Effects of different beta73 amino acids on formation of 14-stranded fibers of Hb S versus double-stranded crystals of Hb C-Harlem

Hb S (alpha(2)beta(2)(6Glu-->Val)) forms polymers, while Hb C-Harlem (alpha(2)beta(2)(6Glu-->Val,73Asp-->Asn)) forms crystals upon oversaturation. Since the only difference between the two is the beta73 amino acid, it follows that this site is a critical determinant in promoting either polymerization or crystallization. Beta73 Asp in Hb S forms a hydrogen bond with beta4 Thr, while beta73 Asn in Hb C-Harlem may inhibit this interaction as well as increase the hydrophobicity at the EF helix beta6 Val acceptor sites. Two new beta73 Hb S variants (beta73 His and Leu) were constructed and analyzed to define other amino acids facilitating formation of Hb S-like polymers versus Hb C-Harlem-like crystals. The two variants that were chosen were expected to either (1) enhance formation of the beta73-beta4 hydrogen bond (beta73 His) or (2) inhibit it and increase the hydrophobicity of the EF helix beta6 Val acceptor sites (beta73 Leu). beta73 His Hb S formed fibers but at a lower concentration than Hb S, while beta73 Leu Hb S formed crystals but at a higher concentration than Hb C-Harlem. The solubility of beta73 His Hb S was (1)/(7) of that of Hb S, while the solubility of beta73 Leu Hb S was similar to that of Hb C-Harlem. The delay time prior to polymer or crystal formation depended on Hb concentration. The delay time for beta73 His Hb S was 10(5)-fold shorter than that for Hb S, while that for beta73 Leu Hb S was 10(5)-fold longer in 1.0 M phosphate buffer. NMR results indicate beta73 amino acid changes induce alteration in the beta-chain heme pocket region, while CD results indicate no change in the helical content of the variants. These results suggest that enhancing the beta73-beta4 hydrogen bond and/or induced changes in the heme pocket by the beta73 Asp to His change facilitate formation of Hb S-like fibers. Our results also suggest that removal of the beta73-beta4 hydrogen bond and enhancing the hydrophobicity of the EF helix beta6 Val acceptor sites by the beta73 Asp to Leu or Asn changes delay nuclei formation and facilitate formation of Hb C-Harlem-like crystals.     

Facilitation of Hb S polymerization by the substitution of Glu for Gln at beta 121

In an effort to clarify the role of Glu-beta 121 of Hb S molecules in polymerization, we studied the solubility and kinetics of polymerization of various mixtures of deoxyhemoglobins S (Glu-beta 6----Val) and D Los Angeles (Glu-beta 121----Gln). It is known that patients with Hb S-D Los Angeles have a relatively severe clinical course. Mixtures of Hb S and Hb D Los Angeles polymerized after a distinct delay time, the length of which depended on the initial hemoglobin concentration and the fraction of Hb S in the mixture. There was a linear relationship between the logarithmic plot of delay time and initial hemoglobin concentration. The line for a 1:1 mixture of Hb S and Hb D Los Angeles shifted to the right of that for deoxy-Hb S by 0.08. This shift is much smaller than the shift of 0.32 for 1:1 AS mixtures. From these data, the probability factor for nucleation of S-D Los Angeles hybrid hemoglobin was calculated to be 1.16, which is higher than that of Hb S (1.0) and AS hybrid hemoglobin (0.5). The degree of co-polymerization of Hb D Los Angeles in S-D Los Angeles mixtures was similar to that of Hb A in AS mixtures. The critical concentration for the polymerization of Hb D Los Angeles was between that of Hb A and Hb Machida, which has the same amino acid substitution (Glu----Gln) at the beta 6 position. These results suggest that the protein interaction of Hb S molecules during nucleation involves at least two steps. First, the Val-beta 6 of a Hb S molecule interacts hydrophobically with the Phe-beta 85 and the Leu-beta 88 of an adjacent Hb S molecule. In the second step, Glu-beta 121 weakens the interaction with His-beta 116 and Pro-alpha 114. The substitution of Glu-beta 121----Gln may strengthen this second reaction and facilitate nucleation as well as polymerization.     

