Nitric oxide formation from the reaction of nitrite with carp and rabbit hemoglobin at intermediate oxygen saturations

The nitrite reductase activity of deoxyhemoglobin has received much recent interest because the nitric oxide produced in this reaction may participate in blood flow regulation during hypoxia. The present study used spectral deconvolution to characterize the reaction of nitrite with carp and rabbit hemoglobin at different constant oxygen tensions that generate the full range of physiological relevant oxygen saturations. Carp is a hypoxia-tolerant species with very high hemoglobin oxygen affinity, and the high R-state character and low redox potential of the hemoglobin is hypothesized to promote NO generation from nitrite. The reaction of nitrite with deoxyhemoglobin leads to a 1 : 1 formation of nitrosylhemoglobin and methemoglobin in both species. At intermediate oxygen saturations, the reaction with deoxyhemoglobin is clearly favored over that with oxyhemoglobin, and the oxyhemoglobin reaction and its autocatalysis are inhibited by nitrosylhemoglobin from the deoxyhemoglobin reaction. The production of NO and nitrosylhemoglobin is faster and higher in carp hemoglobin with high O(2) affinity than in rabbit hemoglobin with lower O(2) affinity, and it correlates inversely with oxygen saturation. In carp, NO formation remains substantial even at high oxygen saturations. When oxygen affinity is decreased by T-state stabilization of carp hemoglobin with ATP, the reaction rates decrease and NO production is lowered, but the deoxyhemoglobin reaction continues to dominate. The data show that the reaction of nitrite with hemoglobin is dynamically influenced by oxygen affinity and the allosteric equilibrium between the T and R states, and that a high O(2) affinity increases the nitrite reductase capability of hemoglobin.     

The effect of potassium chloride on the Bohr effect of human hemoglobin.

The normal and differential titration curves of liganded and unliganded hemoglobin were measured at various KCl concentrations (0.1 to 2.0 M). In this range of KCl concentrations, the curves for deoxyhemoglobin showed no salt-induced pK changes of titratable groups. In the same salt concentration range oxyhemoglobin showed a marked change in titration behavior which could only be accounted for by a salt-induced increase in pK of some titratable groups. These results show that the suppression of the alkaline Bohr effect by high concentrations of neutral univalent salt is not caused by a weakening of the salt bridges in deoxyhemoglobin but is due to an interaction of chloride ions with oxyhemoglobin. Measurements of the Bohr effect at various KCl concentrations showed that at low chloride ion concentration (5 times 10-3 M) the alkaline Bohr effect is smaller than at a concentration of 0.1 M. This observation indicates that at a chloride ion concentration of 0.1 M, part of the alkaline Bohr effect is due to an interaction of chloride ions with hemoglobin. Furthermore, at low concentrations of chloride ions the acid Bohr effect has almost vanished. This result suggests that part of the acid Bohr effect arises from an interaction of chloride ions with oxyhemoglobin. The dependence of the Bohr effect upon the chloride ion concentration can be explained by assuming specific binding of chloride ions to both oxy- and deoxyhemoglobin, with deoxyhemoglobin having the highest affinity.     

A chromatographic study of the reaction sequence and effect of ligand on the reaction of human hemoglobin with negatively charged isothiocyanates: characterization of an intermediate modified only on the amino termini of the alpha chains

A HPLC investigation of the reaction of 4-isothiocyanatobenzoic acid and 4-isothiocyanatobenzenesulfonic acid with oxy- and deoxyhemoglobin was carried out. The initial reaction of aromatic isothiocyanato sulfonates and benzoates with either oxy- or deoxyhemoglobin is with the amino termini of the alpha chains followed by a much slower reaction with the amino termini of the beta chains. Both reactions are much faster with deoxyhemoglobin than with oxyhemoglobin. An intermediate reacted only at the termini of the alpha chains with 4-isothiocyanatobenzoic acid was isolated and purified and its functional properties determined. The intermediate showed a reduced oxygen affinity over a wide pH range and a reduced alkaline Bohr effect in the absence of chloride. The oxygen affinity of the intermediate showed a reduced but still significant response to chloride.     

