Structural analysis of polymers of sickle cell hemoglobin. III. Fibers within fascicles

We have examined the structure of hemoglobin S fibers, which are associated into large bundles, or fascicles. Electron micrographs of embedded and cross-sectioned fascicles provide an end-on view of the component fibers. The cross-sectional images are rotationally blurred as a result of the twist of the fiber within the finite thickness of the section. We have applied restoration techniques to recover a deblurred image of the fiber. The first step in this procedure involved correlation averaging images of cross-sections of individual fibers in order to improve the signal-to-noise ratio. The rotationally blurred image was then geometrically transformed to polar co-ordinates. In this space, the rotational blur is transformed into a linear blur. The linearly blurred image is the convolution of the unblurred image and a point spread function that can be closely approximated by a square pulse. Deconvolution in Fourier space, followed by remapping to Cartesian co-ordinates, produced a deblurred image of the original micrograph. The deblurred images indicate that the fiber is comprised of 14 strands of hemoglobin S. This result provides confirmation of the fiber structure determined using helical reconstruction techniques and indicates that the association of fibers into ordered arrays does not alter their molecular structure.     

Two-step mechanism of homogeneous nucleation of sickle cell hemoglobin polymers

Sickle cell anemia is a debilitating genetic disease that affects hundreds of thousands of babies born each year worldwide. Its primary pathogenic event is the polymerization of a mutant, sickle cell, hemoglobin (HbS); and this is one of a line of diseases (Alzheimer's, Huntington's, prion, etc.) in which nucleation initiates pathophysiology. We show that the homogeneous nucleation of HbS polymers follows a two-step mechanism with metastable dense liquid clusters serving as precursor to the ordered nuclei of the HbS polymer. The evidence comes from data on the rates of fiber nucleation and growth and nucleation delay times, the interaction of fibers with polarized light, and mesoscopic metastable HbS clusters in solution. The presence of a precursor in the HbS nucleation mechanism potentially allows low-concentration solution components to strongly affect the nucleation kinetics. The variations of these concentrations in patients might account for the high variability of the disease in genetically identical patients. In addition, these components can potentially be utilized for control of HbS polymerization and treatment of the disease.     

The growth of sickle hemoglobin polymers

The measurement of polymer growth is an essential element in characterization of assembly. We have developed a precise method of measuring the growth of sickle hemoglobin polymers by observing the time required for polymers to traverse a photolytically produced channel between a region in which polymers are created and a detection region. The presence of the polymer is functionally detected by observing its ability to create new polymers through the well-established process of heterogeneous nucleation. Using this method, we have determined the rate constants for monomer addition to and release from polymer ends, as well as their temperature dependences. At 25°C we find k₊ = 84 ± 2 mM⁻¹ s⁻¹ and k₋ = 790 ± 80 molecules/s from each end. These numbers are in accord with differential interference contrast measurements, and their ratio gives a solubility measured on individual fibers. The single-fiber solubility agrees with that measured in sedimentation experiments. The concentration dependence of the monomer addition rate is consistent with monomer addition, but not oligomer addition, to growing polymers. The concentration dependence suggests the presence of an activation enthalpy barrier, and the rate of monomer addition is not diffusion-limited. Analysis of the temperature dependence of the monomer addition rate reveals an apparent activation energy of 9.1 ± 0.6 kcal/mol.     

Dielectric constant of sickle cell hemoglobin. Dielectric properties of sickle cell hemoglobin in solution and gel

The dielectric constants of sickle cell hemoglobin were determined before and after gelation. The dielectric properties of oxy and deoxy sickle cell hemoglobin in solution are nearly identical to those of oxy and deoxy hemoglobin A. Only in the gel state did deoxy sickle cell hemoglobin display dielectric behavior different from that in solution. Upon gelation of deoxy sickle cell hemoglobin, the dielectric constant showed a marked decrease, and the relaxation frequency shifted towards higher frequencies. This result suggests that dielectric constant measurement can be used for the investigation of the kinetics of polymerization of sickle cell hemoglobin molecules. Despite the marked decrease in the dielectric constant, deoxy sickle cell hemoglobin still showed a well-defined dielectric dispersion even in the gel state. This indicates that individual molecules have considerable freedom of rotation in gels. It was observed that the dielectric properties of gelled deoxy sickle cell hemoglobin were affected by electrical fields at the level of 10 to 20 V/cm. This observation suggests that electrical fields of moderate strengths are able to perturb the gel structure if the system is near the transition region. The non-linear electrical behavior of gelled sickle cell hemoglobin will be discussed further in subsequent papers.     

