Polymerization and Phase Transitions in Deoxy Sickle Cell Hemoglobin

Chapter 16

Polymerization and Phase Transitions in Deoxy Sickle Cell Hemoglobin

Downloaded by NORTH CAROLINA STATE UNIV on October 12, 2012 | http://pubs.acs.org Publication Date: June 10, 1992 | doi: 10.1021/bk-1992-0493.ch016

Muriel S. Prouty Department of Chemistry, University of the District of Columbia, Washington, DC 20008

The polymerization and gelation of deoxygenated sickle cell hemoglobin (HbS) is reviewed here as a classic example of biopolymer self assembly. Under near physiological conditions deoxy HbS polymerizes into multi-stranded fibers, accompanied by an almost simultaneous liquid-to-gel phase transition; and by rheological changes which, in the red blood cell (RBC), form the pathophysiological basis of sickle cell disease. The kinetics and mechanism of polymerization and the gel fiber structure have been described in detail; less is known about alignment and compression of the polymers in concentrated systems. Using the osmotic stress method of Parsegian-Rand, we measured the thermodynamic parameters of liquid-to-gel transitions above solubility in near physiological conditions, and of liquid-to-crystal transitions at higher ionic strengths. The nature of the transition may influence gelation inhibitor testing.

Sickle cell anemia is a genetic disease, but its molecular mechanism and effects are best described in the languages of physical chemistry and of polymer chemistry. Except for genetic therapy, all current therapeutic approaches are based on modifying the physico-chemical behavior of the deoxy sickle cell hemoglobin (HbS) protein molecule, or of its polymeric form; even one of the most promising of the genetic therapies seeks to take advantage of the high degree of non-ideality in concentrated polymer solutions (see "Approaches to Therapy", below). The extensive and sophisticated understanding of the role of the sickle cell hemoglobin molecule in the pathophysiology of sickle cell disease which has been attained in the last fifteen years, is based primarily on physicochemical studies on the macromolecular level. The molecular basis of sickle cell disease is extensively reviewed by Schechter et al. (1). Hofrichter and Eaton have

0097-6156/92/0493-0184$06.00/0 © 1992 American Chemical Society

In Macromolecular Assemblies in Polymeric Systems; Stroeve, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.


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reviewed the physical chemistry of the sickle cell hemoglobin polymer, and its formation and structure (2). O n the whole, the biophysicists (theoreticians and physical chemists) who have worked with biochemists and physicians in this field, have not been polymer chemists; nor has this research been published in journals generally followed by polymer scientists. The symposium on Macromolecular Assembly and this issue of A C S Symposium Proceedings present an opportunity to reaffirm the important commonalities among diverging specialties. We review briefly the molecular basis of sickle cell disease (SCD) and its symptoms, the kinetics and mechanism of polymerization and gelation of the deoxy HbS molecule, the fiber structure and behavior of the gel formed on polymerization, the rheology of the red blood cell (RBC), and the physicochemical basis of therapies, with emphasis on those aspects which most reflect polymer physical chemistry. We describe the "osmotic stress" method (3) for studying the energetics of macromolecular assembly and structure and its use in lipid bilayers, D N A double helices and other systems. In protein solutions we have used this approach to study the thermodynamics of phase transitions, to measure the work of concentrating protein gels, and to bring about crystallization under controlled conditions. We present results of experiments in which we have measured, by osmotic stress, the work of polymerization and gelation of deoxy HbS, and of aligning and compressing the resultant fibers as solvent is removed (4). Recently, we have tested the effect of increasing ionic strength of the phosphate buffer by using 0.5M and 1.0M buffers, and the effect of different anions on the nature of the condensed phase formed when deoxy HbS polymerizes (5). These results have implications for the testing of potential "gelation inhibitors" for their usefulness in the development of drug therapies for sickle cell disease. Sickle Cell Disease The normal red blood cell is a biconcave disc with an extremely flexible membrane; the R B C can squeeze through the capillaries (microvasculature) by thinning out and elongating. The presence of polymerized deoxy HbS causes increased rigidity in the red cell, and changes in rheology which (even in the absence of the morphological changes which gave the disease its name) are responsible, more or less directly, for the wide variety and severity of the symptoms of sickle cell disease. A "crisis" occurs when blood circulation is blocked by accumulation at the capillaries of sickled cells which are more rigid because of the presence of gels of deoxy HbS fibers; these cells are thus not flexible enough to squeeze through the microvasculature, as do normal cells. Blood vessels become blocked, tissues become deprived of oxygen, causing extreme pain in the extremities and joints. Other symptoms may include susceptibility to infection, diminished growth rate in children, anemia (sickle cells have about half the life span of "normal" R B C ) , damage to the spleen and other organs, and shortened life expectancy. Damage to the cell membrane including that caused by oxidative degradation of HbS, imbalance in R B C electrolytes, and other effects (2) have been implicated as causes of S C D pathology.


