Effects of iron nitrosylation on sickle cell hemoglobin solubility

One mechanism by which nitric oxide (NO) has been proposed to benefit patients with sickle cell disease is by reducing intracellular polymerization of sickle hemoglobin (HbS). In this study we have examined the ability of nitric oxide to inhibit polymerization by measuring the solubilizing effect of iron nitrosyl sickle hemoglobin (HbS-NO). Electron paramagnetic resonance spectroscopy was used to confirm that, as found in vivo, the primary type of NO ligation produced in our partially saturated NO samples is pentacoordinate alpha-nitrosyl. Linear dichroism spectroscopy and delay time measurements were used to confirm polymerization. Based on sedimentation studies we found that, although fully ligated (100% tetranitrosyl) HbS is very soluble, the physiologically relevant, partially ligated species do not provide a significant solubilizing effect. The average solubilizing effect of 26% NO saturation was 0.045; much less than the 0.15 calculated for the effect of 26% oxygen saturation. Given the small amounts of NO-ligated hemoglobin achievable through any kind of NO therapy, we conclude that NO therapy does not benefit patients through any direct solubilizing effect.     

The neurotoxic effect of sickle cell hemoglobin

A growing body of experimental evidence suggests that the oxidative neurotoxicity of hemoglobin A may contribute to neuronal loss after CNS hemorrhage. Several hemoglobin variants, including hemoglobin S, are more potent oxidants in cell-free systems. However, despite the increased incidence of hemorrhagic stroke associated with sickle cell disease, little is known of the effect of hemoglobin S on cells of neural origin. In the present study, its toxicity was quantified and directly compared with that of hemoglobin A in murine cortical cell cultures. Reactive oxygen species production, as assessed by cellular fluorescence after treatment with dihydrorhodamine 123, was significantly increased by exposure to 10 μM hemoglobin S for 2-4 h. Neuronal death, as measured by propidium iodide staining and lactate dehydrogenase release, commenced at 4 h; for a 20-h exposure, the EC₅₀ was approximately 0.71 μm. Glial cells were not injured. Cell death was completely blocked by iron chelation with deferoxamine or phenanthroline. Direct comparison of sister cultures exposed to either hemoglobin A or hemoglobin S revealed a similar amount of cell injury in both groups. A significant difference was consistently observed only after treatment with 1 μM hemoglobin for 20 h, which resulted in death of approximately one third more neurons with hemoglobin S than with hemoglobin A. The results of this study suggest that sickle cell hemoglobin is neurotoxic at physiologically relevant concentrations. This toxicity is iron-dependent, oxidative, and quantitatively similar to that produced by hemoglobin A.     

Mechanical stability of hemoglobin subunits: an abnormality in betaS-subunits of sickle hemoglobin

In order to study the mechanism of the ease of precipitation of oxyhemoglobin S by mechanical shaking, the rates of precipitation of α- and β-subunits of oxyhemoglobin A and oxyhemoglobin S were compared. At pH 8.0, the α^A-subunits precipitated rapidly, while the β^A-subunits were very stable, although a part of β^A-bunits converted to the hemichrome form. At pH 6.0, the β^A-subunits precipitated rapidly while the α^A-subunits were stable. Similar studies with β^S-subunits showed that β^S-subunits precipitated rapidly both at acidic and alkaline pHs. The abnormal precipitation of tetrameric oxyhemoglobin S during mechanical shaking may be due to this instability of the β^S-subunits.     

The reactions of myoglobin, normal adult hemoglobin, sickle cell hemoglobin and hemin with hydroxyurea

The kinetics of the reaction of hydroxyurea (HU) with myoglobin (Mb), hemin, sickle cell hemoglobin (HbS), and normal adult hemoglobin (HbA) were determined using optical absorption spectroscopy as a function of time, wavelength, and temperature. Each reaction appeared to follow pseudo-first order kinetics. Electron paramagnetic resonance spectroscopy (EPR) experiments indicated that each reaction produced an FeNO product. Reactions of hemin and the ferric forms of HbA, HbS, and myoglobin with HU also formed the NO adduct. The formation of methemoglobin and nitric oxide-hemoglobin from these reactions may provide further insight into the mechanism of how HU benefits sickle cell patients.     

