blood cells (RBC), promoting sickling and destruction of RBCs. Isoquercitrin, a medicinal bioactive compound found in various medicinal plants, has multiple health benefits. The present study examines the potential of isoquercitrin as an anti-sickle agent, showing a significant decrease in the rate of polymerization as well as sickling of RBCs. Isoquercitrin-induced graded alteration in absorbance and fluorescence of HbS, confirmed their interaction. A negative value of ΔG° strongly suggests that it is a spontaneous exothermic reaction induced by entropy. Negative ΔH° and positive ΔS° predicted that hydrogen and hydrophobic binding forces interfered with a hydrophobic microenvironment of β6Val leading to polymerization inhibition of HbS. HbS-Isoquercitrin complex exhibits helical structural changes leading to destabilization of the HbS polymer as confirmed by CD spectroscopy. MST and DSC results indicate greater changes in thermophoretic mobility and thermal stability of sickle hemoglobin in the presence of isoquercitrin, respectively. These findings were also supported by molecular simulation studies using DOCK6 and GROMACS. Hence, we can conclude that isoquercitrin interacts with HbS through hydrogen bonding, which leads to polymerization inhibition. Consequently, isoquercitrin could potentially be used as a medication for the treatment of sickle cell disease.

Communicated by Ramaswamy H. Sarma     

The entropically favored osmotic "compression" of sickle cell hemoglobin gels

Contrary to the accurate, hard-sphere depiction of monomeric hemoglobin in solution, sickle cell hemoglobin (HbS) polymerization/gelation requires attention to molecular interactions. From the temperature dependence of the osmotic compressibility of HbS gels, we were able to extract the entropy increase for concentrating HbS in this phase. Normalized per mole of water removed, the entropy increase from gel compression DeltaS(gel) is four times the previously measured DeltaS(trans), for the transition from monomeric HbS solution to HbS gel. The positive entropy change cannot emerge from the assembly of hard spheres but can indicate remodeling of HbS fibers driven by release of ordered water. The fourfold difference in DeltaS(gel) and DeltaS(trans) suggests that the act of initial fiber/gel formation from monomeric solution differs from the process of further polymerization due to tighter packing within the gel phase.     

Molecular crowding limits the role of fetal hemoglobin in therapy for sickle cell disease

The dominant assumption central to most treatments for sickle cell anemia has been that replacement of sickle hemoglobin (HbS) by fetal hemoglobin (HbF) would have major clinical benefit. Using laser photolysis, we have measured polymerization kinetics including rates of homogeneous and heterogeneous nucleation on mixtures of 20% and 30% HbF with HbS. We find that the present model for polymerization, including molecular crowding, can accurately predict the rates of such mixtures, by using the single assumption that no significant amount of HbF enters the polymer. The effects of replacing HbS by HbF on the rates of polymer formation are found to be significantly lower than previous measurements appeared to indicate because the impact of the replacement is also highly dependent on the total hemoglobin concentration. This is because the molecular crowding of non-polymerizing HbF offsets substantially the effects of decreasing the concentration of HbS concentration, an effect that increases with concentration. Most strikingly, the demonstrated benefit of hydroxyurea therapy in slowing the kinetics of intracellular polymerization cannot be primarily due to enhanced HbF, but must have some other origin, which could itself represent a promising therapeutic approach.     

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.     

Effects of iron nitrosylation on sickle cell hemoglobin solubility

One mechanism by which nitric oxide (NO) has been proposed to benefit patients with sickle cell disease is by reducing intracellular polymerization of sickle hemoglobin (HbS). In this study we have examined the ability of nitric oxide to inhibit polymerization by measuring the solubilizing effect of iron nitrosyl sickle hemoglobin (HbS-NO). Electron paramagnetic resonance spectroscopy was used to confirm that, as found in vivo, the primary type of NO ligation produced in our partially saturated NO samples is pentacoordinate alpha-nitrosyl. Linear dichroism spectroscopy and delay time measurements were used to confirm polymerization. Based on sedimentation studies we found that, although fully ligated (100% tetranitrosyl) HbS is very soluble, the physiologically relevant, partially ligated species do not provide a significant solubilizing effect. The average solubilizing effect of 26% NO saturation was 0.045; much less than the 0.15 calculated for the effect of 26% oxygen saturation. Given the small amounts of NO-ligated hemoglobin achievable through any kind of NO therapy, we conclude that NO therapy does not benefit patients through any direct solubilizing effect.     

