Effect of nitrite-induced methemoglobinemia on the kinetics of blood deoxygenation

Kinetics of blood deoxygenation was studied during acute hypoxia induced by subcutaneous administration of sodium nitrite using polarographic method. Plasma oxygen tension remained unaltered as the dose of sodium nitrite increased, while the dynamics of oxygen release was dose-dependent. The constant of oxyhemoglobin deoxygenation rate proved to vary with blood deoxygenation. The nitrite-induced deceleration of oxyhemoglobin deoxygenation was due to the inactivation of a fraction of hemoglobin as well as to the increased hemoglobin oxygen affinity and possible changes in the oxygen permeability of erythrocyte membranes during acute methemoglobinemia.     

Studies on cobalt myoglobins and hemoglobins. Preparation of isolated chains containing cobaltous protoporphyrin IX and characterization of their equilibrium and kinetic properties of oxygenation and EPR spectra.

Human hemoglobin containing cobalt protoporphyrin IX or cobalt hemoglobin has been separated into two functionally active alpha and beta subunits using a new method of subunit separation, in which the -SH groups of the isolated subunits were successfully regenerated by treatment with dithiothreitol in the presence of catalase. Oxygen equilibria of the isolated subunit chains were examined over a wide range of temperature using Imai's polarographic method (Imai, K., Morimoto, H., Kotani, M., Watari, H., and Kuroda, M. (1970) Biochim. Biophys. Acta 200, 189-196). Kinetic properties of their reversible oxygenation were investigated by the temperature jump relaxation method at 16 degrees. Electron paramagnetic resonance characteristics of the molecules in both deoxy and oxy states were studies at 77K. The oxygen affinity of the individual regenerated chains was higher than that of the tetrameric cobalt hemoglobin and was independent of pH. The enthalpy changes of the oxygenation have been determined as -13.8 kcal/mol and -16.8 kcal/mol for the alpha and beta chains, respectively. The rates of oxygenation were similar to those reported for iron hemoglobin chains, whereas those of deoxygenation were about 10(2) times larger. The effects of metal substitution on oxygenation properties of the isolated chains were correlated with the results obtained previously on cobalt hemoglobin and cobalt myoglobin. The EPR spectrum of the oxy alpha chain showed a distinctly narrowed hyperfine structure in comparison with that of the oxy beta chain, indicating that the environment around the paramagnetic center (the bound oxygen) is different between these chains. In the deoxy form, EPR spectra of alpha and beta chains were indistinguishable. These observations suggest that one of the inequivalences between alpha and beta chains might exist near the distal histidine group.     

Engineering temperature-sensitive hydrogel nanoparticles entrapping hemoglobin as a novel type of oxygen carrier

Temperature-sensitive oxygen carriers that are responsive to changes in temperature while maintaining colloidal stability would benefit physiological conditions characterized by tissue hypoxia due to decreased body temperature. These conditions are often accompanied with reduced blood flow and vasoconstriction. Temperature-sensitive oxygen carriers should ideally possess increased oxygen affinity when the body temperature is reduced, to selectively target tissues that are hypoxic as a result of temperature drops. This study expands on previous work, which introduced hydrogel based oxygen carriers as a new class of oxygen carrier that can be synthesized within liposomal reactors via photoinitiated free radical polymerization [Patton, J. N.; Palmer, A. F. Biomacromolecules 2005, 6, 414-24]. In addition to the ability of poly(N-isopropylacrylamide) hydrogel nanoparticles encapsulating bovine hemoglobin to swell and shrink in response to physiological changes in temperature, the effect of temperature changes on zeta potential, oxygen affinity, and cooperativity are also examined. The methemoglobin level and hemoglobin encapsulation efficiency of hydrogel-based oxygen carriers are also presented. It was observed that nanoscale hydrogel particles swelled as the temperature decreased from 40 to 29 degrees C, which suggests expansion of the hydrogel matrix and reduced resistance to oxygen transport.     