The binding of chloride ions to ligated and unligated human hemoglobin and its influence on the Bohr effect.

The contribution of the interaction of chloride ions with deoxy and oxyhemoglobin to the Bohr effect can be described by a simple binding model. Applying this model to experiment data reveals that at physiological pH and ionic strength about half of the release of Bohr protons is due to a difference in chloride ion binding to deoxy- and oxyhemoglobin. The chloride-independent part of the Bohr effect corresponds with the shift in pK which His-146 beta shows upon oxygenation. The proton absorptioon by hemoglobin observed upon oxygenation below pH 6 is apparently due to a chloride-ion-induced proton uptake, which is larger for oxyhemoglobin than for deoxyhemoglobin. The analysis of the experimental data indicates the existence of only two oxygen-linked chloride ion binding sites in both deoxy and oxyhemoglobin. In deoxyhemoglobin the binding sites most likely consist of Val-1 alpha of one chain and Arg-141 alpha of the partner chain. The sites in oxyhemoglobin consist of groups with a pK value in the neutral pH range; they do not contain lysyl or arginyl residues.     

Solvent regulation of oxygen affinity in hemoglobin. Sensitivity of bovine hemoglobin to chloride ions

Under physiological conditions of pH (7.4) and chloride concentration (0.15 M), the oxygen affinity of bovine hemoglobin is substantially lower than that of human hemoglobin. Also, the Bohr effect is much more pronounced in bovine hemoglobin. Numerical simulations indicate that both phenomena can be explained by a larger preferential binding of chloride ions to deoxyhemoglobin in the bovine system. Also, they show that the larger preferential binding may be produced by a decreased affinity of the anions for oxyhemoglobin, thereby stressing the potential relevance of the oxy conformation in regulating the functional properties of the protein. The conformation of the amino-terminal end of the beta subunits appears to regulate the interaction of hemoglobin with solvent components. The pronounced sensitivity of the oxygen affinity of bovine hemoglobin to chloride concentration and to pH suggests that in bovine species these are the modulators of oxygen transport in vivo.     

The pH dependence of the binding of D-glycerate 2,3-bisphosphate to deoxyhemoglobin and oxyhemoglobin. Determination of the number of binding sites in oxyhemoglobin.

The number of Bohr protons released upon oxygenation has been measured over a large range of human hemoglobin concentrations (0.02 to 4.5 mM) in the presence of equimolar amounts of D-glycerate 2,3-bisphosphate. From these data the association constants for the binding of this organic phosphate to deoxyhemoglobin and oxyhemoglobin were calculated at different pH values. The maximum number of protons absorbed upon binding to oxyhemoglobin was determined as well. The maximum number of protons bound to deoxyhemoglobin upon binding of D-glycerate 2,3-bisphosphate was measured independently. From the pH dependence of the association constants and the maximum number of protons absorbed it could be concluded that only one D-glycerate 2,3-bisphosphate can be bound to both deoxyhemoglobin and oxyhemoglobin.     

Binding of diphosphoglycerate and ATP to oxyhemoglobin dimers

The relative affinity of diphosphoglycerate and ATP for hemoglobin dimers and tetramers can be measured under conditions where the protein is in large molar excess over the polyphosphate. Binding of both compounds to dimers was about 25 times stronger than to tetramers in the case of the three low-spin hemoglobins, oxyhemoglobin, carboxyhemoglobin and cyanomethemoglobin. The mutation in hemoglobin Kansas leads to an increased dissociation into alpha beta dimers. The increase in diphosphoglycerate binding by this hemoglobin was in good agreement with that expected from the dimer-tetramer dissociation constant over a wide range of hemoglobin concentrations. In contrast to the liganded hemoglobins, both deoxyhemoglobin and aquomethemoglobin bind the two polyanions as tetramers.     

Consequences of chemical modifications on the free radical reactions of human hemoglobin.

