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.     

Interaction of zinc and hemoglobin: binding of zinc and the oxygen affinity.

Stripped human hemoglobin was shown to have a high apparent zinc association constant of 1.3 X 10(7) M-1 with a stoichiometry of one zinc for every two hemes. The saturation of this site produces a dramatic 3.7-fold increase in the oxygen affinity. The effect of zinc on the oxygen affinity is interrelated with the interaction of 2,3-diphosphoglyceric acid (2,3-DPG) and hemoglobin. Thus, a smaller zinc effect is observed in the presence of added 2,3-DPG. Information about the location of the zinc-binding site responsible for the increased oxygen affinity has been obtained by comparing the binding of zinc to various hemoglobins. Blocking the beta93 sulfhydryl group decreases the apparent zinc association constant by an order of magnitude. The substitution of histidine-beta143 in hemoglobin Abruzzo [beta143 (H21) His leads to Arg] and hemoglobin Little Rock [beta143 (H21) His leads to Gln] decreases the apparent zinc association constant by two orders of magnitude. The substitution of histidine-beta143 by other amino acids and the reaction of the beta93 sulfhydryl group are known to produce dramatic increases in the oxygen affinity. The binding of zinc to one or both of these amino acids can, therefore, explain the zinc-induced increase in the oxygen affinity.     

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.     

Iodination-Coupled Tetrazonium and Ferric Ferricya-Nide Reduction Stains for Differentiating Red Blood Cells Containing Fetal or Adult Hemoglobin

Either the iodination-coupled tetrazonium reaction or the ferric ferricyanide reduction procedure can be used to differentiate red blood cells containing fetal hemoglobin (hemoglobin F) from those containing adult hemoglobin (hemoglobin A) in blood smears. Oxalated blood is diluted with 3 parts of physiological saline, and smears are made on slides. The air-dried slides are treated with absolute ethanol for 2 min, dried, and placed in phosphate-citrate buffer of pH 3.2-3.6 for 1 min at 37°C. They are then rinsed in distilled water, and dried for storage or stained at once by either the iodination-coupled tetrazonium or the ferric ferricyanide reduction procedure. Adult hemoglobin is extracted by the buffer, so that red blood cells containing fetal hemoglobin give a much darker stain than those containing adult hemoglobin. The hemoglobin S of patients with sickle-cell anemia behaves like adult hemoglobin.     

Reduced hemoglobin affinity for oxygen in venous blood following hemodilution, independent of changes in pH or PCO2.

A rapidly induced and readily reversible shift in the affinity of hemoglobin for oxygen has been demonstrated. The shift, similar to the Bohr effect, is independent of PCO2 or pH changes. It occurred within 30 min of hemodilution and was seen in portal venous blood but not arterial blood. A hypothesis is suggested involving a phasic alteration in levels of 2,3-diphosphoglycerate (DPG) or ATP binding to hemoglobin. It is proposed that, following hemodilution, the degree of these phosphates to hemoglobin increases on passage through the intestinal vascular bed. The increased DPG binding to hemoglobin results in displacement of additional oxygen. As the blood becomes reoxygenated, the levels of DPG-hemoglobin binding decline and DPG is displaced from the hemoglobin by oxygen.     

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.     

Pharmacological correction of hypothermic P(50) shift does not alter outcome from focal cerebral ischemia in rats

Hypothermia decreases the arterial PO(2) at which hemoglobin is 50% saturated (P(50)), increasing hemoglobin O(2)-binding affinity. We used RSR13, a synthetic allosteric modifier of hemoglobin that increases P(50), to study the role of altered hemoglobin O(2)-binding affinity in mild hypothermic neuroprotection. RSR13 (150 mg/kg iv) restored P(50) to normothermic values. Rats underwent 70 min of middle cerebral artery occlusion (MCAO) at 30.0, 34.0, or 37.5 degrees C with hemoglobin saturation held at 98-100%. The 34.0 degrees C group received RSR13 or vehicle before ischemia. After 7 days of recovery, infarct volumes were reduced in all hypothermic groups, without evidence of a detrimental effect on infarct size or neurological score as a result of P(50) correction. To examine for a beneficial effect of P(50) correction, ischemia duration was increased to 120 min in rats maintained at 34.0 degrees C. Correction of P(50) by RSR13 did not alter cerebral infarct sizes or neurological scores. The decrease in P(50), caused by mild hypothermia, could not be associated with infarct size or neurological deficit resulting from ischemic brain hypoxia in rats.     

