Comparison of nitric oxide-induced oxidation of recombinant oxyhemoglobin subunits using a competition experiment

A low reaction rate with nitric oxide (NO) is one of the important characteristics of hemoglobin (Hb)-based oxygen carriers. The reaction rate between oxyHb and NO is usually measured by stopped-flow spectrophotometry. However, the reported rates vary due to the difficulty of accurately determining the NO concentration and the limit of the instrument dead time. To circumvent these problems, we developed an experiment using oxymyoglobin (oxyMb) to compete with oxyHb for NO that is released from an NO donor. Determination of the rate constants in the competition experiment no longer depends on accurate measurement of time or NO concentration, since this approach instead measures the ratio of rate constants for the reaction of oxyHb and oxyMb with NO. For recombinant mutant Hb α(L29F)β the rates for α(L29F) and β are approximately 15- and 1.6-fold smaller than for wild-type Hb. In conclusion, the competition experiment provides an alternative method for determination of relative reaction rates of recombinant Hb subunits with NO.     

Scavenging of reactive nitrogen species by oxygenated hemoglobin: globin radicals and nitrotyrosines distinguish nitrite from nitric oxide reaction

The reaction of *NO and NO2- with hemoglobin (Hb) is of pivotal importance to blood vessel function. Both species show at least two different reactions with Fe2+ Hb: one with deoxygenated Hb, in which the biological properties of *NO are preserved, and another with oxygenated hemoglobin (oxyHb), in which both species are oxidizes to NO3-. In this study we compared the oxidative reactions of *NO and NO2- and, in particular, the radical intermediates formed during transformation to NO3-. The reaction of NO2- with oxyHb was accelerated at high heme concentrations and produced stoichiometric amounts of NO3-. Direct EPR and spin trapping studies showed that NO2-, but not *NO, induced the formation of globin Tyr-, Trp-, and Cys-centered radicals. MS studies provided evidence of the formation of approximately 2% nitrotyrosine in both the alpha and beta subunits, suggesting that *NO2 diffuses in part away from the heme and reacts with Tyr radicals. No nitrotyrosines were detected in the reaction of *NO with oxyHb. Collectively, these results indicate that NO2- reaction with oxyHb causes an oxidative challenge not observed with *NO. The differences in oxidation mechanisms of *NO and NO2- are discussed.     

Erythrocyte consumption of nitric oxide: competition experiment and model analysis.

It is generally believed that the erythrocyte membrane is highly permeable to nitric oxide (NO). To prevent NO from freely entering and being scavenged by the red blood cell (RBC), it has been suggested that NO consumption is limited by the mass transfer resistance of the diffusion layer adjacent to the erythrocyte membrane. Recently, we (Vaughn et al. (2000). J. Biol. Chem. 275, 2342) presented an experimental technique that overcomes experimental diffusional limitations and showed that RBCs also possess a mechanism to slow nitric oxide uptake. Here, we present a mathematical analysis of this technique by modeling the NO uptake of a single cell. We obtain additional data (n = 33, total) by use of the competition experiment and, through application of the model, show that either the RBC membrane permeability to NO or the intracellular reaction rate between NO and hemoglobin (Hb) is at least 2000-fold lower than previously thought. As a result, RBCs react with NO at a rate three orders of magnitude slower than free oxyHb. This phenomena may play an important role in NO bioavailability.     

Biochemical aspects of the reaction of hemoglobin and NO: implications for Hb-based blood substitutes

The role of Hemoglobin (Hb) on nitric oxide (NO) biology has received much attention. Until recently, the reaction between erythrocytic Hb and NO was generally considered in the context of mechanisms that safely detoxify NO. However, recent insights suggest that properties associated with the red blood cell limit NO-Hb interactions under physiological conditions, and provide some resolution to the question of how NO functions in the presence of blood. Furthermore, Hb-dependent mechanisms that preserve, not destroy NO bioactivity in vivo have also been proposed. The emerging picture suggests that the interplay between NO and erythrocytic Hb is important in regulating the functions of both these molecules in vivo. However, Hb-dependent scavenging and loss of NO function is significant when this heme protein is present outside the red blood cell. This can occur during hemolysis or administration of Hb-based blood substitutes. Scavenging of NO is a significant problem that limits the use of Hb-based blood substitutes in the clinic, and development of Hb molecules that do not efficiently react with NO remains an important area of investigation. In this article, the reactions between NO and erythrocytic Hb or cell-free Hb are described and the effects on NO and Hb function in vivo and development of blood substitutes discussed.     