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Hemoglobin Structure and Polymerization The hemoglobin molecule as visualized in Figure 1 (roughly spherical ~ diameter about 64 A ) is composed of four polypeptide chains (for "normal" adult hemoglobin (HbA), two a, two β) packed in a tetrahedral array. Each chain is coiled compactly in a specific structure and contains a heme group located in a crevice near the exterior; each heme contains an iron atom ( F e r ion) which can bind an oxygen molecule. The difference between H b A and sickle cell hemoglobin (HbS) involves a change in a single base in the gene for the hemoglobin β-chain, which, in turn, causes a change in a single amino acid residue (β-6 L y s - > Val, with a charge change of + l - > 0 ) . In deoxy HbS, the uncharged valine side chain acts as a "sticky spot" on the surface. In the process of giving up oxygen in the tissues, the H b molecule undergoes a conformational change which brings potential complementary contact sites to the surface. Under physiological conditions, the result may be polymerization of the deoxy HbS protein into strands and fibers. The fiber is known to be polymorphic, but the most common structure, determined by electron microscopy, is composed of seven double strands of monomers (Hb molecules) combined into a twisted helical rod as shown in Figure 2 (6). Contact sites in the double strand are accepted to be those found by X-ray diffraction to exist in the deoxy HbS crystal (7). Less is known about inter-strand contacts; the untwisted double strand may conform to the crystal structure; this is the subject of extensive research in X-ray diffraction (8) and electron microscopy (9). The gel formed by deoxy HbS fibers in the red blood cell may be composed of few or many domains, be more or less oriented, and have different effects on cell rheology and morphology, depending on polymerization conditions and kinetics. There have been extensive studies in vitro and in the red blood cell, of the effect of hemoglobin concentration and composition, rapidity and uniformity of deoxygenation, temperature, and other solution conditions on the kinetics and mechanism of polymerization. As polymer chemists would expect, these affect domain formation and size, appearance of spherulitic structures, etc; they are discussed extensively by Hofrichter and Eaton (1).


+

Polymerization Mechanism: Factors in Gelation A l l hemoglobin solutions are highly crowded at red blood cell concentrations (about 340g/L or 0.005 M ) (2). Excluded volume effects result in a large degree of non-ideality (activity coefficients of about 50 at physiological H b concentrations have been calculated (10)). This non-ideality is reflected in the measured osmotic pressures of hemoglobin solutions (see below). Although hemoglobin A remains monomeric and is very soluble both in the oxy and deoxy states under physiological conditions, deoxy HbS polymerizes above its solubility - also called saturating concentration (c^),-- in the red cell, and in phosphate buffer solutions at about physiological ionic strength and p H . The polymerized deoxy HbS solution undergoes a liquid to gel phase transition. Tliere is a delay time (t ) which is very highly dependent on total H b concentration, after which almost complete polymerization to fibers, and gelation occur almost simultaneously. (11,12) (Evidence of the transitory existence of either oligomers or of the liquid solution d



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Figure 1. Arrangement of the four polypeptide chains, two a-(identical) and two B-(identical), and the four hemes, in the hemoglobin molecule. The aand β- chains are similar but distinguishable in sequence and folding. (Copyright Irving Geis, with permission.)