A compartmental model for oxygen transport in brain microcirculation in the presence of blood substitutes

A compartmental model is developed for oxygen (O(2)) transport in brain microcirculation in the presence of blood substitutes (hemoglobin-based oxygen carriers). The cerebrovascular bed is represented as a series of vascular compartments, on the basis of diameters, surrounded by a tissue compartment. A mixture of red blood cells (RBC) and plasma/extracellular hemoglobin solution flows through the vascular bed from the arterioles through the capillaries to the venules. Oxygen is transported by convection in the vascular compartments and by diffusion in the surrounding tissue where it is utilized. Intravascular resistance and the diffusive loss of oxygen from the arterioles to the tissue are incorporated in the model. The model predicts that most of the O(2) transport occurs at the level of capillaries. Results computed from the present model in the presence of hemoglobin-based oxygen carriers are consistent with those obtained from the earlier validated model (Sharan et al., 1989, 1998a) on oxygen transport in brain circulation in the absence of extracellular hemoglobin. We have found that: (a) precapillary PO(2) gradients increase as PO(2) in the arterial blood increases, P(50 p) (oxygen tension at 50% saturation of hemoglobin with O(2) in plasma) decreases, i.e. O(2) affinity of the extracellular hemoglobin is increased, the flow rate of the mixture decreases, hematocrit decreases at constant flow, metabolic rate increases, and intravascular transport resistance in the arterioles is neglected; (b) precapillary PO(2) gradients are not sensitive to (i) intracapillary transport resistance, (ii) cooperativity (n(p)) of hemoglobin with oxygen in plasma, (iii) hemoglobin concentration in the plasma and (iv) hematocrit when accounting for viscosity variation in the flow; (c) tissue PO(2) is not sensitive to the variation of intravascular transport resistance in the arterioles. We also found that tissue PO(2) is a non-monotonic function of the Hill coefficient n(p) for the extracellular hemoglobin with a maximum occurring when n(p) equals the blood Hill coefficient. The results of the computations give estimates of the magnitudes of the increases in tissue PO(2) as arterial PO(2) increases,P(50 p) increases, flow rate increases, hematocrit increases, hemoglobin concentration in the plasma increases, metabolic rate decreases, the capillary mass transfer coefficient increases or the intracapillary transport resistance decreases.     

Vanadate increases oxygen affinity and affects enzyme activities and membrane properties of erythrocytes

Incubation of blood with vanadate markedly increases the affinity of hemoglobin for oxygen, decreases the deformability of erythrocytes, reduces their osmotic fragility and alters their morphology, determining the appearance of equinocytic forms. Since vanadate is easily taken up by the erythrocytes and binds hemoglobin, these effects might result from interactions of vanadate with hemoglobin and with membrane proteins at the glycerate-2, 3-P₂ and/or ATP binding site. In addition, vanadate inhibits phosphoglycerate mutase, phosphoglucomutase and adenylate-kinase activities from hemolysates, suggesting a possible inhibitory effect on erythrocyte metabolism     

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.     

Effects of prostaglandins on the affinity of hemoglobin for oxygen in human whole blood in vitro

The effect of prostaglandin on the affinity of hemoglobin for oxygen was tested on human blood $\text{in vitro}$, using six different prostaglandins at several dosage levels in fresh heparinized blood from normal donors and in stored citrated blood, and using prostaglandin E₂ on the blood from four seriously ill patients. No significant alterations in the affinity of hemoglobin for oxygen were dtected. A very small decrease in oxygen affinity in stored blood with high doses of prostaglandin was not statistically significant and would be of no physiologic significance even if real.We conclude that under the circumstances of this experiment prostaglandins do not alter the affinity of hemoglobin for oxygen in human whole blood $\text{in vitro}$.     

The Histochemical Demonstration of Hemoglobin in Blood Cells and Tissue Smears

The demonstration of intracellular hemoglobin in permanent preparations has long been a problem. The affinity of hemoglobin for iron hematoxylin is well known but this stain also colors yolk, chromatin, and other structures and is therefore not a reliable criterion. The presence of hemoglobin has been associated with an acidophil cytoplasm which stains a characteristic color, but a careful inspection of living cells in early hematopoetic or embryological stages demonstrates that hemoglobin is present in the erythrocytes which are quite basophilic. In the course of some research on the blood of embryonic frogs it became desirable to demonstrate the presence of hemoglobin in cells by means of a specific staining reaction.     

