The effect of 2,3-diphosphoglycerate on the solubility of deoxyhemoglobin S

Although highly charged polyanions, such as inositol hexaphosphate, have been clearly shown to decrease the solubility of deoxyhemoglobin S, the effect of 2,3-diphosphoglycerate (DPG), the endogenous allosteric effector within the red cell, has been more controversial. In this work we have compared the effect of DPG on the solubility of native deoxyhemoglobin S and a derivative in which the DPG binding site is blocked by cross-linking the two beta 82 lysine residues. At pH 6.6 and 30 degrees C the solubility of deoxyhemoglobin S was found to be decreased by 15% (i.e., from 18.8 to 16.0 g/dl) in the presence of saturating concentrations of DPG. Under the same conditions DPG had no effect on the solubility of the cross-linked derivative. This result establishes unequivocally that the binding of DPG within the beta cleft directly facilitates the polymerization of deoxyhemoglobin S. Under physiological conditions, the solubility of deoxyhemoglobin S was found to be decreased by 6% in the presence of an equimolar concentration of DPG. A solubility decrease of this magnitude is sufficient to enhance the tendency of SS cells to sickle and may exacerbate the clinical symptoms of sickle cell disease.     

Kinetics of polymerization of deoxyhemoglobin S and mixtures of hemoglobin A and hemoglobin S at high hemoglobin concentrations

Transverse water proton relaxation times (T₂) have been measured as a function of time after deoxygenation of solutions containing hemoglobin S. The shortened T₂ values observed upon deoxygenation of hemoglobin S result from an increase in the correlation time (τ_c) of the water fraction irrotationally bound to deoxyhemoglobin S as it polymerizes. Therefore, the change in τ_c as a function of time after deoxygenation can be used to measure the rate of polymer formation. The change in τ_c observed is reasonably fit by the first-order equation τ = τ₀ (1 ? e^?kt) + τ_oxy. At a total hemoglobin concentration of approximately 300 mg/ml, the pseudo-first-order rate constant in a heterozygous AS sample is 25 times slower than in a homozygous S sample, k = 0.019 and 0.47 s^?1, respectively. Since the transit time for an erythrocyte in vivo is approximately 15 s, these results suggest that the heterozygous A/S erythrocyte would traverse the circulation and become reoxygenated before extensive polymerization and, therefore, cell sickling could occur. For the homozygous S/S erythrocyte, there is ample time for polymerization and for cell sickling during circulation.     

Refined crystal structure of deoxyhemoglobin S. II. Molecular interactions in the crystal

The refined crystal structure of deoxyhemoglobin S (Padlan, E. A., and Love, W. E. (1985) J. Biol. Chem. 260, 8272-8279) was used to analyze in detail the molecular interactions between hemoglobin tetramers in the crystal. The analysis confirms the close similarity and also the nonequivalence of the molecular interactions involving the two independent tetramers in the asymmetric unit of the crystal. The residue at the site of the hemoglobin S mutation, beta 6, is intimately involved in the lateral contacts between adjacent molecules. The molecular contacts in the crystals of deoxyhemoglobin S, deoxyhemoglobin A, and deoxyhemoglobin F were compared; some contacts involve the same regions of the molecule although the details of the interactions are very different. The effect of introducing an R state tetramer into the deoxyhemoglobin S strands was investigated using the known structure of carbon monoxyhemoglobin A. It was found that substituting a molecule of carbon monoxyhemoglobin A for one of the deoxyhemoglobin S tetramers results in extensive molecular interpenetration.     