Hemoglobin-based oxygen carriers (HBOCs) are candidates for use as blood substitutes and resuscitation fluids. We determined that HBOCs of specific types differ in their ability to generate or interact with free radicals. The differences do not correlate with oxygen affinity. Detailed comparisons with unmodified human hemoglobin, HbA0, were carried out with two cross-linked derivatives: HbA-FMDA, produced by the reaction of human oxyhemoglobin with fumaryl monodibromoaspirin, and HbA-DBBF, produced by the reaction of human deoxyhemoglobin with bis(3,5-dibromosalicyl) fumarate. Both derivatives had lower oxygen affinity than unmodified HbA0. As previously reported, exposure of oxyhemoglobin to H2O2 causes generation of free radicals capable of generating formaldehyde from dimethyl sulfoxide. Relative to the reaction catalyzed by 50 microM HbA (18.0 +/- 3.5 nmol/30 min/ml), the formaldehyde formation was roughly 70% for HbA-DBBF and 50% for HbA-FMDA under comparable conditions. More profound differences are exhibited at lower hemoglobin concentrations. Spectral changes of the HBOCs during the reaction differ qualitatively and occur at different rates. The HBOCs also differ in rates of hemoglobin-catalyzed NADPH oxidation and aniline hydroxylation, reactions mediated by reactive oxygen species. These results show that stereochemical differences brought about by chemical cross-linking alter the ability of HBOCs to generate radicals and to react with activated oxygen species. These studies also show that the ability of hemoglobin to produce activated species of oxygen can be enhanced or suppressed independently of oxygen affinity.     

ESR correlation times of 2,2,6,6-tetramethyl piperidone-N-oxyl (Tempone) in solutions of hemoglobin A and hemoglobin S.

Correlation times for the tumbling motion of the spin probe 2,2,6,6,-tetramethyl piperidone-N-oxyl (Tempone) were obtained in the presence of different concentrations of oxyhemoglobin A, oxyhemoglobin S, and deoxyhemoglobin S and compared to the viscosity of non-gelling hemoglobin solutions. Reorientational motion (or tumbling) of Tempone in gelled solutions of deoxyhemoglobin S is as great as that in non-gelled hemoglobins of the same total concentration. It is concluded that the gel does not exclusively partition Tempone into an aqueous phase of lower solute concentration after gel formation. The gel at room temperature is a highly mobile and dynamic structure on the microscopic level.     

Hemoglobin oxygen fractional saturation regulates nitrite-dependent vasodilation of aortic ring bioassays

Nitrite reacts with deoxyhemoglobin to generate nitric oxide (NO). This reaction has been proposed to contribute to nitrite-dependent vasodilation in vivo and potentially regulate physiological hypoxic vasodilation. Paradoxically, while deoxyhemoglobin can generate NO via nitrite reduction, both oxyhemoglobin and deoxyhemoglobin potently scavenge NO. Furthermore, at the very low O(2) tensions required to deoxygenate cell-free hemoglobin solutions in aortic ring bioassays, surprisingly low doses of nitrite can be reduced to NO directly by the blood vessel, independent of the presence of hemoglobin; this makes assessments of the role of hemoglobin in the bioactivation of nitrite difficult to characterize in these systems. Therefore, to study the O(2) dependence and ability of deoxhemoglobin to generate vasodilatory NO from nitrite, we performed full factorial experiments of oxyhemoglobin, deoxyhemoglobin, and nitrite and found a highly significant interaction between hemoglobin deoxygenation and nitrite-dependent vasodilation (P < or = 0.0002). Furthermore, we compared the effect of hemoglobin oxygenation on authentic NO-dependent vasodilation using a NONOate NO donor and found that there was no such interaction, i.e., both oxyhemoglobin and deoxyhemoglobin inhibited NO-mediated vasodilation. Finally, we showed that another NO scavenger, 2-carboxyphenyl-4,4-5,5-tetramethylimidazoline-1-oxyl-3-oxide, inhibits nitrite-dependent vasodilation under normoxia and hypoxia, illustrating the uniqueness of the interaction of nitrite with deoxyhemoglobin. While both oxyhemoglobin and deoxyhemoglobin potently inhibit NO, deoxyhemoglobin exhibits unique functional duality as an NO scavenger and nitrite-dependent NO generator, suggesting a model in which intravascular NO homeostasis is regulated by a balance between NO scavenging and NO generation that is dynamically regulated by hemoglobin's O(2) fractional saturation and allosteric nitrite reductase activity.     

