non-S Hemoglobins Research Articles

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.

Intracellular Hemoglobin S Polymerization and the Clinical Severity of Sickle Cell Anemia

AbstractRecent work has enabled us to quantitate the four variables (2,3-DPG concentration, pHi, non-S hemoglobin composition, and O2 saturation) that modulate the equilibrium solubility (csat) of Hb S inside sickle erythrocytes (SS RBCs). Using measured values of mean corpuscular hemoglobin concentration (MCHC), 2,3-DPG concentration, and %Hb (F+A2), along with estimates of pHiand the Δcsat due to partial oxygenation of SS RBCs in the microcirculation, we calculated the mean polymer fraction (fp) in erythrocytes from 46 SS homozygotes. Values of fp derived from the conservation of mass equation ranged from 0.30 to 0.59. MCHC and %Hb F were major determinants of the magnitude of fp; 2,3-DPG concentration and pHialso contributed, but to a lesser extent. A clinical severity score (CSS) was assigned to each patient based on mean hospitalization rate. There was a weak, but statistically significant, negative correlation between fp and steady state hematocrit (P = .017), but none between fp and whole blood hemoglobin concentration (P = .218). Although there was no correlation between fp and mean number of hospitalization days per year, patients with the greatest number of admissions and hospitalization days were found only among those who had an fp > 0.45. All five patients who died during the follow-up period (median, 7 years; range, 3 to 10 years) had fp values ≥0.48. However, patients with few admissions, low hospitalization days, and long survivals occurred at all fp levels. These results suggest that the clinical course of homozygous SS disease cannot be predicted by mean fpcalculations, which assume a homogeneous distribution of the five variables that modulate intraerythrocytic polymerization. A heterogeneous distribution is more likely; so the amount of polymerized Hb S could vary considerably among cell populations. Factors such as membrane abnormalities and endothelial cell interactions may also contribute to clinical severity.

Intracellular Hemoglobin S Polymerization and the Clinical Severity of Sickle Cell Anemia

Recent work has enabled us to quantitate the four variables (2,3-DPG concentration, pHi, non-S hemoglobin composition, and O2 saturation) that modulate the equilibrium solubility (csat) of Hb S inside sickle erythrocytes (SS RBCs). Using measured values of mean corpuscular hemoglobin concentration (MCHC), 2,3-DPG concentration, and %Hb (F+A2), along with estimates of pHiand the Δcsat due to partial oxygenation of SS RBCs in the microcirculation, we calculated the mean polymer fraction (fp) in erythrocytes from 46 SS homozygotes. Values of fp derived from the conservation of mass equation ranged from 0.30 to 0.59. MCHC and %Hb F were major determinants of the magnitude of fp; 2,3-DPG concentration and pHialso contributed, but to a lesser extent. A clinical severity score (CSS) was assigned to each patient based on mean hospitalization rate. There was a weak, but statistically significant, negative correlation between fp and steady state hematocrit (P = .017), but none between fp and whole blood hemoglobin concentration (P = .218). Although there was no correlation between fp and mean number of hospitalization days per year, patients with the greatest number of admissions and hospitalization days were found only among those who had an fp > 0.45. All five patients who died during the follow-up period (median, 7 years; range, 3 to 10 years) had fp values ≥0.48. However, patients with few admissions, low hospitalization days, and long survivals occurred at all fp levels. These results suggest that the clinical course of homozygous SS disease cannot be predicted by mean fpcalculations, which assume a homogeneous distribution of the five variables that modulate intraerythrocytic polymerization. A heterogeneous distribution is more likely; so the amount of polymerized Hb S could vary considerably among cell populations. Factors such as membrane abnormalities and endothelial cell interactions may also contribute to clinical severity.

Intracellular Polymerization

The extent of intracellular polymerization of hemoglobin S, leading to loss of erythrocyte deformability and eventual morphological sickling, is primarily determined by oxygen saturation and intracellular hemoglobin concentration and composition. Epidemiological analysis of sickle cell disease severity among the sickle syndromes and studies of the biophysics of intracellular polymerization were used to estimate the potential clinical benefit of various therapeutic strategies. These strategies include those designed to increase deoxyhemoglobin S solubility; to increase erythrocyte volume or water content, thereby reducing the intracellular hemoglobin concentration; or, most recently, to decrease the proportion of hemoglobin S by increasing the amount of non-S hemoglobin. Increasing levels of hemoglobin F is of particular interest due to its "sparing" effect in inhibiting polymerization, the well-characterized epidemiological associations of high levels of hemoglobin F with reduced disease severity, and recent studies of drug-induced increases in hemoglobin F. Our analyses of equilibrium polymer formation at physiological oxygen saturation values suggest that small decreases in polymer formation at intermediate levels of hemoglobin F may give rise to a small decrease in anemia (as associated with homozygous alpha-thalassemia coexistent with sickle cell anemia), but that greater reductions in polymer formation may be necessary to effect a significant improvement in disease severity. Current studies of hydroxyurea-induced increases of hemoglobin F give cautious optimism that therapeutically useful levels may be attainable.