Figure 2. Basic molecular structures in the most common form of polymerized deoxy sickle cell hemoglobin. The twisted 14-strand model of the deoxy HbS fiber (schematic), and the arrangement of 7 pairs of anti-parallel strands in a space-filling model. (Adapted from Ref.9)



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of deoxy HbS polymers, depends on interpretation of extremely exacting measurements.) The delay time is the result of a nucleation mechanism in HbS polymerization; in this sense, the process resembles classical nucleation and crystallization of salts and many small molecules, and that for nucleation and growth in binary mixtures of linear homopolymers (13). Depolymerization occurs spontaneously and without a delay time, when oxygen is introduced into the system or when the temperature is lowered. Figure 3 shows both the homogeneous nucleation mechanism first described by Hofrichter and Eaton (11,12) and the heterogeneous nucleation mechanism of Ferrone (14), which agrees more closely with the observed dependence of the delay time on protein concentration. It should be noted, however, that the discussion of nucleation mechanism arises from kinetic studies, and that therapeutic mechanisms based on increasing the delay time are also kinetically based. Ferrone and his coworkers (15-17) have proposed a theory of the spatial dependence of the polymerization of sickle hemoglobin which should be of interest to polymer physical chemists. Without reference to studies of domain formation in the polymer field, it attempts to reconcile the double (heterogeneous) nucleation mechanism, the kinetics of polymerization, apparent rates of diffusion of monomer into growing polymer domains, and the biréfringent domains and spherulitic forms observed in concentrated HbS gels (18). Whether a gel or crystalline solid results from the phase transition depends more on the thermodynamic state of the system than on kinetics: in the osmotic stress studies of sickle hemoglobin polymerization described below (4,5), the form and the concentration of the "equilibrium solid" depend finally on the chemical potential of the hemoglobin in the solution. Gelation is an endothermic entropie process: both solubility and delay time decrease as temperature increases. Herzfeld and Briehl (19,20) have used statistical mechanical models based on excluded volume and aggregation tendency, to predict phase transitions in reversibly polymerizing systems, and have calculated that repulsive inter-fiber contacts are overcome by the anisotropic crowding in the gel at high hemoglobin concentrations. Gels made from deoxy HbS solutions have been observed to be biréfringent (1,21). Although deoxy HbS gels made under quasi-physiological conditions have been observed to crystallize over long times, they are thermodynamically stable, on the physiological time scale of respiration and metabolism, on the crystallization time scale (months to years), and on that of most experiments. The presence, in sufficient proportion, of non-polymerizing forms of hemoglobin (including oxygenated hemoglobin, normal hemoglobin (HbA), fetal hemoglobin (HbF) (22) or other proteins, may increase solubility ~ if total protein concentration (and thus non-ideality) does not increase. It is for this reason that persons who have "sickle trait" ~ e.g. whose blood contains both H b A (which does not polymerize) and HbS ~ are non-symptomatic. O n the other hand, increased total protein concentration, or changes which lead to copolymerization, hybrid formation, or further crowding in the highly concentrated red cell milieu may cause both deoxy HbS solubility and delay time to decrease.


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Therapeutic Strategies. Most therapeutic approaches seek to modify the physical or chemical factors outlined above, which influence deoxy HbS solubility and/or kinetics. O n the whole, these factors depend on the macromolecular nature of hemoglobin itself, and on the "supra-molecular assemblies" represented by its fiber and gel forms. Some therapeutic designs are outlined in Table I , which shows also the particular property of the sickle hemoglobin system which each attempts to alter. " H P F H " (high persistence of fetal hemoglobin) occurs normally in some individuals, and has been observed to have an ameliorating effect in SCO patients who exhibit it. Currently efforts are under way to use the drug hydroxyurea in sickle patients to modify the regulatory mechanism which "turns off" expression of the H b F β-chain gene at birth, so that the non-polymerizing H b F will continue to be synthesized in the bone marrow ~ using genetics to alter polymer chemistry (23). Although HbS production is not lowered, the cell appears to compensate for the increased total amount of hemoglobin by increasing cell volume (24). The shape of the sickle R B C becomes somewhat more spherical, but any adverse effects on cell rheology appear to be outweighed by the beneficial effect on HbS solubility and polymerization delay time which is gained by decreasing the proportion of HbS in the total hemoglobin content of the cell. The problem addressed in this research project relates to the testing of non-specific "gelation inhibitors"-- compounds which can be expected to prevent polymer formation and sickling by increasing deoxy HbS solubility. The search for peptides which mimic complementary sites and thus compete specifically for contact sites but do not polymerize, has not been successful; however, nonpolar organic compounds, including aromatic amino acids and peptides (25), have been found to act non-specifically to increase the solubility ratio [(Csat)add/( s2Lt)o\ °* HbS. Such an increase has been generally used as a first test of the effectiveness of a potential therapeutic agent (26). Many promising compounds are rare, or relatively insoluble, and HbS itself is not easy to obtain or purify. Therefore, tests have often been carried out in high ionic strength phosphate buffers in which the solubilities of both deoxy HbS and the additive are markedly decreased - even though it has been reported that the form of the condensed phase obtained above c ^ was "aggregate" or "crystal" not "gel" (21). The aim of this research has been to study the effect of high ionic strength buffers on the structure and mechanism of formation of the condensed phase, as well as on HbS solubility, and to assess the importance of the nature of the solvent-determined phase transition on the ability of the added agent to prevent or delay polymerization. c