Helical crystals of sickle cell hemoglobin

Thin ribbon-like crystals are intermediates in the formation of large crystals of deoxyhemoglobin S from many individual fibers. The thin crystals show foldedover regions when observed by electron microscopy. Some crystals are sufficiently long to have several folds each separated by a distance of about 4.4 μm, suggesting that the crystals are helical in solution. The thickness of the crystals varies from 500 to 900 Å as shown by heavy-metal shadowing and by measurements of the thickness at the crossover point where an edge-on view of the crystal is obtained.     

Dielectric constant of sickle cell hemoglobin. Dielectric properties of sickle cell hemoglobin in solution and gel

The dielectric constants of sickle cell hemoglobin were determined before and after gelation. The dielectric properties of oxy and deoxy sickle cell hemoglobin in solution are nearly identical to those of oxy and deoxy hemoglobin A. Only in the gel state did deoxy sickle cell hemoglobin display dielectric behavior different from that in solution. Upon gelation of deoxy sickle cell hemoglobin, the dielectric constant showed a marked decrease, and the relaxation frequency shifted towards higher frequencies. This result suggests that dielectric constant measurement can be used for the investigation of the kinetics of polymerization of sickle cell hemoglobin molecules. Despite the marked decrease in the dielectric constant, deoxy sickle cell hemoglobin still showed a well-defined dielectric dispersion even in the gel state. This indicates that individual molecules have considerable freedom of rotation in gels. It was observed that the dielectric properties of gelled deoxy sickle cell hemoglobin were affected by electrical fields at the level of 10 to 20 V/cm. This observation suggests that electrical fields of moderate strengths are able to perturb the gel structure if the system is near the transition region. The non-linear electrical behavior of gelled sickle cell hemoglobin will be discussed further in subsequent papers.     

Effect of the beta6 Glu replaced by Val mutation on the optical activity of hemoglobin S and of its beta subunits

The carbomonoxy derivatives of hemoglobin A and S showed a different optical activity in the Soret region of the spectrum as measured by circular dichroism. Different optical activity was also measured in the carbomonoxy derivatives of the beta subunits of hemoglobin A and S, the respective deoxy derivatives showed different circular dichroism spectra only in the presence of inositol hexaphosphate. Sedimentation velocity experiments showed that the differences in optical activity are not due to a different state of aggregation of the subunits. Modification of the tertiary structure of the beta subunits seems to be responsible for the phenomenon. Speculation based on the work of Hsu and Woody (Hsu, M.C., and Woody, R.W. (1971) J. Am. Chem. Soc. 93, 3515-3525) suggests the involvement of the beta15 tryptophan in the conformational changes produced by the beta6 Glu-Val mutation in hemoglobin S.     

Effects of amino acids on gelation kinetics and solubility of sickle hemoglobin

Ten amino acids have been studied for their effects on the gelation of sickle hemoglobin using the recently developed assay of Hofrichter, Ross and Eaton. By monitoring kinetics and using high speed sedimentation, the rate and extent of gelation are directly measured. Of the amino acids tested, only phenylalanine significantly inhibited the gelation of sickle hemoglobin. The systematic study of the effects of additives, such as amino acids, on gelation serves as a basis for the study of potential non-covalent inhibitors of sickling.     