Sickle-Cell Trait and Physical Training

Sickle cell anemia is the first disease to be understood on a molecular level. 1 The amino acid valine replaces the normal glutamic acid residue at the sixth position of the β-globin chain, the consequence of a GAG to GTG codon mutation. The resultant sickle hemoglobin, or HbS (α 2 β s 2 ) has a peculiar tendency to self-associate and form long strands of polymer on deoxygenation, which leads to red cell morphologic changes and to abnormal rheologic properties in the microcirculation. 2 See also p 1140. The primary determinants of the rate and extent of hemoglobin S polymerization, at any given oxygen saturation, are the intracellular hemoglobin concentration and hemoglobin composition (fraction of S and non-S hemoglobins). In addition, the ambient temperature, 2,3-diphosphoglycerate concentration, and pH can affect the polymerization tendency, albeit to a lesser extent, under normal physiologic conditions. 2,3 Thus, the hemolytic and overall clinical severity of

Polymerization in erythrocytes containing S and non-S hemoglobins

We analyzed the effects of protein and water nonideality and of erythrocyte heterogeneity on the polymerization of hemoglobin S in cells where there were significant amounts of non-S hemoglobins, sickle trait (AS), and SC disease. For AS erythrocytes, the calculated predicted results were in good agreement with measured polymer formation as previously reported (Noguchi C.T., D.A. Torchia, and A.N. Schnechter, 1981, J. Biol. Chem. 256:4168-4171). Throughout much of the physiologically relevant oxygen saturation region, polymer was not formed in AS erythrocytes. Measurements of polymer formation in SC erythrocytes as a function of oxygen saturation using 13C NMR are reported here and also are in good agreement with the calculated predicted results. As in sickle (SS) erythrocytes, polymer can be detected in SC erythrocytes in the region above 60% oxygen saturation. The increased polymer formation in SC erythrocytes as compared with AS erythrocytes can be explained in terms of hemoglobin composition and concentration in SC erythrocytes, with the concomitant increase in the proportion of dense cells. These findings provide a basis for understanding the pathophysiology of sickle cell and of SC disease, in contrast to benign sickle trait, in terms of intracellular polymer formation.

Solubilization of hemoglobin S by other hemoglobins.

The polymerization of mixtures of Hb S with hemoglobins A, A2, and F has been investigated by analysis of the proportions of S and non-S hemoglobin both in the supernate and in the pellet after centrifugation. In all cases the non-S hemoglobin was incorporated into the polymer even in the absence of hybrids in the order A > A2 > F. The solubility of Hb S is substantially increased by the other hemoglobins, especially by Hb F, which would account for its antisickling effect. It appears that the excluded volume effect of the other hemoglobin on Hb S is largely counterbalanced by the solubilizing effect arising from the interaction between the two hemoglobins in solution. The ability of hybrid hemoglobins to gel was demonstrated directly with tetramers in which alpha beta s dimers were covalently linked to alpha beta A, alpha delta A2, and alpha gamma F dimers.

Thermodynamics of gelation of sickle cell deoxyhemoglobin

This paper describes the thermodynamic behavior of gels of deoxyhemoglobin S. The solubility of the protein with respect to assembled hemoglobin fibers has been measured using a sedimentation technique. The solubility in 0.15 m-potassium phosphate buffer (pH 7.15) is found to decrease with increasing temperature, attain a minimum value of 0.16 g cm −3 at 37 °C, and then increase at higher temperatures. The amount of polymer present at various hemoglobin concentrations and temperatures is presented as part of a phase diagram that may be useful for the calibration of other measurement techniques. The effects of varying pH and urea concentration upon the solubility have also been studied. The heat absorption accompanying gelation has been measured by scanning calorimetry. Using sedimentation data on the amount of polymer formed, molar enthalpy changes are obtained. There is a large negative heat capacity change of − 197 cal deg. mol −1 and ΔH = 0 near 37 °C. Calorimetric molar enthalpy changes are found to agree with those calculated from the temperature dependence of the solubility by the van't Hoff equation. Our previous two-phase, two-component thermodynamic model of gelation is extended to include the effects of solution non-ideality. A large contribution to the activity of the hemoglobin in the solution phase results from the geometric effect of excluded volume. Incorporating solution phase non-ideality permits the calculation of standard state thermodynamic quantities for the gelation process at 37 °C: ΔG O ≅ −3 k cal mol −1, ΔH O ~ 0, ΔS O ~ 10 cal deg. −1 mol −1. The excluded volume effect is also capable of explaining observations of the minimum gelling concentrations of hemoglobin mixtures containing deoxyhemoglobin S without requiring copolymerization of the non-S hemoglobin.

