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.     

Structure of deoxyhemoglobin: ionizable groups and polyethylene glycol

X-ray studies on deoxy-hemoglobin have been reported on crystals grown under conditions of high (about 2.5 M) and low salt (about 0.1 M). The high-salt crystals were grown in water, whereas the low-salt crystals were prepared in polyethylene glycol solutions (m.w. 6000-8000 Da, 10-30% w/v). Oxygen-binding characteristics of hemoglobin in these two environments differ radically. In salt solutions, hemoglobin binds oxygen with a p50 value of about 5 torr of oxygen and in a cooperative manner characterized with an n value of Hill varying from two to three. In polyethylene glycol solutions, hemoglobin crystals are oxygenated with a p50 value of about 270 torr of oxygen, without exhibiting a cooperative effect. I report on a detailed study of the X-ray data defining the dimer interface (alpha1beta2) of these two forms of hemoglobin. The study reveals that the main difference between the two structures lies in the number and arrangement of the water molecules and in distances between ionizable side chains in the dimer interface. I propose that these differences lead to significant shifts in the pK values of the ionizable groups in the dimer interface.     

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)     

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.     

Crystallization and preliminary X-ray crystallographic studies of CO-hemoglobin from the North Atlantic salmon (Salmo salar)

Purification of hemoglobin from North Atlantic salmon (Salmo salar) gave three different types. The CO-complexes of types I and III have been crystallized by the batch method at 4 degrees C from solutions 18% (w/v) in polyethylene glycol 2000, 50 mg/ml in hemoglobin and 0.05 M in phosphate buffer (pH 8.3). Orthorhombic crystals, space group P2(1)2(1)2(1), were obtained for both, with cell dimensions a = 53.9 A, b = 80.4 A, c = 132.4 A, and a = 58.7 A, b = 95.0 A, c = 107.4 A, for types I and III, respectively.     

Water proton magnetic resonance studies of normal and sickle erythrocytes. Temperature and volume dependence.

The temperature and cell volume dependence of the NMR water proton line-width, spin-lattice, and spin-spin relaxation times have been studied for normal and sickle erythrocytes as well as hemoglobin A and hemoglobin S solutions. Upon deoxygenation, the spin-spin relaxation time (T2) decreases by a factor of 2 for sickle cells and hemoglobin S solutions but remains relatively constant for normal cells and hemoglobin A solutions. The spin-lattice relaxation time (T1) shows no significant change upon deoxygenation for normal or sickle packed red cells. Studies of the change in the NMR linewidth, T1 and T2 as the cell hydration is changed indicate that these parameters are affected only slightly by a 10-20% cell dehydration. This result suggests that the reported 10% cell dehydration observed with sickling is not important in the altered NMR properties. Low temperature studies of the linewidth and T1 for oxy and deoxy hemoglobin A and hemoglobin S solutions suggest that the "bound" water possesses similar properties for all four species. The low temperature linewidth ranges from about 250 Hz at -15 degrees C to 500 Hz at -36 degrees C and analysis of the NMR curves yield hydration values near 0.4 g water/g hemoglobin for all four species. The low temperature T1 data go through a minimum at -35 degrees C for measurements at 44.4 MHz and -50 degrees C for measurements at 17.1 MHz and are similar for oxy and deoxy hemoglobin A and hemoglobin S. These similarities in the low temperature NMR data for oxy and deoxy hemoglobin A and hemoglobin S suggest a hydrophobically driven sickling mechanism. The room temperature and low temperature relaxation time data for normal and sickle cells are interpreted in terms of a three-state model for intracellular water. In the context of this model the relaxation time data imply that type III, or irrotationally bound water, is altered during the sickling process.     

Microcrystals of the annexin, p68: paracrystal to crystal transition and molecular packing as determined by electron microscopy and image reconstruction.

The calcium-sensitive, membrane-binding annexin, p68, has been crystallized from solutions of polyethylene glycol and ammonium sulfate. Our electron microscopy and X-ray diffraction data indicate that p68 crystals are tetragonal, in space group P4(1), and have unit cell dimensions of a = b = 68.4 A and c = 209.6 A. The mechanism of crystallization from polyethylene glycol involves a transition from a paracrystalline form to ordered crystals by lateral reordering of chains of molecules extended along the c axis. These chains are directional and might reflect a mechanism whereby the two different ends of (chains of) the p68 molecules interact with different membranes.     

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.     