Ligand recombination to the alpha and beta subunits of human hemoglobin

The rebinding of CO, O2, NO, methyl, ethyl, n-propyl, and n-butyl isocyanide to isolated alpha and beta chains and intact hemoglobin at pH 7, 20 degrees C was examined both during and after a 30-ns dye laser pulse. The resultant absorbance changes were analyzed in terms of a linear three-step reaction scheme: Hb + X in equilibrium with C in equilibrium with B in equilibrium with A or HbX, where A is the final bound state, and C and B are geminate states. Rate constants were assigned for each of the transitions in this mechanism using fitting procedures described previously for analyzing ligand rebinding to sperm whale myoglobin at room temperature (Gibson, Q. H., Olson, J. S., McKinnie, R. E., and Rohlfs, R. J. (1986) J. Biol. Chem. 261, 10228-10239). Five major conclusions were obtained. First, initial geminate recombination phases for the NO and O2 complexes of hemoglobin and its isolated subunits exhibit half-times equal to approximately 12 and approximately 440 ps, respectively. These values are in excellent agreement with more direct, picosecond measurements of the geminate recombination of HbNO (Cornelius, P. A., Hochstrasser, R. M., and Steele, A. W. (1983) J. Mol. Biol. 163, 119-128) and HbO2 (Friedman, J. M., Scott, T. W., Fisanick, G. J., Simon, S. R., Findsen, E. W., Ondrias, M. R., and MacDonald, V. W. (1985) Science 229, 187-229) following extremely short laser pulses. Second, the correspondence between our nanosecond measurements and the published picosecond data suggests strongly that the intrinsic photochemical yield of all ferrous, hexacoordinate heme complexes approaches one. Third, the major differences between the isolated alpha and beta chains involve the rate of ligand migration to the solvent, kC----X and the extent of recombination from the second geminate state, C, as measured by the ratio kC----B/kC----X. Fourth, for both isolated chains and intact hemoglobin, the rate and equilibrium constants for the formation of the initial O2 geminate state starting from ligand in the solvent (i.e. kX----B and KX----B) are 5-10 times greater than the corresponding parameters for the formation of the first CO geminate state. Fifth, the rate-limiting step for NO, O2, and isonitrile binding to hemoglobin and its isolated subunits is ligand migration up to the initial geminate state (i.e. kX----B). In the case of CO binding, both migration to state B and iron-ligand bond formation (kB----A) affect the overall, bimolecular association rate constant.     

The rates of polymerization and depolymerization of sickle cell hemoglobin

The polymerization and depolymerization of concentrated solutions of sickle cell deoxyhemoglobin were initiated by raising and lowering the temperature, and the time courses of the reactions monitored by the change in apparent turbidity. The polymerization reaction exhibits a marked lag phase followed by a rapid increase in turbidity, and is dependent on a very high power of the hemoglobin concentration, roughly the fifteenth. The depolymerization reaction exhibits no such lag, and is much less dependent on concentration. The implications of these results for polymerization models are discussed.     

The N-terminal regions of beta and gamma subunits lower the solubility of adenosylcobalamin-dependent diol dehydratase

Adenosylcobalamin-dependent diol dehydratase is one of essential components of carboxysome-like polyhedral bodies. It exists as a heterohexamer (alphabetagamma)(2), and its activity is recovered in a precipitant fraction of Klebsiella oxytoca and overexpressing Escherichia coli cells. Limited proteolysis of the enzyme with trypsin converted the enzyme into a highly soluble form without loss of enzyme activity. The N-terminal amino acid sequencing of the enzyme thus solubilized indicated that the N-terminal 20 and 16 amino acid residues had been removed from the beta and gamma subunits, respectively. Mutant enzymes with the same N-terminal truncations of either or both of the beta and gamma subunits were expressed on a high level in E. coli cells. All the mutant enzymes obtained were expressed in a soluble, active form. These results indicate that the N-terminal regions of the beta and gamma subunits lower the solubility of diol dehydratase. The mutant enzyme with the N-terminal truncations of both beta and gamma subunits was essentially indistinguishable in catalytic properties from recombinant wild-type enzyme or the enzyme purified from K. oxytoca in a soluble form.     

Oxygen binding to sickle cell hemoglobin.

The extent of oxygen binding and light scattering of concentrated solutions of hemoglobin S have been determined as a function of oxygen partial pressure using a thin film optical cell. Nearly reversible oxygen binding is observed as witnessed by the small hysteresis found between slow deoxygenation and reoxygenation runs. High co-operativity is noted from unusually large concentration-dependent Hill coefficients when aggregated hemoglobin S is present. The application of linkage theory with the inclusion of non-ideal solution properties permits a test of various simple models for oxygen binding to both the monomer (α₂β₂^s) and polymer (aggregated) phase. It is concluded that oxygen binding to the polymer is either negligible or small under present experimental conditions. Phase diagrams of the solution concentration in equilibrium with polymer phase as a function of oxygen partial pressure are derived using best fit values of polymer parameters.     

Beta-sheet aggregation proposed in sickle cell hemoglobin

A β-sheet conformation is predicted at the N-terminal of β chains in sickle cell hemoglobin (Hb S) as a result of the β6 Glu → Val mutation. Since Glu is the weakest and Val is the strongest β-sheet former in the predictive method of Chou and Fasman [Biochemistry $\text{13}$, 211, 222 (1974)], such a substitution greatly increases the β-sheet potential in the β 1–6 region. The similarity in the concentration and temperature dependence of Hb S gelation to β-sheet formation in polyamino acids suggest that a common aggregation mechanism may be involved. Conditions to cause a β → α trans-formation at the β 1–6 region of Hb S is discussed relative to the treatment of sickle cell disease.     