Oxygenation of hemoglobin: Correspondence of crystal and solution properties using diffusion coefficient measurements

Quasi-elastic light scattering has been used to measure the change in the translational diffusion coefficient of hemoglobin upon oxygenation and the difference in the diffusion coefficients of oxy- and methemoglobin. The diffusion coefficients of oxy- and methemoglobin were found to be the same within the experimental accuracy of 0.2%, while the diffusion coefficient of oxyhemoglobin tetramers in solution at 13 mg/ml was found to be 0.8% smaller than that of deoxyhemoglobin at the same concentration, when the reversible dissociation of oxyhemoglobin tetramers into dimers was taken into account. In the limit of zero concentration, the oxyhemoglobin diffusion coefficient was found to be 1.5% ± 1.0% smaller than that of deoxyhemoglobin. This result is in very good agreement with what we predict using atomic coordinates to model the liganded and unliganded hemoglobin molecules as ellipsoids of revolution.     

Observation of allosteric transition in hemoglobin

Two conclusions have been drawn from NMR studies of mixed state hemoglobins. First the α and β subunits in hemoglobin are not equivalent in their conformational properties. Second the mixed state hemoglobin (α^IIICN β^II)₂ can take two different quaternary structures without changing the degree of ligation. One of the two structures is similar to that of deoxyhemoglobin and the other to that of oxyhemoglobin.     

The kinetics of the reactions of Parasponia andersonii hemoglobin with oxygen, carbon monoxide, and nitric oxide

Hemoglobin I was isolated from nodules formed on the roots of Parasponia andersonii inoculated with Rhizobium strain CP 283. The rate of oxygen dissociation from Parasponia hemoglobin increases about 12-fold between pH 4 and 7, with apparent pK 6.4, to reach a limiting value of 14.8s-1. The optical spectrum of oxyhemoglobin in the visible region is also dependent on pH with pK near 6.4. The rate constant for oxygen combination with Parasponia hemoglobin increases about 7-8-fold between pH 4 and 7, with apparent pK 5.37, to reach a value of 1.67 X 10(8) M-1 s-1 at pH 7. The optical spectrum of deoxyhemoglobin in the visible region and the rate constant for carbon monoxide combination are also dependent on pH with apparent pK 5.65 and 5.75, respectively. The rate constant for carbon monoxide dissociation is independent of pH. The oxygen affinity of Parasponia hemoglobin, P50 = 0.049 torr at 20 degrees C, calculated from the kinetic constants at pH 7, is very great. At alkaline pH there is a prominent geminate reaction with oxygen and nitric oxide, with both subnanosecond and tens of nanosecond components. These reactions disappear at acid pH, with pK 6.4, and the effective quantum yield is reduced. In general, the reactions of Parasponia hemoglobin with oxygen and carbon monoxide resemble those of soybean leghemoglobin. In each, great oxygen affinity is achieved by unusually rapid oxygen combination together with a moderate rate of oxygen dissociation. We suggest that protonation of a heme-linked group with pK near 6.4 controls many properties of Parasponia oxyhemoglobin, and protonation of a group with pK near 5.5 controls many properties of Parasponia deoxyhemoglobin.     