O2 dependence of K+ transport in sickle cells: the effect of different cell populations and the substituted benzaldehyde 12C79.

The molecular basis of sickle cell disease (SCD) is well known but the pathophysiology is poorly understood. It remains intractable to therapy. Hyperactivity of several membrane transport systems, including the K+-Cl- cotransporter (termed KCC), cause HbS-containing red cells (termed HbS cells) to dehydrate and sickle, leading to the development of sickle cell crises (SCCs). Contrary to normal red cells (HbA cells), KCC in HbS cells is active at low O2 tensions (PO2s), remaining responsive to low pH or urea. Since these stimuli are usually encountered in hypoxic regions, the abnormal O2 dependence increases the contribution of KCC to dehydration, and hence development of SCCs. These differences with HbA cells may be due to the younger population of cells or to polymerization of HbS. We used 86Rb+ as a K+ congener to investigate the activity of KCC at different PO2s, and density gradient separation to investigate different red cell fractions. We found no correlation of O2 dependence with cell fractions. We also used the substituted benzaldehyde 12C79 to increase the O2 affinity of HbS and found that its effect on HbS O2 saturation and cell sickling correlated with that on both Cl--independent and Cl--dependent K+ transport, implying that, at low PO2s, KCC activity correlated with HbS polymerization. The importance of these results to understanding the pathophysiology of SCD, and for the design of chemotherapeutic agents to ameliorate or prevent SCC, is discussed.     

Metastable polymerization of sickle hemoglobin in droplets

Sickle cell disease arises from a genetic mutation of one amino acid in each of the two hemoglobin beta chains, leading to the polymerization of hemoglobin in the red cell upon deoxygenation, and is characterized by vascular crises and tissue damage due to the obstruction of small vessels by sickled cells. It has been an untested assumption that, in red cells that sickle, the growing polymer mass would consume monomers until the thermodynamically well-described monomer solubility was reached. By photolysing droplets of sickle hemoglobin suspended in oil we find that polymerization does not exhaust the available store of monomers, but stops prematurely, leaving the solutions in a supersaturated, metastable state typically 20% above solubility at 37 degrees C, though the particular values depend on the details of the experiment. We propose that polymer growth stops because the growing ends reach the droplet edge, whereas new polymer formation is thwarted by long nucleation times, since the concentration of hemoglobin is lowered by depletion of monomers into the polymers that have formed. This finding suggests a new aspect to the pathophysiology of sickle cell disease; namely, that cells deoxygenated in the microcirculation are not merely undeformable, but will actively wedge themselves tightly against the walls of the microvasculature by a ratchet-like mechanism driven by the supersaturated solution.     

Influence of semisynthetic modification of the scaffold of a contact domain of HbS on polymerization: role of flexible surface topology in polymerization inhibition

A new variant of HbS, HbS-Einstein with a deletion of segment α_23–26 in the B-helix, has been assembled by semisynthetic approach. B-helix of the α chain of cis αβ-dimer of HbS plays dominant role in the quinary interactions of deoxy HbS dimer. This B-helix is the primary scaffold that provides the orientation for the side chains of contact residues of this intermolecular contact domain. The design of HbS-Einstein has been undertaken to map the influence of perturbation of molecular surface topology and the flexibility of surface residues in the polymerization. The internal deletion exerts a strong inhibitory influence on Val-6 (β)-dependent polymerization, comparable to single contact site mutations and not for complete neutralization of Val-6(β)-dependent polymerization. The scaffold modification in cis-dimer is inhibitory, and is without any effect when present on the trans dimer. The flexibility changes in the surface topology in the region of scaffold modification apparently counteracts the intrinsic polymerization potential of the molecule. The inhibition is close to that of Le Lamentin mutation [His-20 (α) → Gln] wherein a mutation engineered without much change in flexibility of the contact domain. Interestingly, the chimeric HbS with swine–human chimeric α chain with multiple non-conservative mutations completely inhibits the Val-6(β)-dependent polymerization. The deformabilities of surface topology of chimeric HbS are comparable to HbS in spite of the multiple contact site mutations in the α-chain. We conclude that the design of antisickling Hbs for gene therapy of sickle cell disease should involve multiple mutations of intermolecular contact sites.     