Effect of FS (alpha 2 gamma beta s) hybrid hemoglobin on Hb S nucleation and aggregation

Asymmetrical cross-linked FS (alpha 2 gamma beta s) hybrid hemoglobin (Hb FS-fumarate) was prepared by reacting mixtures of hemoglobins F and S with double-headed aspirin, bis(3,5-dibromosalicyl) fumarate. When the molar ratio of hemoglobin to the cross-linking agent was 1 to 2 in a 1:1 FS mixture, the relative ratio of the products, cross-linked hemoglobins F (Hb F-fumarate), FS (HB FS-fumarate), and S (Hb S-fumarate), was 1.0:2.6:2.0, in contrast to a 1:2:1 ratio of cross-linked hemoglobins A, AS, and S in a 1:1 AS mixture. These results suggest that the fumaryl group reacts differently with Hb F, Hb FS and Hb S, and that the difference could be attributed to the difference in the structure in the vicinity of the EF6 Lys of non alpha-chains. The oxygen-binding properties of Hb F-fumarate, Hb FS-fumarate, and Hb S-fumarate were similar, except that the n-value of Hb F-fumarate was slightly lower than n-values of Hb S-fumarate and Hb FS-fumarate. Kinetic studies on aggregation showed that the addition of Hb FS-fumarate to unmodified Hb S did not affect the delay time prior to aggregation, but did increase the total turbidity. Electrophoretic and densitometric scanning analysis of the aggregate phase of this mixture showed the fraction of Hb FS-fumarate to be 19%. Hb F-fumarate's effect on the delay time is concentration-dependent; the greater the concentration of Hb F-fumarate, the longer the delay time. The turbidity after aggregation of the mixture of Hb S and Hb F-fumarate was much less than that of Hb S and Hb FS-fumarate. However, the fraction of Hb F-fumarate in the aggregate phase was 19%, which is similar to that of Hb FS-fumarate. These data suggest that Hb F and FS hybrid hemoglobin cannot participate in nuclei formation, but can participate in aggregation after sufficient amounts of nuclei are formed from Hb S, and that increased levels of Hb F do not have an inhibitory effect on the formation of nuclei but on the growth of aggregates.     

Inhibition of hemoglobin S polymerization in vitro by a novel 15-mer EF-helix beta73 histidine-containing peptide

Our mutational studies on Hb S showed that the Hb S beta73His variant (beta6Val and beta73His) promoted polymerization, while Hb S beta73Leu (beta6Val and beta73Leu) inhibited polymerization. On the basis of these results, we speculated that EF-helix peptides containing beta73His interact with beta4Thr in Hb S and compete with Hb S, resulting in inhibition of Hb S polymerization. We, therefore, studied inhibitory effects of 15-, 11-, 7-, and 3-mer EF-helix peptides containing beta73His on Hb S polymerization. The delay time prior to Hb S polymerization increased only in the presence of the 15-mer His peptide; the higher the amount, the longer the delay time. DIC image analysis also showed that the fiber elongation rate for Hb S polymers decreased with increasing concentration of the 15-mer His peptide. In contrast, the same 15-mer peptide containing beta73Leu instead of His and peptides shorter than 11 amino acids containing beta73His including His alone showed little effect on the kinetics of polymerization and elongation of polymers. Analysis by protein-chip arrays showed that only the 15-mer beta73His peptide interacted with Hb S. CD spectra of the 15-mer beta73His peptide did not show a specific helical structure; however, computer docking analysis suggested a lower energy for interaction of Hb S with the 15-mer beta73His peptide compared to peptides containing other amino acids at this position. These results suggest that the 15-mer beta73His peptide interacts with Hb S via the beta4Thr in the betaS-globin chain in Hb S. This interaction may influence hydrogen bond interaction between beta73Asp and beta4Thr in Hb S polymers and interfere in hydrophobic interactions of beta6Val, leading to inhibition of Hb S polymerization.     

Effect of the beta 73 amino acid on the hydrophobicity, solubility, and the kinetics of polymerization of deoxyhemoglobin S