Isolation and characterization of a new hemoglobin derivative cross-linked between the alpha chains (lysine 99 alpha 1----lysine 99 alpha 2)

Bis(3,5-dibromosalicyl) fumarate and a number of related bifunctional reagents react preferentially with oxyhemoglobin to cross-link the beta chains within the 2,3-diphosphoglycerate-binding site. In this report we describe a new derivative cross-linked between the alpha chains which is formed specifically in the reaction with deoxyhemoglobin. X-ray crystallographic studies show that the cross-link lies between Lys-99 alpha 1 and Lys-99 alpha 2, spanning the central cavity of the tetramer. Lys-99 alpha 1 and Lys-99 alpha 2 are located within a cluster of charged residues very near the middle of the hemoglobin molecule. In oxyhemoglobin, this site is completely inaccessible to the cross-linking agent. Competition experiments with inositol hexaphosphate indicate that the compound enters the central cavity in deoxyhemoglobin through the cleft between the alpha chains. Despite the presence of the cross-link between the alpha chains, the modified hemoglobin remains highly cooperative. The Hill coefficient for HbXL99 alpha is 2.6. The oxygen affinity of the cross-linked derivative is decreased by approximately 2-fold; at pH 7.0 in the presence of 0.1 M NaCl the P50 is 13.9 mm Hg compared to 6.6 mm Hg for HbA. This difference appears to be due to relatively small changes in both KR, the association constant for binding of oxygen to the R state, and the allosteric constant L. Surprisingly, the isoelectric point of oxyHbXL99 alpha is almost identical to that of oxyHbA, whereas in the deoxy form the isoelectric point of the cross-linked derivative is decreased relative to native hemoglobin as expected due to the loss of the two positive charges of the modified amino groups. In agreement with these findings, the alkaline Bohr effect of HbXL99 alpha is decreased by more than 50%. Earlier studies argue strongly against the possibility that Lys-99 alpha is directly responsible for this large fraction of the Bohr effect in HbA. Analysis of the structure suggests that in the cross-linked derivative Glu-101 beta, which is in close proximity to Lys-99 alpha in oxyhemoglobin, becomes an acid Bohr group.     

Transformation of hemoglobin a into methemoglobin by sesamol

Sesamol (3,4-methylenedioxyphenol), a monophenolic antioxidant in sesame iol, produced methemoglobin from hemoglobin A (oxyhemoglobin and deoxyhemoglobin) and from red cells. The activity of the compound was more extensive than the polyphenolic compounds. The profiles of the methemoglobin formation by the compound were compared with those by nitrite and hydroxylamine. The formation of methemoglobin from oxyhemoglobin by the compound was rather slowly progressed, but the amount of methemoglobin formed was proportional to the concentration of oxyhemoglobin even when the concentration of the compound was low. The sesamol-induced methemoglobin formation was influenced by inositol hexaphosphate, an allosteric effector of hemoglobin. Thus, the phosphate enhanced the transformation of oxyhemoglobin and inhibited the transformation of deoxyhemoglobin.     

Fixation of aldehydic dextrans onto human deoxyhemoglobin.

A procedure commonly used to transform native adult human hemoglobin (Hb) into a physiological oxygen carrier consists of a pyridoxylation of the protein to lower its oxygen affinity, followed by its polymerization in the presence of glutaraldehyde, with or without further reduction, to increase its circulating half-life. This series of reactions yields derivatives presenting a great molecular heterogeneity that have to be fractionated for use in vivo. Hemoglobin derivatives with low oxygen affinity and a narrow distribution of molecular weights were obtained by linking a dextran polyaldehydic derivative to deoxyhemoglobin at pH 8. From oxygen-binding measurements carried out in the presence of inositolhexaphosphate, a strong effector of hemoglobin, it appeared that the allosteric site of hemoglobin was blocked, probably by crosslinking bonds, which stabilizes its deoxy structure. On the other hand, when the reaction was performed in the presence of inositolhexaphosphate, the resulting conjugates exhibited an oxygen affinity identical to that of unmodified hemoglobin. After treatment with NaBH4, the polymer-hemoglobin derivatives were stable and possessed a reversible oxygen-carrying capacity similar to that of blood. The conjugates prepared from oxyhemoglobin all possessed a lower P50 than native hemoglobin whatever the reaction conditions.     

