Kinetics of sickle hemoglobin polymerization. I. Studies using temperature-jump and laser photolysis techniques

Using a combination of laser photolysis and temperature-jump techniques, the kinetics of hemoglobin S polymerization have been studied over a wide range of delay times (10(-3) to 10(5)s), concentrations (0.2 to 0.4 g/cm3) and temperatures (5 to 50 degrees C). A slow temperature-jump technique was used to induce polymerization in samples with delay times between 10(2) seconds and 10(5) seconds by heating a solution of completely deoxygenated hemoglobin S. For samples with shorter delay times, polymerization was induced by photodissociating the carbon monoxide complex in small volumes (10(-9) cm3) using a microspectrophotometer equipped with a cw argon ion laser. The photolysis technique is described in some detail because of its importance in studying hemoglobin S polymerization at physiological concentrations and temperatures. In order, to establish conditions for complete photodissociation with minimal laser heating, a series of control experiments on normal human hemoglobin was performed and theoretically modeled. The concentration dependence of the tenth time is found to decrease with increasing hemoglobin S concentration. In the range 0.2 to 0.3 g/cm3, the tenth time varies as the 36th power of the hemoglobin S concentration, while in the range 0.3 to 0.4 g/cm3 it decreases to 16th power. As the tenth times become shorter, the progress curves broaden, with the onset of polymerization becoming less abrupt. For tenth times greater than about 30 seconds, measurements with the laser photolysis technique on small volumes yield highly irreproducible tenth times, but superimposable progress curves, indicating stochastic behavior. The initial part of the progress curves from both temperature-jump and laser photolysis experiments is well fit with an equation for the concentration of polymerized monomer, delta (t) = A[cosh (Bt) -1], which results from integration of the linearized rate equations for the double nucleation mechanism described in the accompanying paper (Ferrone et al., 1985). The dependence of the parameters A and B on temperature and concentration is obtained from fitting over 300 progress curves. The rate B has a large concentration dependence, varying at 25 degrees C from about 10(-4) S-1 at 0.2 g/cm3 to about 100 s-1 at 0.4 g/cm3.     

The interaction of hemoglobin with phosphatidylserine vesicles

The interaction of hemoglobin with phosphatidylserine vesicles at low ionic strength and pH conditions was studied. The fluorescence intensity of a lipid embedded probe was quenched by bound Hb but could not be reversed by an elevation of ionic strength and pH. The irreversibility of the fluorescence quenching is a time-dependent process associated with changes in the heme Soret and visible spectra. The rate of these changes was much faster for methemoglobin than for either cyanomethemoglobin or oxyhemoglobin. Elevation of ionic strength released out of the bound hemoglobin into the water phase most of the globin but only a small fraction of the heme. The data are interpreted as demonstrating the ability of phosphatidylserine vesicles to compete with globin for the heme group. When Hb binds to the liposome, heme is being transferred into the lipid phase and the rate-limiting step is the dissociation of the heme-globin complex. The fact that binding of heme to the lipid vesicles is very strong was demonstrated by the failure of hemin to interact with globin when the two were rapidly mixed in the presence of phosphatidylserine vesicles. A multi-step process is suggested to explain the results of Hb phosphatidylserine interaction.     

The interaction of adenine nucleotides with the red cell membrane: A calorimetric study

The interaction of two adenine nucleotides with the red cell membrane was investigated using highly sensitive differential scanning calorimetry. It was found that ADP and AMP-PNP (an ATP analogue) preferentially modify the A transition, which has been shown to involve the unfolding of a portion of spectrin, an erythrocyte membrane protein complex. The interaction of ADP with spectrin was shown to be reversible and facilitated by the usual cofactor, Mg²⁺. The ADP-induced modification, however, is only observed for membrane associated spectrin; ADP has no effect on extracted spectrin. The results presented are consistent with an ADP-induced conformational change in the spectrin complex which leads to a change in the spectrin-membrane interaction. ADP, but not AMP-PNP, is shown to modify an additional calorimetric transition (B₂) associated with a structural change in the transmembrane protein band 3. This behavior is characteristic of inhibitors of anion transport in the red cell. ADP is also found to be an inhibitor of anion transport in red cells.     