Biochemical characterization of human S-nitrosohemoglobin. Effects on oxygen binding and transnitrosation.

S-Nitrosation of cysteine beta93 in hemoglobin (S-nitrosohemoglobin (SNO-Hb)) occurs in vivo, and transnitrosation reactions of deoxygenated SNO-Hb are proposed as a mechanism leading to release of NO and control of blood flow. However, little is known of the oxygen binding properties of SNO-Hb or the effects of oxygen on transnitrosation between SNO-Hb and the dominant low molecular weight thiol in the red blood cell, GSH. These data are important as they would provide a biochemical framework to assess the physiological function of SNO-Hb. Our results demonstrate that SNO-Hb has a higher affinity for oxygen than native Hb. This implies that NO transfer from SNO-Hb in vivo would be limited to regions of extremely low oxygen tension if this were to occur from deoxygenated SNO-Hb. Furthermore, the kinetics of the transnitrosation reactions between GSH and SNO-Hb are relatively slow, making transfer of NO+ from SNO-Hb to GSH less likely as a mechanism to elicit vessel relaxation under conditions of low oxygen tension and over the circulatory lifetime of a given red blood cell. These data suggest that the reported oxygen-dependent promotion of S-nitrosation from SNO-Hb involves biochemical mechanisms that are not intrinsic to the Hb molecule.     

Erythrocytes possess an intrinsic barrier to nitric oxide consumption

It has been reported that free hemoglobin (Hb) reacts with NO at an extremely high rate (K(Hb) approximately 10(7) M(-1) s(-1)) and that the red blood cell (RBC) membrane is highly permeable to NO. RBCs, however, react with NO 500-1000 times slower. This reduction of NO reaction rate by RBCs has been attributed to the extracellular diffusion limitation. To test whether additional limitations are also important, we designed a competition test, which allows the extracellular diffusion limitation to be distinguished from transmembrane or intracellular resistance. This test exploited the competition between free Hb and RBCs for NO generated in a homogenous phase by an NO donor. If the extracellular diffusion resistance is negligible, then the results would follow a kinetic model that assumes homogenous reaction without extracellular diffusion limitation. In this case, the measured effective reaction rate constant, K(RBC), would remain invariant of the hematocrit, extracellular-free Hb concentration, and NO donor concentration. Results show that the K(RBC) approaches a constant only when the hematocrit is greater than 10%, suggesting that at higher hematocrit, the extracellular diffusion resistance is negligible. Under such a condition, the NO consumption by RBCs is still 500-1000 times slower than that by free Hb. This result suggests that intrinsic RBC factors, such as transmembrane diffusion limitation or intracellular mechanisms, exist to reduce the NO consumption by RBCs.     

Hemoglobin α in the blood vessel wall

Hemoglobin has been studied and well characterized in red blood cells for over 100 years. However, new work has indicated that the hemoglobin α subunit (Hbα) is also found within the blood vessel wall, where it appears to localize at the myoendothelial junction (MEJ) and plays a role in regulating nitric oxide (NO) signaling between endothelium and smooth muscle. This discovery has created a new paradigm for the control of endothelial nitric oxide synthase activity, nitric oxide diffusion, and, ultimately, vascular tone and blood pressure. This review discusses the current knowledge of hemoglobin׳s properties as a gas exchange molecule in the bloodstream and extrapolates the properties of Hbα biology to the MEJ signaling domain. Specifically, we propose that Hbα is present at the MEJ to regulate NO release and diffusion in a restricted physical space, which would have powerful implications for the regulation of blood flow in peripheral resistance arteries.     