The role of nitrite in nitric oxide homeostasis: A comparative perspective

Nitrite is endogenously produced as an oxidative metabolite of nitric oxide, but it also functions as a NO donor that can be activated by a number of cellular proteins under hypoxic conditions. This article discusses the physiological role of nitrite and nitrite-derived NO in blood flow regulation and cytoprotection from a comparative viewpoint, with focus on mammals and fish. Constitutive nitric oxide synthase activity results in similar plasma nitrite levels in mammals and fish, but nitrite can also be taken up across the gills in freshwater fish, which has implications for nitrite/NO levels and nitrite utilization in hypoxia. The nitrite reductase activity of deoxyhemoglobin is a major mechanism of NO generation from nitrite and may be involved in hypoxic vasodilation. Nitrite is readily transported across the erythrocyte membrane, and the transport is enhanced at low O₂ saturation in some species. Also, nitrite preferentially reacts with deoxyhemoglobin rather than oxyhemoglobin at intermediate O₂ saturations. The hemoglobin nitrite reductase activity depends on heme O₂ affinity and redox potential and shows species differences within mammals and fish. The NO forming capacity is elevated in hypoxia-tolerant species. Nitrite-induced vasodilation is well documented, and many studies support a role of erythrocyte/hemoglobin-derived NO. Vasodilation can, however, also originate from nitrite reduction within the vessel wall, and at present there is no consensus regarding the relative importance of competing mechanisms. Nitrite reduction to NO provides cytoprotection in tissues during ischemia-reperfusion events by inhibiting mitochondrial respiration and limiting reactive oxygen species. It is argued that the study of hypoxia-tolerant lower vertebrates and diving mammals may help evaluate mechanisms and a full understanding of the physiological role of nitrite.     

The binding of folyl- and antifolylpolyglutamates to hemoglobin

A binding method that detects only the strongest binding site for a ligand on a protein has been used to show that folates and folate analogs, conjugated with poly-gamma-glutamates, are bound to hemoglobin. When the concentration of hemoglobin is much larger than that of the polyglutamate, as is the case in the red cell, the fraction bound is a direct function of the hemoglobin concentration and is independent of the total polyglutamate concentration. Binding to deoxyhemoglobin tetramers is competitive with 2,3-diphosphoglycerate. In oxyhemoglobin the folyl and methotrexate polyglutamates are bound preferentially by free alpha beta dimers, but removal of the pteridine moiety leads to tetramer binding even in oxyhemoglobin. Changes in the length of the polyglutamate side chain and alterations of the pteridine structure such as reduction and/or methylation have a much larger effect on the constant for binding to deoxyhemoglobin tetramers than on that for oxyhemoglobin dimers. The implications of these results for the storage of pteroylpolyglutamates in the erythrocyte and their release from the red cell under the influence of the degree of oxygenation and variations in the 2,3-diphosphoglycerate level are discussed.     

The Binding of Asparagine,Glutamine and Homoserine to Human Erythrocytes Containing Hemoglobins S and CS and to Soluble Hemoglobins S and CS

Abstract

Tritium labeled asparagine binds to oxyhemoglobin S and to a mixture of hemoglobins C and S in the molar ratio of 3.38:1 and 8.2:1 respectively. From the dialysis equilibrium studies it appears that labeled asparagine does not bind to oxy- or deoxy- hemoglobin A nor to deoxyhemoglobin S. The constant for equilibrium association of asparagine for oxyhemoglobin S is 7.38 × 10⁷ M^?1 and for'oxyhemoglobin CS 4.8 × 10⁴ M^?1 at 23°C. Tritium labeled asparagine is bound to oxyhemoglobin S and CS sufficiently strongly to prevent dissociation under the conditions of gel electrophoresis at pH 9.50. The protein with and without bound asparagine, gluta-mine or homoserine, is indistinguishable in molecular net charge and size by the criteria of quantitative polyacrylamide gel electrophoresis (PAGE). Also there were no significant differences in mobility between hemoglobin S and hemoglobin C in the presence and absence of asparagine, glutamine and homoserine as detectable in agar coated cellulose acetate electrophoresis at pH 6.3. Erythrocytes containing hemoglobin S and CS, after incubation with tritium labeled asparagine and lysis under the conditions of gel electrophoresis at pH 9.5, release hemoglobin S and C with bound tritiated asparagine. No tritiated asparagine remains bound to the ghost.