Ligand binding and the gelation of sickle cell hemoglobin.

The solubility equilibrium between monomer and polymer which has been shown to exist in deoxyhemoglobin S solutions is examined in solutions partially saturated with carbon monoxide. The total solubility is found to increase monotonically with increasing fractional saturation. At low fractional saturations the increase is nearly linear, amounting roughly to an increase of 0.01 g cm^?3 in solubility for each 10% increase in fractional saturation. Linear dichroism measurements on the spontaneously aligned polymer phase are used to examine the composition of the polymer as a function of the fractional saturation of the corresponding solution phase. The dichroism experiments show that the polymer phase contains less than 5% of CO-liganded hemes even at supernatant fractional saturations in excess of 70%. The polymer selects against totally liganded hemoglobin molecules by a minimum factor of 65 and against singly liganded molecules by a factor of at least 2.5. Consequently, polymerized hemoglobin S has a ligand affinity which is significantly lower than that of monomeric hemoglobin S in the deoxy quaternary structure.The kinetics of the polymerization reaction in the presence of CO are similar to those observed in pure deoxyhemoglobin S solutions. The polymerization is preceded by a pronounced delay, the duration of which, t_d, is proportional roughly to the 30th power of the solubility. At low fractional saturations, this amounts to a tenfold increase in t_d for each 10% increase in the fractional saturation.These results show that the polymerization reaction is nearly specific for deoxyhemoglobin. Models for the dependence of the solubility and the polymer saturation on ligand partial pressure demonstrate the importance of solution phase non-ideality in determining the solubility of mixtures. The results require selection against partially liganded species which is significantly greater than is predicted by the two-state allosteric model. The data are compatible with either sequential or allosteric models in which the major polymerized component is the unliganded hemoglobin molecule.     

Alterations of red cell membrane proteins and hemoglobin under natural and experimental oxidant stress

We compared on red cell membrane proteins and hemoglobin (Hb) the effects of (i) natural oxidant stress that has been suggested to occur in a variety of oxidative hemolytic anemias, and (ii) experimental stress induced by hydrogen peroxide. SDS-polyacrylamide gel electrophoresis was used for protein analysis. Under natural conditions (thalassemias, hemoglobinopathies with Hb unstability), a high molecular weight polymer (HMWP) and variable amounts of globin mono- and dimers became apparent. Furthermore, a major 12 kDa polypeptide, its dimer, and conspicuous spectrin degradation products in the band 2.2–2.6 region occurred in a patient carrying the highly unstable Hb Hammersmith. Under experimental conditions, incubation of erythrocyte ghosts with H₂O₂ in the presence of minimal concentration (25 μM) of Hb generated a HMWP at the expense of membrane proteins, mainly spectrin. Incubation of a diluted (200 μM) membrane-free hemolysate with H₂O₂ induced a HMWP, an array of globin oligomers and a 12 kDa polypeptide similar to that mentionned above. Therefore, the damage to the red cell membrane present in various oxidative hemolytic anemias, including polypeptide polymerisation and breakdown, can be produced by experimental oxidant stress. These observations support the view that the alterations described in the patients result directly from oxidative reactions. However, we did not observe in the patient the sharp breakdown of polyunsaturated fatty acids that was triggered in vitro by H₂O₂ in the presence of Hb acting as a catalyst. In most cases, oligo- and polymers were resistant to β-mercaptoethanol, and the chemical nature of the underlying cross-links is discussed. To our knowledge, the 12 kDa polypeptide, that we consider as arising from globin proteolysis, has never been reported under pathological conditions.     

The reduction of nitroblue tetrazolium by red blood cells: a measure of red cell membrane antioxidant capacity and hemoglobin-membrane binding sites