Characteristics of the blood oxygen transport function in angina pectoris patients receiving treatment aimed at correcting the L-arginine-NO pathway

The oxygen transport function of the blood was studied in angina pectoris patients treated with nitrosorbide to correct the L-arginine-NO pathway. A series of parameters (the O₂ content of venous blood, blood oxygen capacity, blood oxygenation level, methemoglobin content, oxygen affinity of methemoglobin, and central hemodynamic parameters) were determined. The oxygen affinity of hemoglobin in patients with functional class I and II stable exertional angina decreased according to the severity of the disease and, therefore, can be considered a criterion of an unfavorable course of the disease. Nitrosorbide therapy resulted in an increase in the oxygen affinity of hemoglobin.     

Oxygen binding by marine fish blood under hypothermic experimental conditions

Influence of temperature in the range of 1-15 degrees C on oxygen binding properties of blood of thermophilic--golden mullet (Liza aurata), anchovy (Engraulis encrasicolus), and cold-tolerant--sardelle (Clupeonella cultriventris) fishes has been investigated under experimental conditions. Heat dependence of oxygenation reaction in thermophilic fish blood at temperature below 10 degrees C considerably increases, which is evidenced by high deltaH values. That is accompanied by a substantial increase of blood oxygen affinity and complicates blood deoxygenation at the tissue level. This reaction is apparently determined by the change of hemoglobin interaction with intraerythrocyte medium. The concentration of NTP in erythrocytes increases, that partially compensates negative changes of blood oxygen affinity (parameter P50 is raised) under long-term maintenance of fishes at 5 degrees C. However this reaction is not observed at low temperatures (1-2 degrees C).     

On the Oxygen Affinity of Bird Blood

In oxygen affinity characteristics bird blood appears to haveseveral features that distinguish it from mammalian blood. Fordomesticated species at least the range of oxygen half saturationvalues is extremely wide. A difference in the shape of the oxygendissociation curve has been recorded by several authors withan increase in sigmoidocity with increasing oxygen saturation.There is evidence that the oxygen affinity determining organicphosphate of bird red blood cells inositol pentaphosphate (IP5)is relatively metabolically inert. This suggests that modulationof blood oxygen affinity is primarily achieved by altering theIP5 hemoglobin interaction rather than varying IP5 levels perse. In contrast to mammals carbon dioxide has no direct effecton whole blood oxygen affinity for some bird species (hen chickgoose) or it may cause the oxygen affinity to increase (pigeonflamingo). Carbon dioxide is a blood oxygen affinity modulatorof some flexibility its effect in both direction and magnitudebeing dependent on the hemoglobin type red cell pH and organicphosphate levels. The physiological significance of these distinguishingfeatures is discussed.     

The involvement of nitric oxide in formation of hemoglobin oxygen-binding properties

The analysis of literature and results of our investigations indicate the possible involvement of L-arginine-nitric oxide (NO) system in formation of blood oxygen-carrying capacity. In reaction with hemoglobin NO forms methemoglobin, nitrosyl-hemoglobin (HbFe2+NO) and S-nitrosohemoglobin (SNO-Hb). The NO-hemoglobin derivatives have the various biological functions (NO transport, storage, elimination etc.) and are involved in the genesis of different pathologic conditions. The presence of different NO-hemoglobin derivatives can differently influence on the whole blood hemoglobin-oxygen affinity (HOA): methemoglobin and SNO-Hb increases, and HbFe2+NO decreases it. Their effect on the blood oxygen-binding properties may be important for the gas exchange processes. At the level of lung capillaries such effect may be the additional mechanism promoting a blood oxygenation, and in the systemic microcirculation it may optimize blood desaturation and hence the tissue oxygen delivery. Blood oxygen-binding properties affect the state of L-arginine-NO system, however this system also may determine HOA through the intraerythrocytic regulatory mechanisms, oxygen-dependent nature of NO generation, regulation of vascular tone and effect of peroxynitrite.     