Insights into the solution structure of human deoxyhemoglobin in the absence and presence of an allosteric effector

We present a nuclear magnetic resonance (NMR) study in solution of the structures of human normal hemoglobin (Hb A) in the deoxy or unligated form in the absence and presence of an allosteric effector, inositol hexaphosphate (IHP), using 15N-1H residual dipolar coupling (RDC) measurements. There are several published crystal structures for deoxyhemoglobin A (deoxy-Hb A), and it has been reported that the functional properties of Hb A in single crystals are different from those in solution. Carbonmonoxyhemoglobin A (HbCO A) can also be crystallized in several structures. Our recent RDC studies of HbCO A in the absence and presence of IHP have shown that the solution structure of this Hb molecule is distinctly different from its classical crystal structures (R and R2). To have a better understanding of the structure-function relationship of Hb A under physiological conditions, we need to evaluate its structures in both ligated and unligated states in solution. Here, the intrinsic paramagnetic property of deoxy-Hb A has been exploited for the measurement of RDCs using the magnetic-field dependence of the apparent one-bond 1H-15N J couplings. Our RDC analysis suggests that the quaternary and tertiary structures of deoxy-Hb A in solution differ from its recently determined high-resolution crystal structures. Upon binding of IHP, structural changes in deoxy-Hb A are also observed, and these changes are largely within the alpha1beta1 (or alpha2beta2) dimer itself. These new structural findings allow us to gain a deeper insight into the structure-function relationship of this interesting allosteric protein.     

Crystal structure of the C-terminal 10-kDa subdomain of Hsc70

The 70-kDa heat shock proteins (Hsp70), including the cognates (Hsc70), are molecular chaperones that prevent misfolding and aggregation of polypeptides in cells under both normal and stressed conditions. They are composed of two major structural domains: an N-terminal 44-kDa ATPase domain and a C-terminal 30-kDa substrate binding domain. The 30-kDa domain can be divided into an 18-kDa subdomain and a 10-kDa subdomain. Here we report the crystal structure of the 10-kDa subdomain of rat Hsc70 at 3.45 A. Its helical region adopted a helix-loop-helix fold. This conformation is different from the equivalent subdomain of DnaK, the bacterial homologue of Hsc70. Moreover, in the crystalline state, the 10-kDa subdomain formed dimers. The results of gel filtration chromatography further supported the view that this subdomain was self-associated. Upon gel filtration, Hsc70 was found to exist as a mixture of monomers, dimers, and oligomers, but the 60-kDa fragment was predominantly found to exist as monomers. These findings suggest that the alpha-helical region of the 10-kDa subdomain dictates the chaperone self-association.     

Human 70-kDa SHP-1L differs from 68-kDa SHP-1 in its C-terminal structure and catalytic activity.

The tyrosine phosphatase SHP-1 functions as a negative regulator in hematopoietic cell development, proliferation, and receptor-mediated cellular activation. In Jurkat T cells, a major 68-kDa band and a minor 70-kDa band were immunoprecipitated by a monoclonal antibody against the SHP-1 protein-tyrosine phosphatase domain, while an antibody against the SHP-1 C-terminal 19 amino acids recognized only the 68-kDa SHP-1. The SDS-gel-purified 70-kDa protein was subjected to tryptic mapping and microsequencing, which was followed by molecular cloning. It revealed that the 70-kDa protein, termed SHP-1L, is a C-terminal alternatively spliced form of SHP-1. SHP-1L is 29 amino acids longer than SHP-1, and its 66 C-terminal amino acids are different from SHP-1. The C terminus of SHP-1L contains a proline-rich motif PVPGPPVLSP, a potential Src homology 3 domain-binding site. In contrast to SHP-1, tyrosine phosphorylation of SHP-1L is not detected upon stimulation in Jurkat T cells. This is apparently due to the lack of a single in vivo tyrosine phosphorylation site, which only exists in the C terminus of SHP-1 (Y564). COS cell-expressed glutathione S-transferase-SHP-1L can dephosphorylate tyrosine-phosphorylated ZAP70. At pH 7.4, SHP-1L was shown to be more active than SHP-1 in the dephosphorylation of ZAP70. At pH 5.4, SHP-1L and SHP-1 exhibited similar catalytic activity. It is likely that these two isoforms play different roles in the regulation of hematopoietic cell signal transduction.     

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.     

