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 pHi and the Deltacsat 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.

Antisickling effects of 2,3-diphosphoglycerate depletion.

Elevation of 2,3-bisphosphoglycerate (2,3-DPG) in sickle erthrocytes (SS RBCs) and concomitant acidification of the cell interior promote polymerization by decreasing the solubility (csat) of deoxyhemoglobin S. The antisickling effect of 2,3-DPG depletion was evaluated after activation of the 2,3-DPG phosphatase activity of bisphosphoglycerate mutase by glycolate-2-phosphate, leading to rapid loss of intracellular 2,3-DPG. To ensure its maximal reduction in a physiologic medium, isosmotic CO2/bicarbonate-buffered saline, pH 7.

Improvement of sickle cell anemia by iron-limited erythropoiesis.

We report the hematologic and clinical features of four adult patients (Pts.) with sickle cell anemia and iron-limited erythropoiesis. Two of the Pts.

Sparing effect of hemoglobin F and hemoglobin A2 on the polymerization of hemoglobin S at physiologic ligand saturations.

Recent interest in therapies for sickle cell anemia based on elevating fetal Hb has made accurate estimates of the sparing effect of fetal Hb (Hb F) and other non-sickle Hbs on sickle Hb (Hb S) polymerization essential. We have developed a technique, using HbCO as surrogate for HbO2, that enables us to assess the solubility of Hb S as a function of ligand saturation under conditions that mimic those of the sickling disorders. Equimolar mixtures of unliganded Hb S with Hb F or normal Hb A2 were isosoluble.

2,3-Diphosphoglycerate and intracellular pH as interdependent determinants of the physiologic solubility of deoxyhemoglobin S.

We have established that 2,3-diphosphoglycerate (2,3-DPG) content and intracellular pH exert separate, but interdependent, effects on the equilibrium solubility (csat) of deoxyhemoglobin S (deoxy-Hb S) that act in concert to modulate intraerythrocytic polymer formation. In a nonphysiologic csat assay system, a steep dependence of csat on pH in the physiologic range 7.0 to 7.

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.

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.

Autologous survival of cyanate-treated cryopreserved sickle erythrocytes.

The effects of carbamylation and frozen storage on the autologous 51Cr survival and metabolic features of sickle erythrocytes (S-RBCs) were determined. Red cells from four patients with sickle hemoglobinopathies were treated with 50 mM sodium cyanate for 2 hr (37 degrees C), glycerolized and frozen (-80 degrees C) for 62-153 days. The mean in vitro loss of S-RBCs from the combination of cyanate treatment and cryopreservation was 23.

Deoxygenated sickle hemoglobin. Modulation of its solubility by 2,3-diphosphoglycerate and other allosteric polyanions.

The effects of 2,3-diphosphoglycerate (DPG) and other allosteric polyanions of the phosphate or sulfate ester class (inositol hexaphosphate (IHP), ATP, pyridoxamine-5'-phosphate (PMP), and inositol hexasulfate (IHS] on the solubility of deoxyhemoglobin S, and the oxygen affinity of Hb A were evaluated. Their effects on the saturation concentration (csat) indicated promotion of gelation in each case, according to the following order of molar effectiveness: IHP greater than IHS greater than DPG greater than ATP much greater than PMP. Four polybasic carboxylic acids (benzenetricarboxylate (trimesic acid), benzenetetracarboxylate (BTC), benzenepentacarboxylate (BPC), and benzenehexacarboxylate (BHC] were evaluated as well.

Noncovalent inhibitors of sickle hemoglobin gelation: effects of tetrasubstituted ammonium salts.

The effects of various tetrasubstituted ammonium compounds on the solubility of deoxygenated sickle hemoglobin were evaluated. These conclusions were drawn from the slopes of the solubility profiles obtained: (1) for the homologous series of tetraalkylammonium chloride salts (R4NCl), antigelling potency increased with increasing chain length of the R group: Ch3 less than C2H5 less than C3H7 less than C4H9; (2) for halide salts of the tetrabutylammonium cation there was no difference in effectiveness among the anions examined (Br-, Cl-, F-), presumably because of the extremely potent salting-in effect of this cation; (3) substitution of a benzyl for an alkyl group in three tetraalkylammonium chloride salts, C6H5CH2(R)3NCl, where R = CH3, C2H5, or C4H9, potentiated the antigelling capacity by as much as eight-fold. Because they are substantially more water soluble than any other class of noncovalent inhibitors examined to date, further manipulation of the aryl substituent on these compounds may contribute to the design of an effective antisickling agent.

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.

Thermodynamic studies of polymerization of deoxygenated sickle cell hemoglobin.

Solubilities of deoxygenated sickle cell hemoglobin (deoxy-Hb S), at varying pH and temperature over a range of concentrations encompassing those found in erythrocytes, were measured. The technique involved ultracentrifugation, which gave values of the supernatant concentration and the mass of the sedimented material. The data establish that the solubility of doexy-Hb S is the saturation concentration and is independent of initial concentration.