The role of Asp-beta 73 on the surface hydrophobicity and solubility of hemoglobin was studied using Hb A, Hb S, Hb C Harlem (alpha 2 beta 2Val-6,Asn-73), and Hb Korle Bu (alpha 2 beta 2Asn-73). The surface hydrophobicity of the oxy form of these hemoglobins increased in the order of Hb A, Hb Korle Bu, Hb S, and Hb C Harlem, coinciding with the change in solubility. The same is not true for deoxyhemoglobins. The solubilities of deoxy-Hb S and deoxy-Hb C Harlem were much lower than that expected from their surface hydrophobicity. Although the hydrophobicity of deoxy-Hb C Harlem is greater than that of deoxy-Hb S, the solubility of deoxy-Hb S is only one-third that of deoxy-Hb C Harlem. This deviation must be caused by the substitution of Asn for Asp at the beta 73 position and its inhibitory effect on hydrogen bonding in Hb S polymers. The kinetics of the polymerization of 1:1 mixtures of the deoxy form of S-C Harlem, A-C Harlem, Korle Bu-S, and Korle Bu-C Harlem were studied in comparison with that of deoxy-Hb S and deoxy-Hb C Harlem alone. All of these binary mixtures polymerized with a distinct delay time prior to polymerization. Based on the results of kinetic studies, the probability factors for nucleation of S-C Harlem, A-S, A-C Harlem, S-Korle Bu, and Korle Bu-C Harlem hybrid hemoglobins were calculated as 0.65, 0.5, 0.5, 0.15, and 0.17, respectively, in comparison with that of Hb S (1.0). The probability factor for Hb C Harlem alone was 0.3. These data suggest that the Asp-beta 73 is directly involved in nucleation during Hb S polymerization and that the beta 73 is always trans to the active Val-beta 6 in the formation of nuclei.     

Enhanced inhibition of polymerization of sickle cell hemoglobin in the presence of recombinant mutants of human fetal hemoglobin with substitutions at position 43 in the gamma-chain

Four recombinant mutants of human fetal hemoglobin [Hb F (alpha2gamma2)] with amino acid substitutions at the position 43 of the gamma-chain, rHb (gammaD43L), rHb (gammaD43E), rHb (gammaD43W), and rHb (gammaD43R), have been expressed in our Escherichia coli expression system and used to investigate their inhibitory effect on the polymerization of deoxygenated sickle cell hemoglobin (Hb S). Oxygen-binding studies show that rHb (gammaD43E), rHb (gammaD43W), and rHb (gammaD43R) exhibit higher oxygen affinity than human normal adult hemoglobin (Hb A), Hb F, or rHb (gammaD43L), and all four rHbs are cooperative in binding O2. Proton nuclear magnetic resonance (NMR) studies of these four rHbs indicate that the quaternary and tertiary structures around the heme pockets are similar to those of Hb F in both deoxy (T) and liganded (R) states. Solution light-scattering experiments indicate that these mutants remain mostly tetrameric in the liganded (R) state. In equimolar mixtures of Hb S and each of the four rHb mutants (gammaD43L, gammaD43E, gammaD43R, and gammaD43W), the solubility (Csat) of each of the pairs of Hbs is higher than that of a similar mixture of Hb S and Hb A, as measured by dextran-Csat experiments. Furthermore, the Csat values for Hb S/rHb (gammaD43L), Hb S/rHb (gammaD43E), and Hb S/rHb (gammaD43R) mixtures are substantially higher than that for Hb S/Hb F. The results suggest that these three mutants of Hb F are more effective than Hb F in inhibiting the polymerization of deoxy-Hb S in equimolar mixtures.     

Effect of amino acid at the beta 6 position on surface hydrophobicity, stability, solubility, and the kinetics of polymerization of hemoglobin. Comparisons among Hb A (Glu beta 6), Hb C (Lys beta 6), Hb Machida (Gln beta 6), and Hb S (Val beta 6)

Surface hydrophobicity, stability, solubility, and kinetics of polymerization were studied using hemoglobins with four different amino acids at the beta 6 position: Hb A (Glu beta 6), Hb C (Lys beta 6), Hb Machida (Gln beta 6), and Hb S (Val beta 6). The surface hydrophobicity increased in the order of Hb C, Hb A, Hb Machida, and Hb S, coinciding with the hydrophobicity of the amino acid at the beta 6 position. Solubility of the oxy-form of these hemoglobins decreased in relation to increases in their surface hydrophobicity, suggesting that the solubility is controlled by the strength of hydrophobicity of the amino acid at the beta 6 position. The solubility of the oxy-form of these hemoglobins is always higher than that of the deoxy-form. There is a similar linear relationship between the solubility and surface hydrophobicity among deoxyhemoglobins A, C, and Machida. However, the solubility of deoxy-Hb S deviated significantly from the expected value, indicating that the extremely low solubility of deoxy-Hb S is not directly related to the hydrophobicity of the beta 6 valine. Kinetic studies on the polymerization of deoxy-Hb Machida revealed a distinct delay time prior to polymerization. This confirms our previous hypothesis that beta 6 valine is not responsible for the delay time prior to gelation. The kinetics of the polymerization of 1:1 mixtures of sickle and non-sickle hemoglobins were similar to those of pure Hb S, suggesting that only one of the two beta 6 valines is involved in an intermolecular contact. In mixtures of equal amounts of Hb S and Hb A, Hb C, or Hb Machida, half of the asymmetrical AS, SC, and S-Machida hybrid hemoglobins behaved like Hb S during nucleation, while the other half behaved like the non-sickle hemoglobin.     

