Solubility Of deoxy-Hb Research Articles

Inhibition of Hb S Polymerization In Vitro by a Novel EF Helix ß73 His Containing Peptide.

Sickle hemoglobin polymerization involves not only ß6 Val in a largely hydrophobic acceptor pocket but other contact sites. We recently found that the ß73 His Hb S variant (ß6 Val and ß73 His) promoted polymerization compared to deoxy Hb S, while ß73 Leu Hb S (ß6 Val and ß73 Leu) inhibited polymerization like deoxy Hb C-Harlem (ß6 Val and ß73 Asn). In fact, ß73 Asp in Hb S makes a hydrogen bond with ß4 Thr in deoxy Hb S polymers, and this is a unique position to promote or inhibit polymerization by amino acid change [Adachi et al., Biochemistry (2003), 42, 4476]. Kinetics of polymerization, solubility and minimum concentration required for polymerization of the ß73 Hb S variants were affected by ß73 amino acid (inhibition of polymerization: His < <Asp <<Asn < Leu). Inhibition of Hb S ß73 Leu polymerization compared to Hb S may be caused by weakening of the hydrogen bond interaction between the hydroxyl group of ß4 Thr and the ß73 amino acid like Hb C-Harlem. Furthermore, kinetics of polymerization of 1:1 Hb S/Hb A ß73 His mixtures were enhanced compared to AS mixtures, while Hb S/Hb A ß73 Leu mixtures showed inhibitory effects similar to FS mixtures. These results suggest that the Hb A ß73His variant promotes Hb S polymerization almost as efficiently as Hb S ß73 His, and that the ß73 His in Hb A and Hb S variants strengthens the hydrogen bond with ß4 Thr, which helps facilitate formation of domains and 14-stranded fibers.Based on these results, we chemically synthesized a 15-mer EF helix peptide containing ß73 His (Lys-Lys-Val-Leu-Gly-Ala-Phe-Ser-His-Gly-Leu-Ala-His-Leu-Asp) and evaluated effects of this peptide on Hb S polymerization. DIC analysis in 1.0 M phosphate buffer showed peptide-induced inhibition of fiber elongation rate for deoxy-Hb S which increased linearly with increasing amounts of peptide (e.g., a 5-fold molar excess of peptide resulted in a 6-fold decrease in fiber elongation rate). Solubility of deoxy Hb S increased linearly with increasing peptide (e.g., a 5-fold molar excess of peptide increased solubility by 1.2-fold). The delay time prior to polymerization of deoxy Hb S in a high phosphate buffer also was increased significantly in the presence of this peptide. In contrast, the same 15-mer peptide containing ß73 Leu instead of His showed no effect on solubility or kinetics of polymerization, suggesting that the ß73 His peptide specifically affects the A-helix of Hb S containing ß4 Thr just like the ß73 His Hb S variant. This and possibly other peptides interrupting the ß73-ß4 interaction which result in inhibition of deoxy Hb S polymerization may be good candidates for inhibitors of Hb S polymerization.

In vitro effects of NIPRISAN (Nix-0699): a naturally occurring, potent antisickling agent.

Among the various potential antisickling agents tested, hydroxyurea (HU) has been the most effective compound used for the treatment of patients with sickle cell disease (SCD). Although HU is effective in many patients, not all patients respond to this drug. In addition, some patients reveal adverse effects, including myelosuppression. In an attempt to find other effective agents with less adverse effects, we investigated the antisickling effect of NIPRISAN (Nix-0699). We found that Nix-0699, an ethanol/water extract from indigenous plants, has a strong antisickling effect. The concentration of Nix-0699 required to inhibit 50% of erythrocyte sickling was about 0.05 mg/ml. As for the kinetics of polymerization, addition of 0.05 microg/ml Nix-0699 caused a sixfold prolongation of the delay time prior to deoxy-Hb S polymerization when compared with that of untreated Hb S samples. The solubility of deoxy-Hb S significantly increased upon treatment with Nix-0699. Analysis of the effect of Nix-0699 on the Hb S oxygen affinity indicated that the drug slightly shifted the oxygen-dissociation curve of Hb S toward the left without any apparent change in the Hill coefficient. These results suggest that the antisickling properties of Nix-0699 may involve direct interaction with Hb molecules. Incubation of red blood cell (RBC) suspensions with various concentrations of Nix-0699 did not dehydrate RBCs, cause haemolysis, increase the amount of denatured Hb, nor form met-Hb. In view of the outcome of this study, Nix-0699 may be a promising option for the treatment of patients with SCD.