Water proton magnetic resonance studies of normal and sickle erythrocytes Temperature and volume dependence

The temperature and cell volume dependence of the NMR water proton linewidth, spin-lattice, and spin-spin relaxation times have been studied for normal and sickle erythrocytes as well as hemoglobin A and hemoglobin S solutions. Upon deoxygenation, the spin-spin relaxation time (T₂) decreases by a factor of 2 for sickle cells and hemoglobin S solutions but remains relatively constant for normal cells and hemoglobin A solutions. The spin-lattice relaxation time (T₁) shows no significant change upon dexygenation for normal or sickle packed red cells. Studies of the change in the NMR linewidth, T₁ and T₂ as the cell hydration is changed indicate that these parameters only slightly by a 10–20% cell dehydration. This result suggests that the reported 10% cell dehydration observed with sickling is not important in the altered NMR properties. Low temperature studies of the linewidth and T₁ for oxy and deoxy hemoglobin A and hemoglobin S solutions suggest that the “bound” water possesses similar properties for all four species. The low temperature linewidth ranges from about 250 Hz at ?15°C to 500 Hz at ?36°C and analysis of the NMR curves yield hydration values near 0.4 g water/g hemoglobin for all four species. The low temperature T₁ data go through a minimum at ?35°C for measurements at 44.4 MHz and ?50°C for measurements at 17.1 MHz and are similar for oxy and deoxy hemoglobin A and hemoglobin S. These similarities in the low temperature NMR data for oxy and deoxy hemoglobin A and hemoglobin S suggest a hydrophobically driven sickling mechanism. The room temperature and low temperature relaxation time data for normal and sickle cells are interpreted in terms of a three-state model for intracellular water. In the context of this model the relaxation time data imply that type III, or irratationally bound water, is altered during the sickling process.     

Triclinic crystals associated with fibers of deoxygenated sickle hemoglobin.

Triclinic crystals have been found in capillaries that initially contained deoxygenated sickled erythrocytes, and in solutions of sickle hemoglobin that were stirred during deoxygenation. In both cases these crystals occur as a phase transition from fibers. They have been observed only as twins; the a-axis of one member is related to that of its twin by 180 degrees rotation about the b* direction. The volume of the triclinic crystal unit cell is half that of the monoclinic crystals that have also transformed from fibers. Analysis of X-ray diffraction data indicates that the two molecules in the triclinic unit cell that repeat at an interval of 64 A form double filaments similar to those found in the monoclinic crystals and in the fiber. The existence of the triclinic crystals which contain only one double filament per unit cell removes a postulated requirement that antipolar double filament pairs be the sole unit of the fiber architecture.     

Hemoglobin precipitation by polyethylene glycols leads to underestimation of membrane pore sizes

The size of pores formed in the plasma membrane by various substances is frequently determined using polyethylene glycols as osmotic protectants. In this work, we have found that the size of pores formed by saponin in the red blood cell membrane determined by hemolysis versus molecular weight of polyethylene glycol was different to that estimated by light dispersion of cell suspensions. After complete swelling of cells induced by saponin in semiisotonic salt media containing 150 mOsm PEG-4000 or PEG-3000, a significant increase in the light absorbance at 640 nm was developed resulting from the formation of hemoglobin precipitates. Easily sedimenting aggregates were also formed when the supernatant of lysed cells was added to the equiosmotic solutions of polyethylene glycols with molecular weight higher than 1000. We suggest that the real size of large pores could be underestimated due to the phenomenon of hemoglobin precipitation by polyethylene glycols.     

Crystallographic characterization and three-dimensional model of yeast Cu,Zn superoxide dismutase

The Cu,Zn superoxide dismutase from yeast was crystallized in the orthorhombic space group P21212 with unit cell dimension a = 105.1 A,b = 142.2 A, c = 62.1 A. The crystals grow in 25 mM citrate, 10 mM phosphate buffer pH 6.5, and 6% (W/V) polyethylene glycol, with a Vm of 3,4 A3/dalton, for two dimers/asymmetric unit. The crystals were unstable in the mother liquor, but were stabilized by transfer to a 35% polyethylene glycol solution. This crystalline form diffracts at high resolution and is suitable for determination of the atomic structure. The three dimensional structure of the yeast enzyme could be model-built by computer graphics techniques using the bovine enzyme atomic coordinates as template. The proposed model requires removal of some salt bridges and non equivalence of the metal-binding sites in the subunits, in line with reported functional properties of the yeast enzyme.     

Purification, crystallization and preliminary crystallographic analysis of porcine aldose reductase

Large crystals of porcine aldose reductase have been grown from polyethylene glycol solutions. The crystals are triclinic, space-group P1, with a = 81.3 A, b = 85.9 A, c = 56.6 A, alpha = 102.3 degrees, beta = 103.3 degrees and gamma = 79.0 degrees. The crystals grow within ten days to dimensions of 0.6 mm x 0.4 mm x 0.2 mm and diffract to at least 2.5 A. There are four molecules in the unit cell related by a set of three mutually perpendicular non-crystallographic 2-fold axes.     

Purification, crystallization, and preliminary X-ray data for porcine fumarase

Single crystals of fumarase purified from pig heart have been prepared from solutions containing polyethylene glycol. The crystals give diffraction data corresponding to Bragg spacings of 2.0 A and contain a single subunit of the enzyme in the asymmetric unit of the C222 unit cell. Therefore, the subunits of this tetrameric molecule are arranged with the point symmetry group 222. The present purification scheme and studies of the NH2-terminal amino acid sequences suggest that only a single form of the enzyme is present, and it is thought to be the mitochondrial enzyme.     