Potential of peroxynitrite to promote the conversion of oxyhemoglobin to methemoglobin

Superoxide anion and NO can react to form the highly oxidizing species peroxynitrite (ONOO-)which can react directly with hemoglobin (Hb) even in the presence of physiological concentration CO:. Thisresearch was to determine the ONOO--mediated oxidation damage to the heme of oxyhemoglobin (oxyHb)under conditions expected in blood. Results showed that 8-10 mol ONOO- was needed to quickly andcompletely convert 1 mol oxyHb to methemoglobin (metHb). ONOO- (20-140 μM) caused raoid andextensive formation of metHb from oxyHb (50 μM) mainly occurring within first 5-20 min of incubation.The conversion efficiency reached 16%, 48%, 60%, 79% and 88% output of metHb after 90 min ofincubation at 0, 20, 40, 100, and 140 μM ONOO- respectively. 1 mM CO2 caused a small decrease in theability of ONOO- to oxidize oxyHb, and ONOO--promoted conversion of oxyHb to metHb increased whenpH decreased from 8.0 to 6.0. Relatively lower temperature in blood condition will inhibit this reaction insome degree. We postulate that ONOO- can mediate oxidation damage to the heme, and cause heme lossfrom the hydrophobic cavity of Hb when its concentration exceeded 90 μM. These results indicated thatONOO- could convert oxyHb to metHb under the conditions expected in blood, and this reaction wasregulated by CO2 concentration, reaction time, temperature and pH value.     

Heme nitrosylation of deoxyhemoglobin by s-nitrosoglutathione requires copper

NO reactions with hemoglobin (Hb) likely play a role in blood pressure regulation. For example, NO exchange between Hb and S-nitrosoglutathione (GSNO) has been reported in vitro. Here we examine the reaction between GSNO and deoxyHb (HbFe(II)) in the presence of both Cu(I) (2,9-dimethyl-1, 10-phenanthroline (neocuproine)) and Cu(II) (diethylenetriamine-N,N,N',N",N"-pentaacetic acid) chelators using a copper-depleted Hb solution. Spectroscopic analysis of deoxyHb (HbFe(II))/GSNO incubates shows prompt formation (<5 min) of approximately 100% heme-nitrosylated Hb (HbFe(II)NO) in the absence of chelators, 46% in the presence of diethylenetriamine-N,N,N',N",N"-pentaacetic acid, and 25% in the presence of neocuproine. Negligible (<2%) HbFe(II)NO was detected when neocuproine was added to copper-depleted HbFe(II)/GSNO incubates. Thus, HbFe(II)NO formation via a mechanism involving free NO generated by Cu(I) catalysis of GSNO breakdown is proposed. GSH is a source of reducing equivalents because extensive GSSG was detected in HbFe(II)/GSNO incubates in the absence of metal chelators. No S-nitrosation of HbFe(II) was detected under any conditions. In contrast, the NO released from GSNO is directed to Cysbeta(93) of oxyHb in the absence of chelators, but only metHb formation is observed in the presence of chelators. Our findings reveal that the reactions of GSNO and Hb are controlled by copper and that metal chelators do not fully inhibit NO release from GSNO in Hb-containing solutions.     

Quantification of Intermediates Formed during the Reduction of Nitrite by Deoxyhemoglobin