Effects of macromolecular polyanions on the solubility of deoxyhemoglobin S.

The effects of four macromolecular polyanions on the equilibrium solubility of deoxy-Hb S and oxygen affinity of Hb A were evaluated. The order of molar effectiveness as gelation inhibitors was: poly-L-aspartate approximately equal to heparin (high M.W.) greater than dextran sulfate greater than heparin (low M.W.). The linear solubility profiles for the two most potent polyanions (poly-L-aspartate and high M.W. heparin) were nearly identical and reached a plateau at roughly the same sub-stoichiometric molar ratio. By contrast, poly-L-lysine, a polycation, strongly promoted gelation, while its succinylated derivative promoted gelation only marginally, as did dextran, a neutral polysaccharide. Among the seven species examined, only the four polyanions affected oxygen affinity appreciably, demonstrating the polyanions exert their solubilizing and oxygen affinity lowering effects via specific interaction with the cluster of cationic groups at the 2,3-diphosphoglycerate binding site in the central cavity of deoxyhemoglobin.

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.

Formation of cross-linked asymmetrical hybrid hemoglobins by double-headed aspirin.

Double-headed aspirin [bis(3,5-dibromosalicyl)fumarate] selectively cross-links hemoglobin molecules between Lys 82 beta 1 and Lys 82 beta 2 and increases solubility of deoxy-Hb S (Walder et al., J. Mol. Biol., 141:195, 1980 and Kikugawa et al., J. Biol. Chem., 257:7525, 1982). We reacted this reagent with the mixture of Hb A and Hb S and the mixture of Hb S and Hb York (beta 146His replaced by Pro). Cross-linked asymmetrical hybrid hemoglobins (alpha 2 beta - beta S and alpha 2 beta Y - beta S) were produced in high yields in addition to the cross-linked parent hemoglobin molecules. Results on electrophoresis, gel electrofocusing, ion exchange column chromatography, mechanical stability and oxygen binding properties showed that the cross-linked asymmetrical hybrid hemoglobins had properties intermediate between those of the cross-linked parent hemoglobins. Oxygen affinities of the cross-linked asymmetrical hybrids were not affected by the addition of 2,3-diphosphoglycerate (DPG) or inositol hexaphosphate, probably due to the presence of a fumaryl group at the DPG binding site.

Polymerization of carbamylated deoxyhemoglobin S in concentrated phosphate buffer.

To study the effect of carbamylation on the polymerization of deoxyhemoglobin (HB) S, three types of NH2-terminal-carbamylated Hb S specifically modified at the NH2 termini at the alpha, beta, or all four subunit chains, alpha c2 beta sc2, and alpha 2 beta sc2, and epsilon-lysine-carbamylated Hb S were prepared by reacting Hb S with cyanate. The solubility of deoxy-Hb S in concentrated phosphate buffer was increased by carbamylation of the NH2-terminal residues of beta chains. Kinetic studies on the polymerization of purified carbamylated deoxy-Hb S showed that the length of the delay time prior to polymerization is related to solubility; carbamylated Hb S with higher solubilities exhibited longer delay times than those with lower solubilities. The delay times for alpha 2 beta sc2 and alpha c2 beta sc2 were prolonged 2-3- and 3-6-fold, respectively, as compared to those of native deoxy-Hb S in 1.8 M phosphate buffer. The logarithmic plot of reciprocal delay time versus hemoglobin concentration of alpha c2 beta s2, alpha 2 beta sc2, and epsilon-lysine-carbamylated deoxy-Hb S showed straight lines with slopes (n values) of 4.0, 3.8, 3.6, and 3.5, respectively. These values are slightly higher than the n value of native deoxy-Hb S (n = 2.7), suggesting that nuclei of carbamylated Hb S are larger than those of native deoxy-Hb S.