The effect of organic compounds on the crystal habit and crystallization of normal and sickle cell hemoglobin in phosphate buffer

Comparative studies were made on the effect of numerous organic compounds in promoting the crystallization of human hemoglobin in 1.9 m phosphate, pH 7.0. It was found that alicyclic or benzenoid structures are essential for promoting crystallization of hemoglobin under these conditions. Hemoglobin crystals prepared in the presence of toluene differed in habit from crystals prepared in its absence. It is suggested that steric factors determine the effectiveness of organic substances in promoting the crystallization of hemoglobin and that the heme group is the binding site involved in the complex formation.The solubility of homozygous sickle cell hemoglobin HbS was found to be less than the heterozygous hemoglobins AS and AC or normal hemoglobin HbA in the presence of organic substances promoting the crystallization of hemoglobin.     

Preliminary X-ray crystallographic study of adenylosuccinate synthetase from Escherichia coli

Adenylosuccinate synthetase, a dimeric enzyme of 96,000 Mr, catalyzes the first committed step toward the de novo biosynthesis of AMP. Large, single crystals of adenylosuccinate synthetase from Escherichia coli grow from solutions of polyethylene glycol and ammonium sulfate. Crystals from ammonium sulfate belong to the orthorhombic space group P212121 with unit cell parameters a = 79.0 A, b = 70.2 A and c = 152.6 A. Crystals from polyethylene glycol belong to the space group P21, having unit cell parameters of a = 71.16 A, b = 71.99 A, c = 82.95 A, and beta = 71.52 degrees. The asymmetric units of both crystal forms probably contain the entire dimeric enzyme.     

Influence of sickle hemoglobin polymerization and membrane properties on deformability of sickle erythrocytes in the microcirculation.

The rheological properties of normal erythrocytes appear to be largely determined by those of the red cell membrane. In sickle cell disease, the intracellular polymerization of sickle hemoglobin upon deoxygenation leads to a marked increase in intracellular viscosity and elastic stiffness as well as having indirect effects on the cell membrane. To estimate the components of abnormal cell rheology due to the polymerization process and that due to the membrane abnormalities, we have developed a simple mathematical model of whole cell deformability in narrow vessels. This model uses hydrodynamic lubrication theory to describe the pulsatile flow in the gap between a cell and the vessel wall. The interior of the cell is modeled as a Voigt viscoelastic solid with parameters for the viscous and elastic moduli, while the membrane is assigned an elastic shear modulus. In response to an oscillatory fluid shear stress, the cell--modeled as a cylinder of constant volume and surface area--undergoes a conical deformation which may be calculated. We use published values of normal and sickle cell membrane elastic modulus and of sickle hemoglobin viscous and elastic moduli as a function of oxygen saturation, to estimate normalized tip displacement, d/ho, and relative hydrodynamic resistance, Rr, as a function of polymer fraction of hemoglobin for sickle erythrocytes. These results show the transition from membrane to internal polymer dominance of deformability as oxygen saturation is lowered. More detailed experimental data, including those at other oscillatory frequencies and for cells with higher concentrations of hemoglobin S, are needed to apply fully this approach to understanding the deformability of sickle erythrocytes in the microcirculation. The model should be useful for reconciling the vast and disparate sets of data available on the abnormal properties of sickle cell hemoglobin and sickle erythrocyte membranes, the two main factors that lead to pathology in patients with this disease.     

Preliminary crystallographic studies on duck ovotransferrin

Crystals of duck ovotransferrin and duck apo-ovotransferrin have been grown from polyethylene glycol solutions. For both crystals, the space group is P2(1)2(1)2(1), the unit cell dimensions for the ovotransferrin are a = 49.6 A, b = 85.6 A, c = 178.7 A and for the apo-ovotransferrin a = 77.6 A, b = 98.8 A, c = 127.0 A, giving four molecules in the unit cell.     

Crystallization of rat intestinal fatty acid binding protein. Preliminary X-ray data obtained from protein expressed in Escherichia coli

Rat intestinal fatty acid binding protein has been expressed in Escherichia coli, purified with bound long chain fatty acids and crystals grown from solutions of polyethylene glycol 4000. The crystals are monoclinic, space group P2(1), a = 3638 A, b = 57.2 A, c = 31.9 A, and beta = 113.9 degrees. Each unit cell contains two monomers of this 132-residue, 15.1-kDa polypeptide. The crystals are remarkably resistant to x-ray damage. X-ray diffraction data have been observed to 2.0 A resolution. Platinum chloride was used to generate a potential isomorphous heavy atom derivative.     

Oxygen equilibrium of emulsified solutions of normal and sickle hemoglobin.

Emulsions containing microdroplets of concentrated solutions of normal or sickle hemoglobin dispersed in a continous oil phase have been prepared, and the aggregation of sickle hemoglobin within microdroplets in a deoxygenated emulsion demonstrated. The equilibrium oxygen saturation of hemoglobin in the emulsions has been measured as a function of the partial pressure of oxygen by a novel spectrophotometric technique which corrects for the scattering of light in the emulsion. The half-saturation oxygen pressure and cooperativity of oxygen binding are substantially greater in concentrated (27 g/dl) solutions of sickle hemoglobin than in solutions of non-aggregating hemoglobin. The shape of the oxygen equilibrium curve of concentrated sickle hemoglobin is qualitatively discussed in terms of a previously proposed model (1).