Nitric oxide (NO) plays a crucial role in human physiology by regulating vascular tone and blood flow. The short life-span of NO in blood requires a mechanism to retain NO bioactivity in the circulation. Recent studies have suggested a mechanism involving the reduction of nitrite back to NO by deoxyhemoglobin in RBCs. A role for RBCs in transporting NO must, however, bypass the scavenging of NO in RBCs by hemoglobin. To understand how the nitrite reaction can deliver bioactive NO to the vasculature, we have studied the intermediates formed during the reaction. A reliable measure of the total concentration of heme-associated nitrite/NO intermediates formed was provided by combining filtration to measure free nitrite by chemiluminescence and electron paramagnetic resonance to measure the final product Hb(II)NO. By modifying the chemiluminescence method used to detect NO, we have been able to identify two intermediates: 1) a heme-associated nitrite complex that is released as NO in acid solution in the presence of ascorbate and 2) an intermediate that releases NO at neutral pH in the presence of ferricyanide when reacted with an Fe(III) ligand like azide. This species designated as “Hb(II)NO⁺ ⇆ Hb(III)NO” has properties of both isomeric forms resulting in a slower NO dissociation rate and much higher stability than Hb(III)NO, but provides a potential source for bioactive NO, which can be released from the RBC. This detailed analysis of the nitrite reaction with deoxyHb provides important insights into the mechanism for nitrite induced vasodilation by RBCs.Nitric oxide (NO), also known as the endothelium-derived relaxing factor, is an important messenger molecule involved in the regulation of vascular tone and blood flow (1). The primary source for the synthesis of NO in the circulatory system involves endothelial nitric-oxide synthase (2). This enzyme requires oxygen for the synthesis of NO and is, therefore, less effective in the microcirculation where hypoxic vasodilation regulates the delivery of oxygen. Because nitric oxide has a life-time in blood of <2 ms (3), a mechanism is required to allow for more distal and sustained effects of NO at the reduced oxygen pressures found in the microcirculation. Recent studies have suggested that the bioactivity of NO can be conserved in the blood by the uptake of NO and/or nitrite by red blood cells (RBCs)² and its interaction with hemoglobin (4–7). However, any role for the red cell in transporting nitric oxide must be able to avoid the very efficient scavenging of nitric oxide by both oxyhemoglobin (oxyHb) and deoxyhemoglobin (deoxyHb) that destroy and trap NO, respectively, preventing a physiological role for RBC NO.In a series of studies, Stamler and co-workers (7–10) have hypothesized that NO can bypass this difficulty by being transferred to the β-93 thiol group of hemoglobin (Hb) forming S-nitrosylated hemoglobin (SNO-Hb) when partially heme nitrosylated hemoglobin (Hb(II)NO) is oxygenated. The allosteric quaternary conformational change of hemoglobin at low oxygen pressure destabilizes the β-93 nitrosylated thiol and results in the transfer of NO to membrane thiol groups facilitating the release of the NO to the plasma and the vasculature. However, the extremely low levels of SNO-Hb (11) found in human blood and its instability (12) as a result of intracellular reducing conditions within the RBCs do not support the SNO-Hb hypothesis as the major mechanism for NO transport (11–13).The 2003 studies by Rifkind and Gladwin and their collaborators (4, 5, 14, 15) proposed an alternative mechanism that involved the reduction of nitrite, formed by the oxidation of NO, back to NO by a reaction with deoxyHb. Nitrite is present in the blood at fairly high levels (0.1–0.5 μmol/liter) (4, 16–18), and it is much more stable than NO or S-nitrosothiols (6), making nitrite an ideal storage pool that can be converted to NO. However, the mechanism by which the NO produced in the red cell by nitrite reduction is exported without being trapped or destroyed is still unclear. Recent studies by Rifkind and co-workers (5, 13, 19) have suggested that the trapping of NO by deoxyHb and/or oxyHb can be bypassed by the formation of a metastable intermediate(s) that retains the NO in a state that is not quenched by reacting with oxyHb or deoxyHb.In this report, we quantitate the two intermediate species that are formed during the reduction of nitrite by deoxyHb when an excess of hemoglobin is present. We also demonstrate that one of the intermediate species designated as “Hb(II)NO⁺ ⇆ Hb(III)NO” has properties of Hb(II)NO⁺ and Hb(III)NO, respectively. This species has a slower NO dissociation rate and a much higher stability than Hb(III)NO. This intermediate is a potential source for bioactive NO that can be released from RBCs.     

The binding of hemoglobin to red cell membrane lowers its oxygen affinity

The mode of interaction of human hemoglobin (Hb) with the red cell membrane was investigated with special reference to the effect on oxygen binding properties and Hb-membrane binding constants. Compared to free native Hb, the membrane-bound native Hb showed a strikingly lowered oxygen affinity and smaller response to organic phosphates such as 2,3-diphosphoglycerate and inositol hexaphosphate. Similar effects of membrane binding were also observed for intermediately cooperative Hbs such as N-ethylmaleimide-treated Hb (NES-Hb) and iodoacetamide-treated Hb (AA-Hb), but very small effects were observed for non-cooperative Hb, i.e., carboxypeptidase A-treated Hb (des-His-Tyr Hb). The magnitude of the affinity lowering was in the order: NES-Hb greater than native Hb greater than AA-Hb much greater than des-His-Tyr Hb. In the presence of inositol hexaphosphate, the three chemically modified Hbs showed an increased oxygen affinity when bound to the red cell membrane, probably due to partial replacement of bound inositol hexaphosphate by membrane. The binding to membrane caused a slight decrease in cooperativity for native Hb, but no distinct change in cooperativity was observed for the three modified Hbs. These results imply: a) the red cell membrane binds to deoxyHb more strongly than to oxyHb; b) the difference in membrane binding affinity between oxyHb and deoxyHb is closely related to the quaternary structure change in the Hb molecule occurring upon oxygenation. The higher affinity of the membrane for deoxyHb than for oxyHb apparently disagrees with the conclusion drawn by earlier investigators. However, the present binding experiments by means of ultrafiltration proved that the red cell membrane actually binds to deoxyHb much more strongly than to oxyHb, validating the present conclusion based on oxygenation experiments. Our results are consistent with those obtained recently by other investigators using a synthetic peptide or the cytoplasmic fragment of red cell membrane band 3.     