The hand of the helix of deoxyhemoglobin S fibers.

Electron micrographs of deoxyhemoglobin S fiber cross sections provide an end-on view of the fiber whose appearance is sensitive to small changes in orientation. We have developed a procedure to exploit this sensitivity in order to determine the hand of these particles. In a sickle hemoglobin fiber the hemoglobin molecules form long pitch helical strands which twist about the particle axis with a pitch of about 3000 A. Tilting a 400-A-thick cross section by a few degrees aligns one of the long pitch helices so that it is nearly parallel to the direction of view. When a strand of hemoglobin molecules in a fiber is aligned in this manner it appears as a strongly contrasted bright spot. It is this spot, rather than the fiber axis, which appears to be the apparent center of rotation of the cross section. The direction of the displacement of the spot from the particle axis depends upon the particle hand and tilt direction. We have used this property to determine that sickle hemoglobin fibers are right-handed particles. This method may be applicable to other particles with long pitch helices as well.     

Refined crystal structure of deoxyhemoglobin S. I. Restrained least-squares refinement at 3.0-A resolution

The crystal structure of deoxyhemoglobin S has been refined at 3.0-A resolution using the Hendrickson-Konnert restrained least-squares method. Comparison with the structure of deoxyhemoglobin A reveals a hingelike movement of the beta-chain A helices, which are involved in molecular contacts, toward the EF corners of their respective subunits. This movement brings the amino termini of the beta-chains closer to the molecular dyad. The A helices remain alpha-helical throughout their entire lengths. No other major structural difference is found between deoxyhemoglobin A and deoxyhemoglobin S.     

Effect of pH, carbamylation and other hemoglobins on deoxyhemoglobin S aggregation inside intact erythrocytes as detected by proton relaxation rate measurements.

Measurement of the transverse water proton relaxation rate has been used to study the effect of pH, carbamylation, and other hemoglobins on the aggregation of deoxyhemoglobin S inside intact erythrocytes. Upon complete deoxygenation, cyanate-treated ($\text{S}\text{S}$) erythrocytes and erythrocytes heterozygous with respect to hemoglobin S ($\text{A}\text{S}$, $\text{C}\text{S}$, and $\text{S}\text{D}$) have high transverse water proton relaxation rates very similar to the values obtained with homozygous ($\text{S}\text{S}$) erythrocytes. These results suggest extensive intermolecular interactions between deoxyhemoglobin S molecules and a resultant increase in the correlation time for the small fraction of “irrotationally bound” water. When the transverse relaxation rate in deoxygenated ($\text{S}\text{S}$) erythrocytes was measured as a function of pH, the maximum rate was observed between pH 7.0 and 7.5. Upon increasing the pH beyond this range the observed relaxation rate decreases as does the number of sickled cells. Upon decreasing the pH, the observed transverse relaxation rate also decreases but the ratio of values from $\text{deoxy}\text{oxy}$ ($\text{S}\text{S}$) erythrocytes remains in the normal range of 4–6 and the number of sickled cells does not change. Therefore, the deoxyhemoglobin S aggregate inside sickled erythrocytes, as observed by water proton relaxation rates, is not altered by carbamylation or by the presence of nongelling hemoglobins. In addition, the enhancement of the relaxation rates as a function of pH is consistent with the number of sickled forms observed.     

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.     

Crystallization of deoxyhemoglobin S by fiber alignment and fusion.