Hemoglobin S-G (S-D) syndrome

Two subjects are described who had positive sickle cell preparations and routine alkaline electrophoretic patterns showing only hemoglobins S, F and A 2. Although this combination suggests sickle cell anemia, their hemoglobin separated into two major bands on agar gel electrophoresis using a citrate buffer, pH 6.2. The non-S hemoglobins were identified as Osu-Christiansborg [β52 Asp → Asn (D3)] in one instance and as G Galvestonton [β43 Glu → Ala (CD2)] in the other. By most criteria, including exercise testing for one and red cell survival studies for both, these patients resembled patients with sickle cell trait. Careful study and diagnosis are essential to avoid unfairly stigmatizing subjects with benign laboratory phenomena as having sickle cell anemia or even sickle cell disease.

Conformational requirements for the polymerization of hemoglobin S: Studies of mixed liganded hybrids

Gelation experiments with artificially formed half-liganded hybrid tetramers of hemoglobin S demonstrate that when either the α chains or the β s chains are fixed in the cyanmet (CNmet) liganded state, gelation occurs upon deoxygenation of the ferrous chains. The minimum concentration of hemoglobin required for gelation is equivalent for both hybrids ( α 2 cnmet β 2 s and α 2 β 2 scnmet ), is considerably higher than the concentration required to gel deoxy-Hb S ( α 2 β 2 s ), and can be restored to the lower minimum gelling point of α 2 β 2 s by reduction of the CNmet chains with dithionite. These results suggest that the most important conformational determinant of the deoxy state for polymerization of Hb S is the quaternary deoxy structure rather than the tertiary structural effect of the ligand state of the α or the β s chains, and are furthermore consistent with the notion that asymmetric deoxy-CNmet hybrid tetramers assume a conformation which resembles, but is not identical to that of deoxyhemoglobin. The results of gelation experiments with mixtures of hemoglobins S and A in which selected chains of one or both hemoglobins are in the CNmet form support the concept that certain non-S hemoglobins may participate in the sickling process by forming hybrid tetramers with Hb S (such as α 2 β a β s ). The conformational requirement for participation of these hybrids in polymers also appears to be a quaternary deoxy-like structure.

The interaction of sickle hemoglobin with DPG, CO 2 and with other hemoglobins: formation of asymmetrical hybrids.

In either the red cell or in concentrated solution, deoxyhemoglobin S (α2β2 6 val) possesses the unique property of aggregating into microfilaments, leading to increased viscosity and ultimately to gelation. This property may be strongly influenced by the presence of non-S hemoglobins or by a second amino acid sub-stitution on either the β chain (hb C-Harlem (α2β2 6 val 73 asn) (Bookchin et. Al., 1967) or the α chain (hb Memphis (α2 23 gln β 6 val) (Kraus et al., 1966). Furthermore, the sickling phenomenon may be affected by intracellular modifiers of hemoglobin function such as carbon dioxide (CO2) and 2,3-diphosphoglycerate (DPG). Both of these co-factors bind to hemoglobin at N-terminal amino groups of globin polypeptide chains. Carbamino formation occurs at the N-terminal amino groups of both the α and the β chains (Kilmartin and Rossi-Bernardi, 1969) while DPG binds to positively charged sites on the β chains including the α amino group of NA1 valine, the imidazole of H21 histidine and the ɛ amino group of EF6 lysine (Perutz, 1970). It is of interest that sickling is inhibited by cyanate, an agent which reacts preferentially with N-terminal amino groups of proteins (Cerami and Manning, 1971).