Properties of a recombinant human hemoglobin double mutant: sickle hemoglobin with Leu-88(beta) at the primary aggregation site substituted by Ala.

A recombinant double mutant of hemoglobin (Hb), E6V/L88A(beta), was constructed to study the strength of the primary hydrophobic interaction in the gelation of sickle Hb, i.e., that between the mutant Val-6(beta) of one tetramer and the hydrophobic region between Phe-85(beta) and Leu-88(beta) on an adjacent tetramer. Thus, a construct encoding the donor Val-6(beta) of the expressed recombinant HbS and a second mutation encoding an Ala in place of Leu-88(beta) was assembled. The doubly mutated beta-globin gene was expressed in yeast together with the normal human alpha-chain, which is on the same plasmid, to produce a soluble Hb tetramer. Characterizations of the Hb double mutant by mass spectrometry, by HPLC, and by peptide mapping of tryptic digests of the mutant beta-chain were consistent with the desired mutations. The absorption spectra in the visible and the ultraviolet regions were practically superimposable for the recombinant Hb and the natural Hb purified from human red cells. Circular dichroism studies on the overall structure of the recombinant Hb double mutant and the recombinant single mutant, HbS, showed that both were correctly folded. Functional studies on the recombinant double mutant indicated that it was fully cooperative. However, its gelation concentration was significantly higher than that of either recombinant or natural sickle Hb, indicating that the strength of the interaction in this important donor-acceptor region in sickle Hb was considerably reduced even with such a conservative hydrophobic mutation.     

The kinetics of nucleation and growth of sickle cell hemoglobin fibers

Polymerization of sickle cell hemoglobin (HbS) in deoxy state is one of the basic events in the pathophysiology of sickle cell anemia. For insight into the polymerization process, we monitor the kinetics of nucleation and growth of the HbS polymer fibers. We define a technique for the determination of the rates J and delay times theta of nucleation and the fiber growth rates R of deoxy-HbS fibers, based on photolysis of CO-HbS by laser illumination. We solve numerically time-dependent equations of heat conductance and CO transport, coupled with respective photo-chemical processes, during kinetics experiments under continuous illumination. After calibration with experimentally determined values, we define a regime of illumination ensuring uniform temperature and deoxy-HbS concentration, and fast (within <1 s) egress to steady conditions. With these procedures, data on the nucleation and growth kinetics have relative errors of <5% and are reproducible within 10% in independent experiments. The nucleation rates and delay times have steep, exponential dependencies on temperature. In contrast, the average fiber growth rates only weakly depend on temperature. The individual growth rates vary by up to 40% under identical conditions. These variations are attributed to instability of the coupled kinetics and diffusion towards the growing end of a fiber. The activation energy for incorporation of HbS molecules into a polymer is E(A)=50 kJ mol(-1), a low value indicating the significance of the hydrophobic contacts in the HbS polymer. More importantly, the contrast between the strong theta(T) and weak R(T) dependencies suggests that the homogenous nucleation of HbS polymers occurs within clusters of a precursor phase. This conclusion may have significant consequences for the understanding of the pathophysiology of sickle cell anemia and should be tested in further work.     

The dissociation of carbon monoxide from the alpha and the beta subunits of human carbonmonoxy hemoglobin

We have measured the intrinsic CO dissociation rates from the subunits of the human hemoglobin tetramers (alpha CO beta NO)2 and (alpha NO beta CO)2 using microperoxidase and a stopped-flow spectrophotometer. The dissociation of NO is negligible. The rate constants for the and the subunits are similar (0.014 s-1 vs. 0.011 s-1, respectively, at pH 7, 20 C; and 0.016 s-1 for both in the presence of inositol hexaphosphate), indicating that they are equivalent in the first step of the CO dissociation. Therefore, the chain unequality observed in the third and fourth steps (Samaja, M., Rovida, E., Niggeler, M., Perrella, M., and Rossi-Bernardi, L. (1987). J. Biol. Chem.: 262, 4528-4533) are not due to the intrinsic properties of the subunits, but to the conformational state of the molecule.