Studies on cobalt myoglobins and hemoglobins, XIII. A consequence of the occurrence of glutamine at the E7 (58) site of alpha subunits in opossum hemoglobin

To investigate the mode of interactions between heme metal, bound oxygen and the distal residue at the E7 site, we have measured accurate oxygen equilibrium curves, oxygen binding relaxations following temperature-jump, and electron paramagnetic resonance spectra of natural and cobalt-substituted opossum hemoglobin, which has glutamine and histidine at the E7 site of the α chain and the β chain, respectively, and compared them with those of natural and cobalt-substituted human hemoglobin, which has histidine at the E7 site of both the α and β chains.Natural opossum hemoglobin has a lower oxygen affinity, slightly smaller and pH-dependent co-operativity, a somewhat greater Bohr effect, and a smaller effect of organic phosphates such as 2,3-diphosphoglycerate and inositol hexaphosphate on oxygen affinity as compared to natural human hemoglobin. Upon substitution of cobalt for iron, these oxygenation characteristics of opossum hemoglobin relative to those of human hemoglobin were preserved well. The behavior of the intrinsic oxygen association constants pertaining to the four oxygenation steps (i.e. the Adair constants) upon addition of the organic phosphates or pH changes indicates that the allosteric equilibrium in opossum hemoglobin is biased towards the T state as compared with that in human hemoglobin, and that the oxygen affinity of the R structure is lower for opossum hemoglobin than for human hemoglobin. The temperature-jump kinetic data indicate that the lower oxygen affinity of opossum cobalt-hemoglobin in comparison with that of human cobalt-hemoglobin can be ascribed to a decreased oxygen association rate constant. The electron paramagnetic resonance experiments on oxy and deoxy opossum and human cobalt-hemoglobins in buffered H₂O and ²H₂O, including their photolysed products at a low temperature, provided the following information. The cobaltous ion of the α subunits of deoxy opossum cobalt-hemoglobin is in an environment that is similar to that for cobaltous ions of deoxy human cobalt-hemoglobin in the T state. The hydrogen bond between the bound oxygen and the residue at E7, which has been shown to exist in oxy human cobalt-hemoglobin and oxy sperm whale cobalt-myoglobin, is absent or, at least, significantly altered in the α subunits of oxy opossum cobalt-hemoglobin, probably resulting in a lower oxygen affinity. Interference by isoleucine at E11α with an oxygen molecule is suggested as an explanation for the lowered affinity of opossum iron-hemoglobin. However, no straightforward structural explanation is available for the lower oxygen affinity of the R structure and the allosteric equilibrium biased towards the T state in opossum iron-hemoglobin.     

Oxygen equilibrium properties of highly purified human adult hemoglobin cross-linked between 82 beta 1 and 82 beta 2 lysyl residues by bis(3,5-dibromosalicyl) fumarate

We investigated oxygen equilibrium properties of highly purified human adult hemoglobin cross-linked between lysine-82 beta 1 and lysine-82 beta 2 by a fumaryl group, which is prepared by reaction of the CO form with bis(3,5-dibromosalicyl) fumarate. The cross-linked hemoglobin preparation isolated by the previous purification method, namely, gel filtration in the presence of 1 M MgCl2 followed by ion-exchange chromatography, was found to be contaminated with about 20% of an electrophoretically silent impurity that shows remarkably high affinity for oxygen. This impurity was separated from the desired cross-linked hemoglobin by a newly developed purification method, which utilizes a difference between the authentic hemoglobin and the impurity in reactivity of the sulfhydryl groups of cysteine-93 beta toward N-ethylmaleimide under a deoxygenated condition. After this purification procedure, the oxygen equilibrium properties of purified cross-linked hemoglobin in the absence of organic phosphate became very similar to those of unmodified hemoglobin with respect to oxygen affinity, cooperativity, and the alkaline Bohr effect. The functional similarity between the cross-linked hemoglobin and unmodified hemoglobin allows us to utilize this cross-linking for preparing asymmetric hybrid hemoglobin tetramers, which are particularly useful as intermediately liganded models. Previous studies on this type of cross-linked hemoglobin should be subject to reexamination due to the considerable amount of the impurity.     

Functional and biochemical properties of the hemoglobins of the burrowing brittle star Hemipholis elongata say (Echinodermata,Ophiuroidea)

The burrowing brittle star Hemipholis elongata (Say) possesses hemoglobin-containing coelomocytes (RBCs) in its water vascular system. The RBCs, which circulate between the arms and body, are thought to play a role in oxygen transport. The hemoglobin of adult animals has a moderate affinity for oxygen (P(50) = 11.4 mm Hg at pH 8, 20 degrees C, measured in cellulo) and exhibits cooperativity (Hill coefficient > 1.7). The hemoglobin of juveniles has a higher affinity (P(50) = 2.3 mmHg at pH 8.0, 20 degrees C) and also exhibits cooperativity. The oxygen-binding properties of the hemoglobin are relatively insensitive to pH, temperature, and hydrogen sulfide. Adult hemoglobin is a heterogeneous mixture composed of three major fractions. The combined results of electrospray mass spectrometry and oxygen-binding experiments performed on purified fractions indicate that the native hemoglobin is in the form of homopolymers. A partial amino acid sequence (about 40 amino acids) of adult hemoglobin reveals little homology with holothurian hemoglobins.     