Dithionite Tube Test—A Rapid, Inexpensive Technique for the Detection of Hemoglobin S and Non-S Sickling Hemoglobin

Abstract An inexpensive, rapid, convenient screening tube test for the detection of hemoglobin S and non-S sickling hemoglobins has been developed, which has a molecular basis. The reagents consist of potassium phosphate, sodium dithionite, and saponin. When sickling red cells are introduced into such a solution, the red cells lyse immediately, the hemoglobin deoxygenates, the beta globin chains of each hemoglobin tetramer are displaced laterally, complementarity of steric fit between interacting hemoglobin tetramers is achieved in accordance with the recently modified Murayama hypothesis for the molecular mechanism of sickling, a nematic liquid crystal system is formed, and in the presence of hemoglobin S or non-S sickling hemoglobins, the system becomes turbid. On addition of urea, those nematic liquid crystal systems dependent upon hydrophobic bonds are dispersed.

Automated Dithionite Test for Rapid, Inexpensive Detection of Hemoglobin S and Non-S Sickling Hemoglobinopathies

Abstract Automated adaptations of dithionite and urea—dithionite tube tests are accurate, reliable, inexpensive methods for detecting hemoglobin S. More than 3,000 individuals have been screened (120 determinations per hour; reagent cost, 2 to 4 cents per test). The dithionite reagent consists of potassium phosphate, sodium dithionite, and saponin. When S hemoglobin contacts this reagent, the red cells lyse, and the hemoglobin deoxygenates and sickles, forming a hydrophobic-bond-dependent nematic liquid crystal system that is manifested as turbidity. The resulting AutoAnalyzer curve is strikingly and diagnostically different from that produced by hemoglobin A in the same reagent. Specificity of the automated dithionite test may be enhanced by use of the automated urea—dithionite test, which consists of a specimen set of two aliquots: one traverses a dithionite line, the other a urea—dithionite line. A comparison of transmittance in the two lines yields typical diagnostic curves because the urea disperses the sickling, with a consequently increased transmittance over that of the dithionite aliquot. Methods are discussed for recognizing non-S sickling hemoglobins and a few other rare hemoglobinopathies.

Biphasic blood oxygen dissociation curves in hemoglobin S hemoglobinopathies. Sickle cell heterozygotes

The oxygen binding curves of whole cells of heterozygous sickle cell hemoglobinopathies were measured. Under the conditions of measurement (T=25°, pH=6.4, pCO 2=0) the curves are biphasic. Samples in which 2,3-diphosphoglycerate (2,3-DPG) is very low or absent are monophasic, suggesting that there is a differential binding of 2,3-DPG to S and non-S hemoglobin in cells of heterozygotes. The biphasic curve has been confirmed in a hemoglobin SC patient, under physiologic conditions (T=37°, pH=7.4, pCO 2=44 mm).

Modified Sickledex tube test: a specific test for S hemoglobin

Summary 1. The Sickledex test is a screening method for the detection of S hemoglobin. However, the principle of this test has never been disclosed by the developers. On the basis of analytical studies conducted in our laboratory, we have concluded that the Sickledex test is a version of the Itano Solubility test which is predicted on the extraordinary insolubility of deoxygenated hemoglobin S in phosphate buffer systems. 2. We show that certain non-S sickling hemoglobins and also unstable hemoglobins may and can give false positive Sickledex tests. We have modified the Sickledex test by the reversal of a positive test in the presence of urea when the specimen is S hemoglobin. 3. If the test specimen is a non-S sickling hemoglobin, it is expected that a positive test will remain positive in the presence of urea. This differential property of the clearance of turbidity (the disbursal of the nematic liquid crystal system of sickled S hemoglobin) with urea under the conditions of this test is shown to occur only in the presence of unique, specific hydrophobic bonds between interacting tetramers, a condition which Murayama has shown to be essential to the sickling event in S hemoglobin. 4. The modified Sickledex tube test as herein described will thus identify specifically not only S hemoglobin but should also detect non-S hemoglobin as well.

Studies on abnormal hemoglobins. VII. The composition of the non-S hemoglobin fraction in sickle-cell anemia bloods; a comparative quantitative study by the methods of electrophoresis and alkali denaturation.

Abstract A comparative study, by means of electrophoresis and alkali denaturation, of the non-S hemoglobin fraction of typical sickle-cell anemia hemolysates was performed. It could be shown that this fraction is almost entirely composed of the alkali resistant fetal (F) hemoglobin. Whether small amounts of normal adult (A) hemoglobin also exist in this fraction cannot be definitely decided due to the relative insensitivity of the methods employed. For practical purposes, the quantity of S hemoglobin in typical sickle-cell anemia hemolysates may be expressed as the difference between the total hemoglobin and the percentage of the F pigment. Two atypical cases of sickle-cell disease whose bloods contain relatively large amounts of A and F pigments, besides S hemoglobin, are presented.