Oxygen binding to sickle cell hemoglobin.

The extent of oxygen binding and light scattering of concentrated solutions of hemoglobin S have been determined as a function of oxygen partial pressure using a thin film optical cell. Nearly reversible oxygen binding is observed as witnessed by the small hysteresis found between slow deoxygenation and reoxygenation runs. High co-operativity is noted from unusually large concentration-dependent Hill coefficients when aggregated hemoglobin S is present. The application of linkage theory with the inclusion of non-ideal solution properties permits a test of various simple models for oxygen binding to both the monomer (α₂β₂^s) and polymer (aggregated) phase. It is concluded that oxygen binding to the polymer is either negligible or small under present experimental conditions. Phase diagrams of the solution concentration in equilibrium with polymer phase as a function of oxygen partial pressure are derived using best fit values of polymer parameters.     

Structural basis and dynamics of the fiber-to-crystal transition of sickle cell hemoglobin

The kinetics of the assembly of structurally distinct, polymeric aggregates constituting the fiber-to-crystal transition of sickle cell hemoglobin in slowly stirred, deoxygenated solutions has been studied with the use of electron microscopy as a function of pH, as a function of the crystal structures of mutant forms of human deoxyhemoglobins employed as nucleating seeds, and as a function of hemoglobin S chemically modified at the Cys F9 (beta 93) position. The temporal order of appearance of fibers of approximately 210 A diameter, bundles of aligned fibers, macrofibers of greater than or equal to 650 A diameter, and microcrystals is observed. Microscopic fragments of end-stage crystals formed under slowly stirred conditions and introduced as nucleating seeds enhance the rate of crystallization only when added prior to the formation of large bundles of aligned fibers, while microscopic seed crystals added after the formation of bundles of aligned fibers do not alter the rate of crystallization. Over the pH range 6.3 to 7.1, the presence of macrofibers does not influence modulation of the kinetics of the transition with seed crystal fragments. Microscopic seed crystals of deoxyhemoglobin S and deoxyhemoglobin C formed under acidic conditions (pH less than 6.5) have a comparable influence on the kinetics of the fiber-to-crystal transition to that of end-stage crystals. Microscopic seed crystals of deoxyhemoglobin C formed under alkaline conditions (pH greater than 6.5) enhance the formation of macrofibers but do not alter the rate of crystallization. Under conditions associated with enhanced formation of macrofibers, metastable microscopic crystals having axial periodicities of approximately 64 A and approximately 210 A are observed in the intermediate phase of the transition, while end-stage crystals have axial unit cell dimensions identical to those of deoxyhemoglobin S crystallized from polyethylene glycol solutions of pH less than 6.5. Although the metastable crystals may arise from fragments of macrofibers, it is shown that they cannot be transformed directly into end-stage crystals under slowly stirred conditions without undergoing dissolution. These results stipulate that the pathway of the fiber-to-crystal transition proceeds according to the reaction: (Formula: see text) wherein the rate-limiting step is the alignment of fibers into large bundles, and macrofibers are not an intermediate of the fiber-to-crystal transition.(ABSTRACT TRUNCATED AT 400 WORDS)     

X-ray diffraction studies of 14-filament models of deoxygenated sickle cell hemoglobin fibers. II. Models based on the deoxygenated sickle hemoglobin crystal structure

The calculated transforms of a number of crystal-based models of the deoxygenated sickle cell hemoglobin fiber have been compared with X-ray diffraction data of 15 A (1 A = 0.1 nm) resolution. The fiber models consist of 14 single strands of sickle cell hemoglobin (HbS) molecules, which associate into seven protofilaments arranged similarly to those present in the crystal structure. Six of the protofilaments are arranged in three crystallographic until cells extending in the c-axis direction with the seventh protofilament positioned so as to provide an elliptical cross-section when the assemblage is viewed down the fiber axis. Models were generated by systematically and independently translating each of the model's three subcells in steps of 3.5 A along the fiber axis. The seventh protofilament was kept fixed as a point of reference. Each translation of a subcell corresponded to a different fiber model whose transform was then compared with observed data. In all, over 46,000 transforms were computed; of these, three models with minimal residuals were identified. The free energy of packing for all crystal-based models was evaluated to find configurations of protofilaments possessing minimal free energies. The results of the calculations support the subcell configurations of two of the three models with minimal residuals.     