Fibers of deoxyhemoglobin S obtained directly from lysed sickled red blood cells have been compared with fibers from chromatographically pure deoxyhemoglobin S solutions of known chemical composition. Electron micrographs of negatively stained specimens reveal that the molecular packing within the fibers remains largely invariant with changes in pH, ionic strength, Mg²⁺ concentration, 2,3-diphosphoglycerate concentration, temperature or the method of deoxygenation.When solutions of chromatographically pure deoxyhemoglobin S are stirred, the fibers align into well defined fascicles. After several hours of stirring, long needles and twisted ribbons develop and in a relatively short time replace the fascicles in solution. With continued stirring all forms are replaced by small crystals. By use of electron microscopy and low-angle X-ray diffraction we have found these crystals to have cell parameters indistinguishable from those of crystals grown in polyethylene glycol and citrate/phosphate buffer at pH 5 to 6 (Wishner et al., 1975a).Our evidence indicates that crystal formation in stirred solutions of deoxyhemoglobin S is the result of a progressive alignment and fusion of the fibers, and that the molecular arrangement within the fibers is closely related to that within the crystal. The remarkable pH invariance of the molecular packing within the fiber and crystal structures is consistent with the dominance of hydrophobic bonding between molecules. The β6-valine contact observed by Wishner et al. (1975b) is apparently the pathological contact responsible for the polymerization of deoxyhemoglobin S in vivo. On the basis of our observations and knowledge of the crystal structure we propose that the deoxyhemoglobin S fiber consists of eight molecular double strands, four of which run in each direction along the length of the fiber.     

Orthophosphate determination in the presence of high concentrations of ATP

A method to measure orthophosphate which contaminates samples of ATP was developed. Concentrations of orthophosphate as low as 0.4% of the ATP concentration were determined using a zinc-molybdate reagent [D. A. Bencini, J. R. Wild, and G. A. O'Donovan, Anal. Biochem. 132, 254-258 (1983)] and continuous spectrophotometric monitoring of chromophore formation. Since the rate of ATP hydrolysis was pseudo-first order and was slow compared to the rate of chromophore formation, the initial concentration of phosphate could be readily determined by extrapolation to zero time. The method is rapid and reproducible, and requires a single, stable reagent.     

Hydrogen exchange kinetics of human hemoglobins. The pH dependence of solvent accessibility in cyanomet-, oxy-, and deoxyhemoglobin.

The hydrogen exchange kinetics of human oxy-, deoxy-, and cyanomethemoglobin have been measured as a function of pH by the tritium tracer method. At 5 degrees C and in phosphate buffer both liganded and unliganded forms of ferrohemoglobin exhibit deviations from the regular pH dependence of exchange that is characteristic of cyanomethemoglobin. In oxyhemoglobin, the deviation from the normal exchange pattern is centered at pH 7.4 and is in the direction of increased exchange or solvent accessibility. The effect in deoxyhemogloin, while occurring at the same pH and being of the same order of magnitude, is in the opposite direction, thus suggesting a pH-induced conformational transition leading to a less accessible structure. The width of these pH-induced deviations in solvent accessibility is approximately 1 pH unit in both cases. We propose a model in which specific interactions between charged groups in both froms of ferrohemoglobin account for these deviations.     

Circular dichroism of aggregated deoxyhemoglobin S and the effect of amino acids.

It is well known that deoxyhemoglobin S (deoxy Hb S) aggregates at 37 °C and that it disaggregates at 1–5 °C. In this study solutions of pure Hb S at concentrations of 20–22 g/100 ml exhibit a normal circular dichroic spectrum in the range 250–650 nm at the temperature 1 °C. However, by the proper manipulation of the following parameters: temperatures of 1, 24 and 37 °C as well as the times required to change temperature and periods of maintaining at a certain temperature, five stages with different circular dichroic spectra can be produced. Not only the dichroic spectra of these stages are different but the kinetic behavior and stability of each of these stages are different. The evidence suggests that the mechanism of aggregation is similar to crystallization; that is, it exhibits a period of nucleation followed by growth. The overall kinetics of circular dichroic changes are described. At representative solution conditions the circular dichroic changes have been compared and found to parallel gel formation with pure Hb S. Also, the effect of certain anti-sickling amino acids (Sophianopoulos, A. J., et al. (1974) Clin. Biochem.7, 112–118) on the minimum Hb S concentration at which circular dichroic changes occur has been studied, and arginine chloride and arginine aspartate were found to raise this minimum concentration appreciably.