The solubility of sickle and non-sickle hemoglobins in concentrated phosphate buffer.

A new turbidimetric method for the direct measurement of the solubility of oxy- and deoxyhemoglobins (Hb) in concentrated phosphate buffer has been established. The principle of the method is the formation of a homogeneous emulsion when hemoglobin is introduced in concentrated phosphate buffer. The solubility of the oxy and deoxy forms of Hb A, Hb S, Hb C, Hb F, and Hb CHarlem (beta 6Glu leads to Val, beta 73Asp leads to Asn) has been studied. The solubility of deoxy-Hb S was the lowest and the solubility curve was broader than those of the other hemoglobins indicating that the aggregates of deoxy Hb S require more water to be dissolved. The solubility of oxy- and deoxyhemoglobins depends on temperature and pH. The solubility of hemoglobins is increased as the temperature is lowered and the pH is raised. The pH dependency of the solubility of deoxy-Hb S in high phosphate buffer was opposite to that of the minimum gelling concentration of deoxy-Hb S. The order of the solubility of Hb CHarlem, Hb FS, Hb AS, Hb CS, and Hb S in concentrated phosphate buffer corresponds to the order of minimum gelling concentration of these hemoglobins or hemoglobin mixtures. Solubility studies of a 1:1 mixture of deoxy-Hb A and deoxy-Hb S show that deoxy-Hb A aggregates in 2.42 M phosphate buffer in which pure deoxy-Hb A is totally soluble. This result indicates that deoxy-Hb S interacts with deoxy-Hb A and decreases its solubility.     

A biochemical and biophysical characterization of recombinant mutants of fetal hemoglobin and their interaction with sickle cell hemoglobin.

Three recombinant mutants of human fetal hemoglobin (Hb F) have been constructed to determine what effects specific amino acid residues in the gamma chain have on the biophysical and biochemical properties of the native protein molecule. Target residues in these recombinant fetal hemoglobins were replaced with the corresponding amino acids in the beta chain of human normal adult hemoglobin (Hb A). The recombinant mutants of Hb F included rHb F (gamma 112Thr --> Cys), rHb F (gamma 130Trp --> Tyr), and rHb F (gamma 112Thr --> Cys/gamma 130Trp --> Tyr). Specifically, the importance of gamma 112Thr and gamma 130Trp to the stability of Hb F against alkaline denaturation and in the interaction with sickle cell hemoglobin (Hb S) was investigated. Contrary to expectations, these rHbs were found to be as stable against alkaline denaturation as Hb F, suggesting that the amino acid residues mentioned above are not responsible for the stability of Hb F against the alkaline denaturation as compared to that of Hb A. Sub-zero isoelectric focusing (IEF) was employed to investigate the extent of hybrid formation in equilibrium mixtures of Hb S with these hemoglobins and with several other hemoglobins in the carbon monoxy form. Equimolar mixtures of Hb A and Hb S and of Hb A(2) and Hb S indicate that 48-49% of the Hb exists as the hybrid tetramer, which is in agreement with the expected binomial distribution. Similar mixtures of Hb F and Hb S contain only 44% hybrid tetramer. The results for two of our recombinant mutants of Hb F were identical to the results for mixtures of Hb F and Hb S, while the other mutant, rHb F (gamma 130Trp --> Tyr), produced 42% hybrid tetramer. The sub-zero IEF technique discussed here is more convenient than room-temperature IEF techniques, which require Hb mixtures in the deoxy state. These recombinant mutants of Hb F were further characterized by equilibrium oxygen binding studies, which indicated no significant differences from Hb F. While these mutants of Hb F did not have tetramer-dimer dissociation properties significantly altered from those of Hb F, future mutants of Hb F may yet prove useful to the development of a gene therapy for the treatment of patients with sickle cell anemia.     