Studies on electron transfer between mercury electrode and hemoprotein.

The electrochemical behaviour of ferricytochrome c, metmyoglobin and methemoglobin was studied using d.c., a.c. and differential pulse polarography, and controlled potential electrolysis. 1. The three hemoproteins yield d.c. polarographic steps, and peaks in differential pulse polarograms, the height of which is proportional to concentration. The charge transfer is influenced by strong adsorption. 2. The concentration dependence of the a.c. polarograms indicates structural changes in the adsorbed molecules. 3. The reduction products of controlled potential electrolysis of metmyoglobin and methemoglobin have absorption spectra identical with the native control samples. The affinity for oxygen and the cooperativity in hemoglobin are not affected by the reaction at the electrode. 4. The charge transfer proceeds via adsorbed, already reduced, molecules to freely diffusible proteins.     

Oxygen-binding properties of hemoglobins from estivating and active African lungfish.

The oxygen-binding characteristics and the multiplicity of the stripped hemoglobiin from active lungfish Protopterus amphibius, are the same as in specimens that have been estivating for about 30 months, showing that alteration in the hemoglobin molecules is not involved in the earlier reported increase in oxygen affinity of whole blood during estivation (Johansen et al., '76). At pH 7.0 and 26 degrees C the hemolysates show a high oxygen affinity (P50 = 3.1 Torr), a Bohr factor (delta log P50/delta pH) of - 0.33, and a cooperativity coefficient (n) of 1.7. Between 15 and 26 degrees C, the apparent heat of oxygenation (delta H) is - 8.6 Kcal-mole-1 at pH 7.0, corresponding with data for other fish. A low sensitivity of oxygen affinity to urea appears to be adaptive to the high urea concentrations in estivating lungfish. The salt sensitivity is, however, similar to human hemoglobin. The hemoglobin consists of two major (electrophoretically anodal) components, which differ slightly in oxygen affinity but are both sensitive to pH and nucleoside triphosphates (NTP). Guanosine triphosphate (GTP), the major erythrocytic organic phosphate, however, depresses the oxygen affinity of the composite and separated hemoglobins more effectively than ATP suggesting that GTP is the primary modulator of oxygen affinity. Comparative measurements reveal only one major hemoglobin component in P. annectens which has a markedly lower oxygen affinity and phosphate sensitivity than P. amphibius hemoglobins and thus seems less pliable to phosphate-mediated variation in oxygen affinity. The data are discussed in relation to the hemoglobin systems of other fish.     

The primary structure of hemoglobin from reindeer (Rangifer tarandus tarandus) and its functional implications

The primary structures of alpha- and beta-chains of hemoglobin from reindeer (Rangifer tarandus tarandus) were determined. Comparison of the reindeer hemoglobin sequence with those of human and bovine hemoglobins showed 50 and 29 substitutions per alpha beta dimer, respectively. The influence of replacements on the modulation of hemoglobin oxygen affinity by heterothopic ligands and temperature, as well as their importance on the structure-function relationships in hemoglobin are discussed.     

Hemoglobin Richmond. Subunit dissociation and oxygen equilibrium properties.

In hemoglobin Richmond (beta102 leads to Lys), amino acid substitution has occurred at the same site as the mutation in hemoglobin Kansas (beta102 Asn leads to Thr), a variant with very low oxygen affinity. Although hemoglobin Richmond has been shown to have increased tetramer-dimer dissociation, its oxygen affinity has been inferred to be normal from studies on hemolysates of carriers. We have isolated hemoglobin Richmond and have further studied its properties. We confirm that the oxygen affinity of pure hemoglobin Richmond under conditions similar to those found in vivo is normal. However, the Bohr effect of the variant hemoglobin is markedly abnormal. Its oxygen affinity is low at high pH and high at low pH, relative to hemoglobin A. The tetramer-dimer equilibrium displays a strong pH dependence such that protons promote dissociation. A model is presented in which the structural change in hemoglobin Richmond results in low oxygen affinity, like hemoglobin Kansas. However, the close linkage between tetramer-dimer dissociation and proton concentration seen with hemoglobin Richmond results in normal oxygen affinity at intracellular pH and hemoglobin concentration, and carriers display no hematological abnormalities.     