A recombinant human hemoglobin with anti-sickling properties greater than fetal hemoglobin

A new recombinant, human anti-sickling beta-globin polypeptide designated beta(AS3) (betaGly(16) --> Asp/betaGlu(22) --> Ala/betaThr(87) --> Gln) was designed to increase affinity for alpha-globin. The amino acid substitutions at beta22 and beta87 are located at axial and lateral contacts of the sickle hemoglobin (HbS) polymers and strongly inhibit deoxy-HbS polymerization. The beta16 substitution confers the recombinant beta-globin subunit (beta(AS3)) with a competitive advantage over beta(S) for interaction with the alpha-globin polypeptide. Transgenic mouse lines that synthesize high levels of HbAS3 (alpha(2)beta(AS3)(2)) were established, and recombinant HbAS3 was purified from hemolysates and then characterized. HbAS3 binds oxygen cooperatively and has an oxygen affinity that is comparable with fetal hemoglobin. Delay time experiments demonstrate that HbAS3 is a potent inhibitor of HbS polymerization. Subunit competition studies confirm that beta(AS3) has a distinct advantage over beta(S) for dimerization with alpha-globin. When equal amounts of beta(S)- and beta(AS3)-globin monomers compete for limiting alpha-globin chains up to 82% of the tetramers formed is HbAS3. Knock-out transgenic mice that express exclusively human HbAS3 were produced. When these mice were bred with knock-out transgenic sickle mice the beta(AS3) polypeptides corrected all hematological parameters and organ pathology associated with the disease. Expression of beta(AS3)-globin should effectively lower the concentration of HbS in erythrocytes of patients with sickle cell disease, especially in the 30% percent of these individuals who coinherit alpha-thalassemia. Therefore, constructs expressing the beta(AS3)-globin gene may be suitable for future clinical trials for sickle cell disease.     

Evidence for carbon monoxide binding to sickle cell polymers during melting.

The melting of sickle cell hemoglobin (HbS) polymers was induced by rapid dilution using a stopped-flow apparatus. The kinetics of polymer melting were monitored using light scattering. Polymer melting in the absence of any hemoglobin ligand was compared to melting when the diluting buffer was saturated with carbon monoxide (CO). In this way the role of CO in polymer melting could be assessed. The data were analyzed using models that assumed that melting occurs only at the ends of polymers. It was further assumed that CO could only bind to HbS in the solution phase. However, our data could not be fitted to this model, where CO cannot bind directly to the polymer. Thus, CO probably binds directly to the polymers during our melting experiments. This result is discussed in terms of oxygen induced polymer melting and polymerization processes in sickle cell disease     

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.     

A role for the alpha 113 (GH1) amino acid residue in the polymerization of sickle hemoglobin. Evaluation of its inhibitory strength and interaction linkage with two fiber contact sites (alpha 16/23) located in the AB region of the alpha-chain

A cluster of amino acid residues located in the AB-GH region of the alpha-chain are shown in intra-double strand axial interactions of the hemoglobin S (HbS) polymer. However, alphaLeu-113 (GH1) located in the periphery is not implicated in any interactions by either crystal structure or models of the fiber, and its role in HbS polymerization has not been explored by solution experiments. We have constructed HbS Twin Peaks (betaGlu-6-->Val, alphaLeu-113-->His) to ascertain the hitherto unknown role of the alpha113 site in the polymerization process. The structural and functional behavior of HbS Twin Peaks was comparable with HbS. HbS Twin Peaks polymerized with a slower rate compared with HbS, and its polymer solubility (C(sat)) was found to be about 1.8-fold higher than HbS. To further authenticate the participation of the alpha113 site in the polymerization process as well as to evaluate its relative inhibitory strength, we constructed HbS tetramers in which the alpha113 mutation was coupled individually with two established fiber contact sites (alpha16 and alpha23) located in the AB region of the alpha-chain: HbS(alphaLys-16-->Gln, alphaLeu-113-->His), HbS(alphaGlu-23-->Gln, alphaLeu-113-->His). The single mutants at alpha16/alpha23 sites were also engineered as controls. The C(sat) values of the HbS point mutants involving sites alpha16 or alpha23 were higher than HbS but markedly lower as compared with HbS Twin Peaks. In contrast, C(sat) values of both double mutants were comparable with or higher than that of HbS Twin Peaks. The demonstration of the inhibitory effect of alpha113 mutation alone or in combination with other sites, in quantitative terms, unequivocally establishes a role for this site in HbS gelation. These results have implications for development of a more accurate model of the fiber that could serve as a blueprint for therapeutic intervention.