Electron paramagnetic resonance of Hb St Louis beta28 (B10) Leu replaced by Gln.

Hemoglobin St Louis beta28 (B10) Leu replaced by Gln is a new mutant which occurs as a natural valency hybrid (alpha2beta+2), or hemoglobin M (Cohen-Solal, M., Seligmann, M., Thillet, J. and Rosa, J. (1973) FEBS Lett. 33, 37-41). The electron paramagnetic resonance (EPR) spectrum of native Hb St Louis at pH 6.2 shows a mixture of three species. Two are high spin, one with tetragonal symmetry, like Hb+ A, the other with rhombic distortion. The third is a low-spin form corresponding to a hemichrome with the distal (E7) histidine as the sixth ligand of the ferric iron. The hemichrome is also found in red blood cells. After oxidation to the alpha+2beta+2 form, three EPR species are seen. Surprisingly, there remains only one high-spin signal, with almost tetragonal symmetry. Besides the low-spin hemichrome, a hydroxy signal is observed even at pH 6.2. These observations imply interactions between the alpha and beta hemes.     

Polymer structure and solubility of deoxyhemoglobin S in the presence of high concentrations of volume-excluding 70-kDa dextran. Effects of non-s hemoglobins and inhibitors

Earlier observations indicated that volume exclusion by admixed non-hemoglobin macromolecules lowered the polymer solubility ("Csat") of deoxyhemoglobin (Hb) S, presumably by increasing its activity. In view of the potential usefulness of these observations for in vitro studies of sickling-related polymerization, we examined the ultrastructure, solubility behavior, and phase distributions of deoxygenated mixtures of Hb S with 70-kDa dextran, a relatively inert, low ionic strength space-filling macromolecule. Increasing admixture of dextran progressively lowered the Csat of deoxyHb S. With 12 g/dl dextran, a 5-fold decrease in apparent Csat ("dextran-Csat") was obtained together with acceptable sensitivity and proportionality with the standard Csat when assessing the effects of non-S Hb admixtures (A, C, and F) or polymerization inhibitors (alkylureas or phenylalanine). The volume fraction of dextran excluding Hb was 70-75% of total deoxyHb-dextran (12 g/dl) volumes. Electron microscopy showed polymer fibers and fiber-to-crystal transitions indistinguishable from those formed without dextran. Thus when Hb quantities are limited, as with genetically engineered recombinant Hbs or transgenic sickle mice, the dextran-Csat provides convenient and reliable screening of effects of Hb S modifications on polymerization under near-physiological conditions, avoiding problems of high ionic strength.     

Mutational analysis of phenylalanine beta 85 in the valine beta 6 acceptor pocket during hemoglobin S polymerization.

Hemoglobin (Hb) S containing Leu, Ala, Thr, or Trp substitutions at beta 85 were made and expressed in yeast in an effort to evaluate the role of Phe-beta 85 in the acceptor pocket during polymerization of deoxy Hb S. The four Hb S variants have the same electrophoretic mobility as Hb S, and these beta 85 substitutions do not significantly affect heme-globin interactions and tetramer helix content. Hb S containing Trp-beta 85 had decreased oxygen affinity, whereas those with Leu-, Ala-, and Thr-beta 85 had increased oxygen affinity. All four supersaturated beta 85 variants polymerized with a delay time as does deoxy Hb S. This is in contrast to deoxy Hb S containing Phe-beta 88, Ala-beta 88, Glu-beta 88, or Glu-beta 85, which polymerized with no clear delay time (Adachi K, Konitzer P, Paulraj CG, Surrey S, 1994, J Biol Chem 269:17477-17480; Adachi K, Reddy LR, Surrey S, 1994, J Biol Chem 269:31563-31566). Leu substitution at beta 85 accelerated deoxy Hb S polymerization, whereas Ala, Thr, or Trp substitution inhibited polymerization. The length of the delay time and total polymer formed for these beta 85 Hb S variants depended on hemoglobin concentration in the same fashion as for deoxy Hb S: the higher the concentration, the shorter the delay time and the more polymer formed. Critical concentrations required for polymerization of deoxy Hb SF veta 85L, Hb SF beta 85A, Hb SF beta 85T, and Hb SF beta 85W are 0.65-, 2.2-, 2.5- and 3-fold higher, respectively, than Hb S.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature and domain size dependence of sickle cell hemoglobin polymer melting in high concentration phosphate buffer.