Polymerization of deoxyhemoglobin C Harlem (β6 Glu → Val, β73 Asp → Asn) : The effect of β73 asparagine on the gelation and crystallization of hemoglobin

The kinetics of aggregation and the solubility of deoxy Hb† C Harlem ( α 2 β 2 6 Val, 73 Asn) in concentrated phosphate buffers were studied in comparison with those of deoxy Hb S and deoxy Hb A. Deoxy Hb C Harlem aggregated with a clear exhibition of a delay time. The length of the delay and aggregation times and the degree of the aggregation depended upon the initial hemoglobin concentration. The initial hemoglobin concentration required for the aggregation of deoxy Hb C Harlem was approximately 200% of its solubility, a value much higher than that required for the aggregation of deoxy Hb S (120%). With the same hemoglobin concentration, the delay time for the aggregation of deoxy Hb C Harlem was approximately 100 times longer than that of deoxy Hb S. The logarithmic plotting of the delay time versus hemoglobin concentration in 1.8 m-phosphate buffer (pH 7.4) showed linear lines with a slope ( n) of 4.0 for deoxy Hb C Harlem. In contrast to the results for the aggregation of deoxy Hb S, n values for deoxy Hb C Harlem were unchanged with phosphate concentrations varying from 1.2 m to 2.0 m. The solubilities of deoxy Hb S and deoxy Hb C Harlem were increased exponentially by lowering the pH of the medium, with the increase being more conspicuous for Hb C Harlem. The gels (or aggregates) of Hb C Harlem were converted to crystals at a rate much faster than were those of Hb A and Hb S. The kinetics for gelation and crystallization of deoxy Hb C Harlem can be explained by the following scheme, where nuclei G and nuclei C are formed before gelation and crystallization, respectively. Monomenc deoxy Hb ▪ The hemoglobin concentration required for the crystallization of deoxy Hb C Harlem was about ten times lower than that required for deoxy Hb A. The solubility of deoxy Hb C Harlem after aggregation was about twice that of deoxy Hb S, suggesting that the substitution of Asn for Asp at the β73 residue inhibits the formation of nuclei G and accelerates the formation of nuclei C.

Nucleation-controlled aggregation of deoxyhemoglobin S effect of organic phosphates on the kinetics of aggregation of deoxyhemoglobin S in concentrated phosphate buffer

The kinetics of the aggregation of deoxyhemoglobin S in concentrated phosphate buffer were studied turbidimetrically in the presence of organic phosphates. The addition of inositol hexaphosphate shortened the delay time by 35–80%, the range depending on the hemoglobin concentration. The logarithmic plot of delay time versus hemoglobin concentration in 1.85 M potassium phosphate buffer, pH 7.34, showed a linear relationship with a slope ( n value) of 2.5 ± 0.14 in the absence of inositol hexaphosphate, while the slope was decreased to 2.1 in the presence of inositol hexaphosphate. The turbidity ( A 700) of aggregates per g deoxy Hb S was found to be lower in the presence of inositol hexaphosphate than in the absence of inositol hexaphosphate. The solubility of deoxy Hb S was decreased by inositol hexaphosphate. 2,3-Diphosphoglyceric acid also showed an effect similar to that of inositol hexaphosphate. However, if the pentacyclohexylammonium salt of diphosphoglyceric acid were used, the delay time was increased significantly. The opposite effect of the pentacyclohexylammonium salt of diphosphoglyceric acid on the delay time was found to be attributed to the anti-gelation effect of the pentacyclohexoxylammonium ion. The difference in the effect of inositol hexaphosphate and diphosphoglyceric acid on the delay time appears to be related to their effect on the solubility of deoxy Hb S.

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.

Effect of 2, 3-diphosphoglycerate on the solubility of deoxy-sickle hemoglobin.

We have examined the effect of 2, 3-diphosphoglycerate (DPG) on the solubility of deoxy-sickle hemoglobin (deoxy-Hb S) under conditions such that concentration, pH, and osmolarity of deoxy-Hb S solutions approached physiological. The range of DPG/Hb molar ratios encompassed the extremes found for this ratio in erythrocytes from individuals with sickle cell anemia. After monomer-polyer equilibrium had been established, the phases were separated by centrifugation and assayed for concentrations of Hb and DPG. DPG had no effect on the solubility of deoxy-Hb S. Furthermore, at DPG/Hb molar ratios less than one, there was no preferential incorporation of deoxy-Hb S containing bound DPG into polymers. At DPG/Hb molar ratios greater than one, concentrations of free DPG in monomer and polymer phases were virtually identical. Thus, under the specified equilibrium conditions, DPG is not a determining factor in the polymerization of deoxy-Hb S.