The reduction of nitroblue tetrazolium (NBT) with intact Red Blood Cells (RBCs) is biphasic with an initial rapid reduction followed by a slower second phase. This biphasic kinetics has been explained with the initial rapid phase attributed to antioxidants in the red cell which reduce membrane bound NBT and the slower phase associated with the reaction of NBT with membrane bound hemoglobin. This model has been confirmed by a utilization of a number of red cell modifications which either increase the red cell antioxidants (vitamin C and vitamin E) or damage the red cell membrane (cumene hydroperoxide and N-ethylmaleimide). The utilization of this assay for human blood samples was investigated by studying a series of 20 human subjects ranging between 34 and 87 years of age. It was possible to fit all of these samples with two adjustable parameters which reflect the red cell membrane antioxidant capacity (x) and the hemoglobin membrane interactions (m). The antioxidant capacity shows a significant (p < 002; R = -.67) decrease with age. This finding is consistent with a decrease in the level of antioxidants in aged subjects. In addition, the number of hemoglobin membrane sites are negatively correlated with the antioxidant capacity (p < .02; R = -.52) suggesting that the oxidative stress associated with reduced antioxidants results in increased hemoglobin-membrane interactions.     

The interaction of folylpolyglutamates with deoxyhemoglobin. Identification of the binding site

Previous studies have shown that pteroylheptaglutamate (PteGlu7) can form a 1:1 complex with deoxyhemoglobin. The solution and crystallographic studies reported in this paper delineate the nature of the PteGlu7 binding site. We find that the three structural elements of PteGlu7 (the pteridine moiety, the p-aminobenzoyl portion, and the glutamate groups) each contribute to the binding energy by interacting with residues in the central cavity between the beta subunits and with residues at the alpha 1 beta 1 interface. Identification of the 2,3-diphosphoglycerate (DPG) binding site as part of the PteGlu7 binding site was accomplished in two ways; first by the demonstration of reduced PteGlu7 binding to hemoglobin selectively modified by pyridoxylation at this site, and second by the finding that DPG and PteGlu7 bind to deoxyhemoglobin in a competitive manner. In addition, since analogs of PteGlu7 in which the pteridine moiety is modified display reduced binding, it can be concluded that the pteridine group also contributes significantly to the binding energy. The crystallographic studies are completely consistent with the results determined in solution. A difference electron density image at 4.3 A resolution shows that the pteridine and p-aminobenzoyl groups are nestled against an interior edge of the alpha 1 beta 1 interface with the pteridine ring interacting with Phe 36 alpha 1 and the p-aminobenzoyl group positioned against a portion of the H helix between residues Lys 132 beta 1 and Ala 135 beta 1. The difference density for the glutamate residues is less well resolved (for reasons described in the text), but it is clear that some of the carboxylate side chains must interact with residues at the DPG binding site.     

The production of activated oxygen species by an interaction of methemoglobin with ascorbate

Ascorbate reacts with methemoglobin to produce reactive oxygen species, most probably hydroxyl radicals. The main features of this system are: a) disappearance of ascorbate; b) consumption of oxygen with an ascorbate/O2 stoichiometry of 2:1; c) requirement of unliganded heme iron; d) formation of H2O2. The proposed mechanism involves an ascorbate-mediated interconversion of methemoglobin and oxy-hemoglobin, resulting in the production of H2O2. This product is decomposed by hemoglobin to produce hydroxyl radicals according to a Fenton-like reaction in which ascorbate recycles methemoglobin to hemoglobin. Alternative pathways of formation and of decomposition of H2O2 in this system appear to play a minor role.     

Comparison of cholesterol-phospholipid interaction in bilayers of liposomes and the red cell membrane

The energetics of interactions of cholesterol with phospholipid in simple liposome bilayers were compared with those in the bilayer of the human erythrocyte membrane, by measuring cholesterol distribution between erythrocytes and liposomes prepared from their whole phospholipid extract. With liposomes of a range of initial cholesterol contents, the equilibrium value for $\text{r}$, the ratio of cholesterol/phospholipid in the liposomes to that in the cells, is in the range 1.1–1.2. The closeness of this value to 1.0 indicates that overall cholesterol-phospholipid interaction in the cell membrane is similar to that in liposomes. However, while the deviation from 1.0 is small, and could arise from average cholesterol-phospholipid interactions in the membrane being only 0.06 to 0.1 kcal · mol^?1 weaker than in liposomes, it could also result from 10 to 20% of the cell membrane phospholipid being unavailable to mix with cholesterol.     