Modification of hemoglobin A with dimethyl adipimidate. Contribution of individual reacted subunits to changes in oxygen affinity.

The effect of dimethyl adipimidate, a bifunctional imidoester, on the oxygen affinity of hemoglobin A has been studied. Treatment of human oxyhemoglobin with 5 mM dimethyl adipimidate at pH 8.5, room temperature is accompanied by an increase in oxygen affinity in the presence and absence of 2,3-diphosphoglyceric acid. Circular dichroism measurements in the ultraviolet region indicate that dimethyl adipimidate-treated hemoglobin exhibits a reduced conformational change upon deoxygenation. In order to study the contribution of reacted individual subunits, alpha and beta subunits of dimethyl adipimidate-treated and untreated hemoglobin have been separated and reconstituted to form hybrid tetramers containing either the alpha-treated (alpha t beta c) or the beta-treated subunits (alpha c beta t). Electrophoresis on sodium dodecyl sulfate polyacrylamide gels of isolated alpha and beta globin subunits as well as hybrid tetramers from dimethyl adipimidate-treated hemoglobin reveals that 20% of the globin subunits are cross-linked. In the absence of 2,3-diphosphoglyceric acid, modification of alpha subunits increases the oxygen affinity and reduces the conformational change of the tetramer upon deoxygenation whereas modification of beta subunits has no effect. However, treatment of beta subunits decreases the effect of 2,3-diphosphoglyceric acid on the oxygen affinity of the hybrids and reduces the 2,3-diphosphoglyceric acid-induced spectral changes in oxyhemoglobin. Therefore the interaction of dimethyl adipimidate with both the alpha and beta subunits contributes to regulating the oxygen affinity of human hemoglobin.     

Studies on the interaction of zinc with human hemoglobin

Zn has previously been shown to increase the oxygen affinity of both normal and sickle red blood cells. Experiments are presented which demonstrate that the oxygen affinity effect of Zn is due to a Zn-hemoglobin binding mechanism rather than a Zn-2,3 diphosphoglycerate binding mechanism. Further a large shift (6 mm Hg) in the oxygen affinity of a red cell-saline suspension occurs with a low Zn/hemoglobin (tetramer) molar ratio (0.4). Zn had no influence on the Bohr effect of hemoglobin but it did decrease the Hill coefficient. Hemoglobin binding experiments using partially purified hemoglobin indicated that Zn can bind to more than one amino acid residue but it appears that the amino acid residue with the highest binding capacity for Zn is also the residue involved in the oxygen affinity effect of Zn. Hydrogen ion concentration (pH 5–8) had no influence on the Zn/hemoglobin ratios obtained in these binding experiments. The possible (and the improbable) Zn binding sites on the hemoglobin molecule are discussed.     

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.     

Acylation of human hemoglobin with polyoxyethylene derivatives

Monomethoxypolyoxyethylene (Mw = 5000) was covalently linked to human hemoglobin via an amide bond formed between amino groups of the protein and a carboxylic group introduced onto the polymer. The conjugates thus obtained have a molecular size corresponding to that of a globular protein with a molecular weight of about 190 000. Their oxygen-binding properties depend upon the initial conformation of the hemoglobin and reaction pH: hemoglobin modified in the deoxy state exhibited a lower oxygen affinity than that modified in the oxy state, and the lower the reaction pH, the lower the oxygen affinity of polymer-linked hemoglobin. However, the affinity of modified hemoglobin is always higher than that of native hemoglobin. On the other hand, when deoxyHb was complexed with organic phosphates during the condensation reaction, the resulting conjugates exhibited oxygen-binding characteristics quite similar to those of native hemoglobin, i.e., the same oxygen affinity, modified cooperativity and the same alkaline Bohr effect. Finally, in order to decrease the oxygen affinity of hemoglobin conjugates, the polymer was coupled to deoxy hemoglobin previously covalently modified with pyridoxal phosphate. The oxygen affinity of such conjugates was in fact as low as that of the initial pyridoxylated hemoglobin.     