The Osmotic Stress Method Applied to Proteins We have adapted the "osmotic stress" method, developed by Parsegian and Rand for the study of intermolecular forces in lipids (3), to the study of the thermodynamics of phase transitions in protein solutions. In systems such as lipid bilayers, D N A double helices, and other macromolecular assemblies whose structural parameters may be readily determined by such techniques as x-ray


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Table I. Approaches to Therapy PHYSICQCHEMICAL PROPERTY

THERAPEUTIC STRATEGY

LModification of HbS

-Increase deoxyHbS solubility

A . Contact Sites B. Non-specific (hydrophobic) solvent effects C. Deoxy HbS conformation

-Sterospecific inhibitors -Non-specific non-polar gelation inhibitors -Covalent reagents bind to amino terminal (increase 0 affinity) -Replacement transfusion -decrease HbS cone, same total H b cone. 2

D . Non-polymerizing hemoglobins ( H b A for HbS) /J. Modification of Red Cell A . Increase cell volume B. Modify Cell Membrane //J. Genetic Modifications Decrease relative cone, of HbS in total H b cone.

-Decrease HbS concentration to increase delay time -Decrease salt concentration (hyponatremia) -Modify ion transport •Alter genes to modify pattern of hemoglobin synthesis -Hydroxyurea treatment to maintain expression of fetal hemoglobin

HETEROGENEOUS NUCLEATION

Figure 3. Double nucleation mechanism for deoxy HbS polymerization. (Adapted from Ref.2, based on data in Ref.9 and 14).



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diffraction (27), osmotic stress has been used to measure inter- and intra-lamellar forces and to test model structures. This "secondary osmometry" technique is useful for determining the osmotic pressure (and thus macromolecular chemical potentials) in concentrated solutions and in gels and solids in which such measurements have not been practical previously. As applied to protein studies, the method involves equilibration of a number of solutions of protein in dialysis sacs against inert polymer solutions of known, and increasing, osmotic pressure, forming a series of "secondary osmometers". For liquid solution conditions, these yield protein solution osmotic pressures in good agreement with directly measured values. We have shown that protein solutions, brought to concentrations greater than their solubility by equilibration against inert polymers in this way, undergo the same phase transitions as when a temperature-jump (or other unstabilizing change) is used to induce phase transitions in liquid solutions. We are able to measure the osmotic pressure of gels and solids (which we refer to as condensed phases in this context), and thus to calculate the thermodynamic parameters of the phase transition. On a practical level, if it is necessary to prepare condensed phases with large domains, osmotic stress can be used to increase polymer solution concentrations to values much greater than their solubility slowly and regularly, in such a manner that the expected phase transition can be brought about under controlled conditions. This is preferable to such measures as temperature jumps, which often lead to a large number of very small domains, and which may not succeed for the most concentrated solutions. We may predict that the response of protein solutions to being concentrated by increasing osmotic stress will depend on the intermolecular forces present (protein-protein, protein-solvent, and solvent-solvent), and on excluded volume considerations. Where repulsive forces or hard sphere conditions exist, high non-ideality leads to sharply increasing osmotic pressures (or, stated another way, a limiting solution concentration with increasing osmotic pressure) as shown by the liquid solution curves in Figures 4 and 6. We plot concentration versus osmotic pressure as the independent variable. Above saturation, we have seen two kinds of phase transitions: in the first the product is a single non-compressible condensed phase - this is formed by proteins (such as lysozyme) which in a test tube, yield a solid crystalline phase in equilibrium with a saturated liquid solution. The product in osmotic stress cells prepared at stresses greater than (the osmotic pressure of a saturated solution), is always a solid phase only, with no liquid solution present. The saturated liquid solution can never be in equilibrium with the stressing solution: solvent is always being withdrawn from the saturated stressed solution, which undergoes a continuing phase transition to the condensed phase until there is no saturated solution left. Figure 4 shows actual data for lysozyme (4) for which the product found in all tubes above π^ was a white powder. Alternatively, we find (4) a compressible (gel) phase as in deoxy HbS (Figure 6), in which the final protein concentration is determined by its equilibrium osmotic pressure (imposed by the stressing inert polymer solution). Where attractive forces dominate (eventually causing phase separation), clustering of proteins may occur in the liquid solution, and may decrease osmotic pressures observed below that expected for the hard sphere χ