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.     

Helical crystals of sickle cell hemoglobin

Thin ribbon-like crystals are intermediates in the formation of large crystals of deoxyhemoglobin S from many individual fibers. The thin crystals show foldedover regions when observed by electron microscopy. Some crystals are sufficiently long to have several folds each separated by a distance of about 4.4 μm, suggesting that the crystals are helical in solution. The thickness of the crystals varies from 500 to 900 Å as shown by heavy-metal shadowing and by measurements of the thickness at the crossover point where an edge-on view of the crystal is obtained.     

Kinetics of sickle hemoglobin polymerization. II. A double nucleation mechanism

A double nucleation mechanism for the polymerization of sickle hemoglobin is described. The mechanism accounts for all of the major kinetic observations: the appearance of a delay, the high concentration dependence of the delay time, and the stochastic behavior of slowly polymerizing samples in small volumes. The mechanism postulates that there are two pathways for polymer formation: polymerization is initiated by homogeneous nucleation in the solution phase, followed by nucleation of additional polymers on the surface of existing ones. This second pathway is called heterogeneous nucleation. Since the surface of polymers is continuously increasing with time, heterogeneous nucleation provides a mechanism for the extreme autocatalysis that is manifested as an apparent delay in the kinetic progress curves. In this mechanism, each spherulitic domain of polymers is considered to be initiated by a single homogeneous nucleation event. The mechanism explains the irreproducibility of the delay time for single domain formation as arising from stochastic fluctuations in the time at which the homogeneous nucleus for the first polymer is formed. Integration of the linearized rate equations that describe this model results in a simple kinetic form: A[cosh(Bt)-1] (Bishop & Ferrone, 1984). In the accompanying paper (Ferrone et al., 1985) it was shown that the initial 10 to 15% of progress curves, with delay times varying from a few milliseconds to over 10(5) seconds, is well fit by this equation. In this paper, we present an approximate statistical thermodynamic treatment of the equilibrium nucleation processes that shows how the nucleus sizes and nucleation equilibrium constants depend on monomer concentration. The equilibrium model results in expressions for B and B2A as a function of monomer concentration in terms of five adjustable parameters: the bimolecular addition rate of a monomer to the growing aggregate, the fraction of polymerized monomers that serve as heterogeneous nucleation sites, the free energy of intermolecular bonding within the polymer, and two parameters that describe the free energy change as a function of size for the bonding of the heterogeneous nucleus to a polymer surface. This model provides an excellent fit to the data for B and B2A as a function of concentration using physically reasonable parameters. The model also correctly predicts the time regime in which stochastic behavior is observed for polymerization in small volumes.     

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.     

Calorimetric and optical characterization of sickle cell hemoglobin gelation.

We have used two techniques to characterize the gelation of deoxyhemoglobin S, a high sensitivity heat-flow calorimeter to measure the heat of gelation and a simple light-transmission method to measure the optical birefringence resulting from the alignment of deoxyhemoglobin S fibers in the gel. A theory for the interpretation of the birefringence measurements is presented. We combine the results of the calorimetric and optical measurements with those of sedimentation experiments to obtain enthalpy changes for gelation. The enthalpy change obtained from scanning and isothermal calorimetric measurements (0.25 m-potassium phosphate, 0.05 m-sodium dithionite, pH 6.9) varies from 4000 to 2200 cal mol⁻¹ hemoglobin between 16 and 25 °C. There is a large apparent heat capacity change of −130 to −190 cal deg.⁻¹ mol⁻¹. The apparent enthalpy change estimated from solubility measurements and birefringence melting experiments is 2200 ± 500 cal mol⁻¹ in qualitative agreement with the calorimetric results. Analysis of the time dependence of the calorimetric and optical progress curves at 20 °C leads to a rough estimate of 1800 to 4000 and −800 to 1500 cal mol⁻¹ hemoglobin for the enthalpies of polymerization and alignment of fibers, respectively. The small magnitude of the observed enthalpy change is in accord with the view that no large conformational change takes place in the deoxyhemoglobin S molecule upon gelation.     

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.     

Gelation of sickle hemoglobin. III. Nitrosyl hemoglobin.