Osmotic tolerance limits of red blood cells from umbilical cord blood

Effective methods for long-term preservation of cord red blood cells (RBCs) are needed to ensure a readily available supply of RBCs to treat fetal and neonatal anemia. Cryopreservation is a potential long-term storage strategy for maintaining the quality of cord RBCs for the use in intrauterine and neonatal transfusion. However, during cryopreservation, cells are subjected to damaging osmotic stresses during cryoprotectant addition and removal and freezing and thawing that require knowledge of osmotic tolerance limits in order to optimize the preservation process. The objective of this study was to characterize the osmotic tolerance limits of cord RBCs in conditions relevant to cryopreservation, and compare the results to the osmotic tolerance limits of adult RBCs. Osmotic tolerance limits were determined by exposing RBCs to solutions of different concentrations to induce a range of osmotic volume changes. Three treatment groups of adult and cord RBCs were tested: (1) isotonic saline, (2) 40% w/v glycerol, and (3) frozen–thawed RBCs in 40% w/v glycerol. We show that cord RBCs are more sensitive to shrinkage and swelling than adult RBCs, indicating that osmotic tolerance limits should be considered when adding and removing cryoprotectants. In addition, freezing and thawing resulted in both cord and adult RBCs becoming more sensitive to post-thaw swelling requiring that glycerol removal procedures for both cell types ensure that cell volume excursions are maintained below 1.7 times the isotonic osmotically active volume to attain good post-wash cell recovery. Our results will help inform the development of optimized cryopreservation protocol for cord RBCs.     

The interaction of leucocidin with the cell membrane of the polymorphonuclear leucocyte

1. When leucocidin is incubated with leucocytes it is inactivated in solution and only a little adsorption takes place. This reaction has been used to purify the cell membrane. 2. The interaction of the membrane with leucocidin is very complex and at least three phenomena occur: (a) An inactivation of leucocidin in solution by large amounts of membrane which is synergistic between the two components of leucocidin, is thermolabile and is not inhibited by electrolyte. (b) An adsorption of leucocidin which is synergistic between the two components of leucocidin, does not proceed to the same extent as the inactivation in solution and is a function of the phospholipid components. Phospholipids isolated from the membrane adsorb leucocidin but the adsorption requires the presence of several molecular species. (c) Polymerization of leucocidin induced by tenfold smaller amounts of membrane than are required to bring about the first two interactions. The polymerization is reversed by adjustment of the ionic strength. It is due to the presence of the lipid components of the membrane. Different lipids are equally effective in inducing the polymerization. 3. Each component of leucocidin will polymerize in the absence of membranes and lose biological activity at low ionic strength. This is reversed by electrolyte and it does not proceed to the same extent as in the presence of membranes. 4. The nature of the interaction of leucocidin with cells, membranes and lipids and the spontaneous polymerization indicate that each component of leucocidin can adopt different isomeric forms. 5. The relationship of the interaction with the membrane to the cytotoxic effect of leucocidin is discussed.     

Low resolution crystal structure of Arenicola erythrocruorin: influence of coiled coils on the architecture of a megadalton respiratory protein

Annelid erythrocruorins are extracellular respiratory complexes assembled from 180 subunits into hexagonal bilayers. Cryo-electron microscopic experiments have identified two different architectural classes. In one, designated type I, the vertices of the two hexagonal layers are partially staggered, with one hexagonal layer rotated by about 16 degrees relative to the other layer, whereas in the other class, termed type II, the vertices are essentially eclipsed. We report here the first crystal structure of a type II erythrocruorin, that from Arenicola marina, at 6.2 A resolution. The structure reveals the presence of long continuous triple-stranded coiled-coil "spokes" projecting towards the molecular center from each one-twelfth unit; interdigitation of these spokes provides the only contacts between the two hexagonal layers of the complex. This arrangement contrasts with that of a type I erythrocruorin from Lumbricus terrestris in which the spokes are broken into two triple-stranded coiled coils with a disjointed connection. The disjointed connection allows formation of a more compact structure in the type I architecture, with the two hexagonal layers closer together and additional extensive contacts between the layers. Comparison of sequences of the coiled-coil regions of various linker subunits shows that the linker subunits from type II erythrocruorins possess continuous heptad repeats, whereas a sequence gap places these repeats out of register in the type I linker subunits, consistent with a disjointed coiled-coil arrangement.