Improvement of oxygen-carrying properties of human hemoglobin by chemical modification with a benzene hexacarboxylate-monosubstituted polyoxyethylene

Benzene hexacarboxylate-monosubstituted polyoxyethylene on contact with Hb decreases its oxygen affinity, probably because it specifically interacts with the amino groups of the phosphate-binding site. This site specificity was used to direct the covalent coupling of this polymeric reagent with hemoglobin, in the vicinity of this cleft in order to obtain conjugates with low oxygen affinity and well-defined molecular weight. Such conjugates could thus be regarded as potential candidates for blood substitutes. Covalent fixation of this polymeric site-labeling reagent onto hemoglobin was carried out with the oxy and the deoxy form in the presence of a water soluble carbodiimide. It turns out that the oxygen-binding properties of the resulting hemoglobin derivatives depend on the reaction conditions, yet in all cases the oxygen affinity of the modified protein was lower than that of native hemoglobin and was no longer affected by organic phosphates. These results indicate that phosphate-binding site amines are probably involved in the covalent coupling, although in some conjugates (especially those prepared with high ratios of reagents) other amino groups participate also in the linking to the polymer. Chromatographic analysis and trypic peptide mapping of some conjugates evidenced that the -terminal valine residue was in fact the preferential binding site of hexacarboxylate-monosubstituted polyoxyethylene.     

Reaction of S-nitrosoglutathione with the heme group of deoxyhemoglobin

The mechanism of interaction between S-nitrosoglutathione (GSNO) and hemoglobin is a crucial component of hypotheses concerning the role played by S-nitrosohemoglobin in vivo. We previously demonstrated (Patel, R. P., Hogg, N., Spencer, N. Y., Kalyanaraman, B., Matalon, S., and Darley-Usmar, V. M. (1999) J. Biol. Chem. 274, 15487-15492) that transnitrosation between oxygenated hemoglobin and GSNO is a slow, reversible process, and that the reaction between GSNO and deoxygenated hemoglobin (deoxyHb) did not conform to second order reversible kinetics. In this study we have reinvestigated this reaction and show that GSNO reacts with deoxyHb to form glutathione, nitric oxide, and ferric hemoglobin. Nitric oxide formed from this reaction is immediately autocaptured to form nitrosylated hemoglobin. GSNO reduction by deoxyHb is essentially irreversible. The kinetics of this reaction depended upon the conformation of the protein, with more rapid kinetics occurring in the high oxygen affinity state (i.e. modification of the Cysbeta-93) than in the low oxygen affinity state (i.e. treatment with inositol hexaphosphate). A more rapid reaction occurred when deoxymyoglobin was used, further supporting the observation that the kinetics of reduction are directly proportional to oxygen affinity. This observation provides a mechanism for how deoxygenation of hemoglobin/myoglobin could facilitate nitric oxide release from S-nitrosothiols and represents a potential physiological mechanism of S-nitrosothiol metabolism.     

Hemoglobin Abruzzo (beta143 (H21) His replaced by Arg). Consequences of altering the 2,3-diphosphoglycerate binding site.

Hemoglobin Abruzzo is an abnormal human hemoglobin with a substitution at a residue known to be involved in the binding of 2,3-diphosphoglyceric acid. It has increased oxygen affinity and reduced heme-heme interaction in the absence of organic or inorganic phosphate cofactors. In inorganic phosphate buffers the Bohr effect and heme-heme interaction are normal, but the oxygen affinity remains higher than that of hemoglobin A. CO combination in inorganic phosphate is more strongly autocatalytic than in normal hemoglobin and a slower rate of oxygen dissociation is observed. Although many of the functional differences of this variant may be attributed to the high oxygen affinity of the mutant beta chains, the interactions between subunits are also affected by the histidine to arginine substitution at beta143. Stripped hemoglobin Abruzzo appears to be significantly more dissociated than hemoglobin A. Kinetic studies indicate that interaction with organic or inorganic phosphates decreases its subunit dissociation. In all of the functional properties examined, hemoglobin Abruzzo is more sensitive to the allosteric influence of organic and inorganic anions than is hemoglobin A.     

Solution of the spatial structure of dimeric transmembrane domains of proteins by heteronuclear NMR spectroscopy and molecular modeling

The goal of the work was to asses the effect of peroxynitrite on the affinity of hemoglobin for oxygen in in vitro experiments. It was demonstrated that the incubation of whole venous blood with peroxynitrite increased the affinity of hemoglobin for oxygen. Presumably, this effect is realized through generation of various forms of hemoglobin: heme-oxidized and modified at amino acid residues of the protein. The dependence of the results of hemoglobin-peroxynitrite reactions on carbon dioxide tension and the degree of hemoglobin oxygenation is discussed.