Deoxygenated sickle cell hemoglobin (Hb S) in 1.8 M phosphate buffer, and carbon monoxide (CO) saturated buffer were rapidly mixed using a stopped-flow apparatus. The binding of the CO to the Hb S polymers and the polymer melting was measured by time resolved optical spectroscopy. Polymer melting was associated with decreased turbidity, and CO binding to deoxy-Hb S was monitored by observation of changes in the absorption profile. The reaction temperature was varied from 20 degrees C to 35 degrees C. Polymer domain size at 20 degrees C was also varied. The data for mixtures involving normal adult hemoglobin (Hb A) fit well to a single exponential process whereas it was necessary to include a second process when fitting data involving Hb S. The overall Hb S-CO reaction rate decreased with increasing temperature from 20 degrees C to 35 degrees C, and increased with decreasing domain size. In comparison, Hb A-CO reaction rates increased uniformly with increasing temperature. Two competing reaction channels in the Hb S-CO reaction are proposed, one involving CO binding directly to the polymer and the other involving CO only binding to Hb molecules in the solution phase. The temperature dependence of the contribution of each pathway is discussed.     

Stability of asymmetrical hybrid hemoglobins. Determination by anaerobic high performance liquid chromatography

Asymmetrical hybrid hemoglobins formed in mixtures of Hb A and Hb S, Hb F and Hb S, Hb S and Hb York(beta 146 His----Pro), and Hb A and Hb York were separated by high performance liquid chromatography on cation and anion exchange columns under anaerobic conditions. The ratio of the hybrid hemoglobin to the total mixture was consistently lower than that theoretically expected and decreased with longer elution times. The hybrid tetramer appears to be unstable even under anaerobic conditions and dissociates into alpha beta dimers. The time course of dissociation of the hybrid hemoglobins was determined by varying the separation programs and thus separating the hybrid hemoglobin at different elution times. The rate of the dissociation of the hybrid hemoglobins studied follows first order kinetics. The lines representing the time course of dissociation of hybrid hemoglobins were extrapolated to time 0 to determine the fraction of the hybrid hemoglobin in the mixture prior to separation. The values obtained for equimolar mixtures of Hb A and Hb S and Hb York and Hb S or Hb A were in agreement with the expected theoretical value (50%). In contrast, the value obtained for hybrid hemoglobin FS was slightly less (about 40%). AY and SY hybrid hemoglobins dissociated into dimers at a considerably faster rate than did AS and FS hybrid hemoglobins, possibly because of the mutation at the beta 146-position in hybrid hemoglobins containing alpha beta Y dimers. This mutation hinders the formation of salt bridges that normally stabilize the "T" quaternary conformation. Since such hybrid hemoglobins have a partial "R" conformation even when deoxygenated, their rate of dissociation to dimers is expected to increase.     

Characterization of chromosomes with hybrid genes for Hb Lepore-Washington,Hb Lepore-Baltimore,Hb P-Nilotic,and Hb Kenya

Summary This study concerns the characterization of chromosomes with hybrid genes for Hb Lepore-Washington (44 chromosomes), for Hb Lepore-Baltimore (5 chromosomes), for Hb P-Nilotic (8 chromosomes), and for Hb Kenya (7 chromosomes) by determining a relatively large number of restriction enzyme polymorphism. Two, and possibly three, different Hb Lepore-Washington chromosomes were identified by specific haplotypes, while the haplotype of the Hb Lepore-Baltimore chromosome had its own characteristic pattern. A likely conclusion is that the crossovers leading to the formation of these chromosomes have occurred as independent events within the populations. Chromosomes with the -Lepore-Washington hybrid gene maintained specific characteristies (such as increased Hb F levels in heterozygotes, and high or low ^G values in this Hb F) which have been observed in normal individuals with chromosomes having comparable haplotypes. Only one haplotype was observed for each of the chromosomes carrying either the -P-Nilotic hybrid gene or the ^A hybrid gene of Hb Kenya.     

Simulated formation of polymer domains in sickle hemoglobin.