Effect of Cl- and H+ on the oxygen binding properties of glutaraldehyde-polymerized bovine hemoglobin-based blood substitutes

Bovine hemoglobin was cross-linked with glutaraldehyde, resulting in high oxygen affinity polymeric hemoglobin dispersions of varying molecular weight distributions. High oxygen affinity acellular oxygen carriers were designed in order to exhibit oxygen release profiles closer to that of human red blood cells (RBCs), without exhibiting the inherent increased vasoactivity that occurs with low oxygen affinity acellular oxygen carriers (1, 2). Oxygen dissociation curves were measured for polymerized hemoglobin dispersions at various pH values (7.0, 7.4, and 8.0) and chloride ion concentrations. Unmodified hemoglobin showed an increase in oxygen affinity with increased chloride ion concentration and a decrease in oxygen affinity with increased pH, as was previously demonstrated in the literature (3). For glutaraldehyde-polymerized hemoglobin dispersions, the ability of the oxygen affinity to respond to changes in Bohr H+ and Cl- concentration was weakened. However, at acidic physiological pH (pH = 7), the Bohr effect was still present at high Cl- concentrations. Thus, the Bohr effect maintained some dependency on the Cl- concentration.     

Functional characterization of the single hemoglobin of the migratory bird Ciconia ciconia

Hemolysate from white stork displayed a single hemoglobin component, thus resulting into two bands and two globin peaks in dissociating PAGE and reversed phase-HPLC, respectively. Stripped hemoglobin showed an oxygen affinity higher than that of human HbA, a small Bohr effect, and a cooperative oxygen binding. A small decrease of oxygen affinity, of the same extent in all the pH range examined, was observed by addition of chloride, thus indicating an unusual chloride-independent Bohr effect (DeltalogP50/Deltalog pH=-0.24). Saturating amounts of inositol hexakisphosphate, largely decreased hemoglobin-oxygen affinity (DeltalogP(50)=1.17 at pH 7.0), and increased the extent of its Bohr effect (DeltalogP50/DeltalogpH=-0.45). The phosphate binding curve allowed to measure a very high overall binding constant (K=1.18 x 10(5) M(-1)). The effect of temperature on the oxygen affinity was measured, and the enthalpy change of oxygenation resulted almost independent on pH. Structural-functional relationships are discussed by considering some amino acid residues situated at alpha1/beta1 and alpha1/beta2 interfaces, such as alpha38 and alpha89 positions. The presence of only one hemoglobin component, a rare event among birds, and its functional properties have been related to the physiological oxygen requirements of this soaring migrant bird and to its technique of flight during migration.     

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.     

Reciprocal interaction of hemoglobin with oxygen and protons. The influence of allosteric polyanions.

The interaction of three inositol esters, inositol hexaphosphate (IHP), inositol pentaphosphate (IPP), and inositol hexasulfate (IHS), with hemoglobin has been investigated. The proton uptake method was used to obtain the six binding constants for deoxy- and oxyhemoglobin. These data combined with oxygen binding curves over a range of cofactor concentrations were used to test theoretical and empirical equations relating the affinity of hemoglobin for oxygen and allosteric effectors. The Bohr and Haldane coefficients in the presence of the inositol esters are unequal at low, but not at high, concentration of the cofactors. The maximum value reached by both parameters increases with the number of negative charges of the polyanion. 2,3-Diphosphoglycerate (DPG) differs sharply from the inositol esters since even at high concentrations of this cofactor, the Haldane coefficient remains elevated. This is a reflection of the negligible affinity of DPG for fully oxygenated hemoglobin.     

The role of oxygen-binding properties of hemoglobin in the development of oxygen deficiency in acute exogenous hyperthermia

Acid-base balance and oxygen-binding hemoglobin properties in mixed venous blood have been studied in 25 mongrel rabbits with acute environmental hyperthermia. As oxygen-hemoglobin affinity at standard pH, pCO2 and temperature increases, the effect of heat on oxygen-hemoglobin interaction is considerably attenuated. The Bohr effect increases. The mechanisms of changes in oxygen-binding properties of hemoglobin and their role in development of oxygen deficiency are discussed.     

Modulation of oxygen affinity in hemoglobin by solvent components. Interaction of bovine hemoglobin with 2,3-diphosphoglycerate and monatomic anions

In the absence of Cl- in Hepes buffer at pH 7.4, the oxygen affinity of bovine and human hemoglobin is equally sensitive to 2,3-diphosphoglyceric acid. The low oxygen affinity measured for bovine hemoglobin at physiological salt concentration can be explained by the high affinity of Cl- anions for oxygen-linked sites that are absent in human hemoglobin. Bovine hemoglobin can discriminate between the different halogens in the sense that different halide concentrations are necessary to produce the same P50. Competition experiments indicate that the halogens interact with the same oxygen-linked sites. In agreement with the different affinities for halides, the Bohr effect of bovine hemoglobin is larger in the presence of Cl- than in that of Br- and there is good agreement between the number of protons and anions exchanged with the solvent upon oxygenation of bovine hemoglobin.