Sickle cell nitrosyl hemoglobin was examined for gelation by an ultracentrifugal method previously described (Briehl &; Ewert, 1973) and by birefringence. In the presence of inositol hexaphosphate gelation which exhibited the endothermic temperature dependence seen in gels of deoxyhemoglobin S was observed by both techniques. In the absence of inositol hexaphosphate no gelation was observed, nor did nitrosyl hemoglobin A exhibit gelation. On the assumption that gelation is dependent on the deoxy or T (low ligand affinity) as opposed to the oxy or R (high ligand affinity) quaternary structure this supports the conclusion that nitrosyl hemoglobin S in inositol hexaphosphate assumes the T structure, in contrast to the other liganded ferrohemoglobin derivatives oxy and carbon monoxide hemoglobin. Assuming further that the quaternary structures and isomerizations are the same in hemoglobins A and S it can also be concluded that nitrosyl hemoglobin A in inositol hexaphosphate assumes the T state. Since no gelation was seen in stripped nitrosyl hemoglobin S, inositol hexaphosphate serves to effect an R to T switch in this derivative. Thus R-T isomerization in nitrosyl hemoglobin occurs without change in ligand binding at the sixth position of the heme group confirming the conclusion of Salhany (1974) and Salhany et al. (1974).Lowering of the pH toward 6 favors gelation of NO hemoglobin S as it does of deoxy and aquomethemoglobin S (Briehl &; Ewert, 1973,1974), consistent with a favoring of the T structure due to strengthening of the interchain salt bridges and the binding of inositol hexaphosphate and/or changes in site-to-site interactions on which gelation depends.     

Beta-sheet aggregation proposed in sickle cell hemoglobin

A β-sheet conformation is predicted at the N-terminal of β chains in sickle cell hemoglobin (Hb S) as a result of the β6 Glu → Val mutation. Since Glu is the weakest and Val is the strongest β-sheet former in the predictive method of Chou and Fasman [Biochemistry $\text{13}$, 211, 222 (1974)], such a substitution greatly increases the β-sheet potential in the β 1–6 region. The similarity in the concentration and temperature dependence of Hb S gelation to β-sheet formation in polyamino acids suggest that a common aggregation mechanism may be involved. Conditions to cause a β → α trans-formation at the β 1–6 region of Hb S is discussed relative to the treatment of sickle cell disease.     

High-resolution proton nuclear magnetic resonance studies of sickle cell hemoglobin.

High-resoluiton proton nuclear magnetic resonance spectroscopy at 250 MHz has been used to investigate sickle cell hemoglobin. The hyperfine shifted, the ring-current shifted, and the exchangeable proton resonances suggest that the heme environment and the subunit interfaces of the sickle cell hemoglobin molecule are normal. These results suggest that the low oxygen affinity in sickle cell blood is not due to conformational alterations in the heme environment or the subunit interfaces. The C-2 proton resonances of certain histidyl residues can serve as structural probes for the surface conformation of the hemoglobin molecule. Several sharp resonances in sickle cell hemoglobin are shifted upfield from their positions in normal adult hemoglobin. These upfield shifts, which are observed in both oxy and deoxy forms of the molecule under various experimental conditions, suggest that some of the surface residues of sickle cell hemoglobin are altered and they may be in a more hydrophobic environment as compared with that of normal human adult hemoglobin. These differences in surface conformation are pH and ionic strength specific. In particular, upon the addition of organic phosphates to normal and sickle cell hemoglobin samples, the differences in their aromatic proton resonances diminish. These changes in the surface conformation may, in part, be responsible for the abnormal properties of sickle cell hemoglobin.     

Interpretation of the osmotic behavior of sickle cell hemoglobin solutions: Different interactions among monomers and polymers

It has long been known that a simple hard particle model quantitatively explains the osmotic properties of monomeric hemoglobin near its isoelectric point. However, we find that a hard particle model is not consistent with the osmotic properties of polymerized hemoglobin and that substantial soft repulsions are indicated. With allowance for different interactions among monomers and among polymers, a self-consistent quantitative fit to the experimental data is obtained. The results suggest that the decreasing “solubility” of deoxy sickle cell hemoglobin with increasing temperature from 20 to 37°C is due to weaker repulsions between polymers at higher temperatures rather than stronger polymerization. The temperature dependence of these variables indicates that the aggregation of monomers is enthalpically and entropically driven (the latter effect being stronger), while the approach of polymers toward each other is enthalpically disfavored and entropically favored (with the former dominating). In both cases, the entropic contribution suggests that water is released. © 1998 John Wiley & Sons, Inc. Biopoly 45: 299–306, 1998