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excluded volume case. Tardieu et al (28) have reported this effect in 7-crystallin solutions.


Experimental Methods Sickle cell hemoglobin is purified from whole blood using standard methods, as described in Ref.4. The inert polymer used for stressing is Dextran (T500 and T70) from Pharmacia. Osmotic stress cells are prepared from dialysis tubing and test tubes (Figure 5). (Generally about 0.5 m L of hemoglobin solution is immersed in 7-8 m L of Dextran solution in the same buffer.) Osmotic cells are assembled in the cold in a glove box under nitrogen. A s an oxygen scavenger, sodium dithionite (final concentration = 0.02M) is added to both H b and degassed Dextran solutions. Dextran solutions are made up by weight in the desired buffer; their osmotic pressures are determined before and after equilibration. Equilibration is carried out in a thermostatted shaker bath, and has been shown to be complete in four days. (Deoxy hemoglobin is a stable protein below about 45°C and does not show deterioration under osmotic stress, using standard tests.) Gelation of the hemoglobin is tested by cessation of flow. After equilibration the cells are opened, the gelled or crystallized hemoglobin is "melted" in air at 3°C., and hemoglobin concentrations are measured spectrophotometrically as cyanomet hemoglobin (29). In our previous work, as well as in most other osmotic stress studies, final equilibrium osmotic pressures of the inert polymer solutions have been routinely calculated from concentrations obtained by refractive index or viscosity measurements, using calibration curves from carefully carried out standardization experiments. It has been found that, because of the high degree of non-ideality of the dextran or polyethylene glycol solutions used, neither the molecular weight of the polymer used, the temperature or the nature of the aqueous solvent has a significant effect on the polymer solution osmotic pressure. For dextran in 0.15M phosphate buffers, we used data from measurements in distilled water with confidence (4). This assumption has been shown to be valid also in the work of Parsegian, Rau and Rand and others on lipid bilayers (3), muscle and T M V (30), and D N A ' s (27), where pressures of several atmospheres or more are common. However, in the present research, we are primarily concerned with solvent and salt effects, using phosphate buffers up to 2.0 M , and also achieving high ionic strengths with other anions. In these media, the solubilities of both hemoglobin and dextran are reduced, as is the osmotic pressure at which the hemoglobin phase transition occurs, so that most of our critical measurements are at osmotic pressures below one-fourth of an atmosphere. This requires use of dextran solutions at quite low concentrations. We now use a U I C Osmomat 050 electronic membrane osmometer (with cellulose membranes from U I C , or Amicon membranes) to measure directly the osmotic pressure of the stressing dextran solutions. We find experimentally significant differences in dextran osmotic pressures in the high ionic strength media. We have not yet attempted to analyze these, but use the directly measured osmotic pressure in each osmotic stress equilibration experiment. One of the advantages of the osmotic stress method in thermodynamic studies is that non-ideality is built in: osmotic pressures


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Polymerization and Phase Transitions in Deoxy Sickle Cell Hemoglobin

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