Using experimentally observed processes of linear growth, heterogeneous nucleation, and polymer bending, with no additional assumptions, we have been able to model the two-dimensional formation of polymer domains by sickle hemoglobin. The domains begin with twofold symmetry and proceed toward closure into spherulites at a constant rate. Relationships derived from the simulations presented and the requirements of scaling result in simple expressions for the sensitivity of the closure times to the model input parameters and allow the results to be extended to regions not actually simulated. For concentrations above approximately 25 g/dl, closure times are longer than the time required for the conclusion of the polymerization reaction, and thus incomplete spherulites will be the dominant geometry at high concentrations. Moreover, spherulites are not predicted to form in times less than a few seconds, implying that spherulites will not form during the transit of erythrocytes through the capillaries. Polymer-polymer exclusion, surface nucleation, and monomer exhaustion were also explored and found to have only weak effects on the results.     

Protein complexation with acrylic polyampholytes

The interaction of dilute mixtures of proteins and ABC triblock methacrylic polyampholytes at different values of pH was investigated turbidimetrically. The onset of interaction was manifested by large changes in turbidity at certain critical pHs which lie close to the isoelectric points of the two interacting components. Protein precipitation yields in protein-polyampholyte binary mixtures followed the corresponding turbidity profiles and varied from 10% to 90%. The synthetic polyampholytes self-aggregate around their isoelectric point. The kinetics of precipitation of one of the same polymer with soybean trypsin inhibitor were studied, with turbidity-based characteristic times (exponential fit) of 2-3 min. The kinetics of precipitation of the protein-polymer mixture are slower than that of pure polymer because a small, but steady, long-term increase in turbidity is observed in the former case. The pH-dependence of the turbidity of binary mixtures of one protein and one synthetic polyampholyte, as well as a tertiary mixture of two proteins and one polyampholyte, were measured 30 min after the pH adjustment. The observations in these experiments along with the measured protein precipitation yields in the binary mixtures and the polyampholyte self-aggregation can be used for polymer removal and recycling. The latter constitutes a significant advantage over the use of homopolyelectrolytes which cannot easily be recycled. (c) 1994 John Wiley & Sons, Inc.     

Similarities in the thermodynamics and kinetics of aggregation of disease-related Abeta(1-40) peptides

Increasing evidence indicates that polypeptide aggregation often involves a nucleation and a growth phase, although the relationship between the factors that determine these two phases has not yet been fully clarified. We present here an analysis of several mutations at different sites of the Abeta(1-40) peptide, including those associated with early onset forms of the Alzheimer's disease, which reveals that the effects of specific amino acid substitutions in the sequence of this peptide are strongly modulated by their structural context. Nevertheless, mutations at different positions perturb in a correlated manner the free energies of aggregation as well as the lag times and growth rates. We show that these observations can be rationalized in terms of the intrinsic propensities for aggregation of the Abeta(1-40) sequence, thus suggesting that, in the case of this peptide, the determinants of the thermodynamics and of the nucleation and growth of the aggregates have a similar physicochemical basis.     

Contact inhibition within hemoglobin S polymer by thiol reagents

N-Ethylmaleimide, a thiol reagent, increases the solubility of deoxyhemoglobin S. We investigated which of the two reacted beta 93 cysteine residues of the Hb tetramer was responsible for the inhibition of Hb S polymerization. Accordingly we compared the solubility of equal mixtures of HbA + HbS, HbA NEM + HbS and HbA + HbS NEM. Upon deoxygenation these mixtures contain about 50% a stable and asymmetrical hybrid alpha 2A beta A beta S, alpha 2A beta A,NEM beta S or alpha 2A beta A beta S,NEM respectively and 25% parental molecules as confirmed by ion-exchange HPLC performed in anaerobic conditions. Within the hybrid molecule, beta A or beta A,NEM chain has to be present in the alpha beta dimer located in trans to the dimer which contains the only beta 6 valine residue participating in intermolecular contacts (dimer in cis), while beta S or beta S,NEM must be in cis position in the hybrid molecule. The solubility of mixtures increases 4% for HbA NEM + HbS and 20% for HbA + HbS NEM mixtures compared to HbA + HbS mixture, indicating that the inhibitory effect of N-ethylmaleimide is more effective in cis than in trans position. The absence of a major role played by N-ethylmaleimide located in trans was supported by the solubility study of a mixture of HbS + Hb Créteil beta 89 Ser----Asn. The beta 89 residue in trans next to the cysteine beta 93 modified the T structure similarly to N-ethylmaleimide, and did not affect intermolecular contacts. Crystallographic studies of molecular contacts within deoxyHbS crystals suggest that the cis inhibitory effect of N-ethylmaleimide can be explained by direct inhibition of 'external' contacts between double strands involving the CD corner of the alpha chains.