S-Nitrosohemoglobin: an allosteric mediator of NO group function in mammalian vasculature

Since the discovery of NO as the endothelium-derived relaxing factor, there has been considerable interest in how NO interacts with hemoglobin (Hb). Numerous investigations have highlighted the possibility that rather than operating as a sink to consume NO, the vasculature can operate as a delivery mechanism for NO. The principal hypothesis proposed to explain this phenomenon is that Hb can transport NO on the conserved cysteine residue beta93 and deliver that NO to the tissues in an allosterically dependent manner. This proposal has been termed the S-Nitrosohemoglobin (SNO-Hb) Hypothesis. This review addresses the experimental evidence that led to development of this hypothesis and examines much of the research that resulted from its generation. Specifically it covers the evidence concerning NO in the vasculature, the SNO-Hb Hypothesis itself, the biochemical and biophysical data relating to NO and Hb interactions, SNO-Hb in human physiology, and alternative vascular forms of NO. Finally a model of NO in the vasculature in which SNO-Hb forms the central core is proposed.     

Active nitric oxide produced in the red cell under hypoxic conditions by deoxyhemoglobin-mediated nitrite reduction

Recent studies have generated a great deal of interest in a possible role for red blood cells in the transport of nitric oxide (NO) to the microcirculation and the vascular effect of this nitric oxide in facilitating the flow of blood through the microcirculation. Many questions have, however, been raised regarding such a mechanism. We have instead identified a completely new mechanism to explain the role of red cells in the delivery of NO to the microcirculation. This new mechanism results in the production of NO in the microcirculation where it is needed. Nitrite produced when NO reacts with oxygen in arterial blood is reutilized in the arterioles when the partial pressure of oxygen decreases and the deoxygenated hemoglobin formed reduces the nitrite regenerating NO. Nitrite reduction by hemoglobin results in a major fraction of the NO generated retained in the intermediate state where NO is bound to Hb(III) and in equilibrium with the nitrosonium cation bound to Hb(II). This pool of NO, unlike Hb(II)NO, is weakly bound and can be released from the heme. The instability of Hb(III)NO in oxygen and its displacement when flushed with argon requires that reliable determinations of red blood cell NO must be performed on freshly lysed samples without permitting the sample to be oxygenated. In fresh blood samples Hb(III)NO accounts for 75% of the red cell NO with appreciably higher values in venous blood than arterial blood. These findings confirm that nitrite reduction at reduced oxygen pressures is a major source for red cell NO. The formation and potential release from the red cell of this NO could have a major impact in regulating the flow of blood through the microcirculation.     

Encapsulation of hemoglobin inside liposomes surface conjugated with poly(ethylene glycol) attenuates their reactions with gaseous ligands and regulates nitric oxide dependent vasodilation

Acellular hemoglobin (Hb)-based O2 carriers (HBOCs) are being investigated as red blood cell (RBC) substitutes for use in transfusion medicine. However, commercial acellular HBOCs elicit both vasoconstriction and systemic hypertension which hampers their clinical use. In this study, it is hypothesized that encapsulation of Hb inside the aqueous core of liposomes should regulate the rates of NO dioxygenation and O2 release, which should in turn regulate its vasoactivity. To test this hypothesis, poly(ethylene glycol) (PEG) conjugated liposome-encapsulated Hb (PEG-LEHs) dispersions were prepared using human and bovine Hb. In this study, the rate constants for O2 dissociation, CO association, and NO dioxygenation were measured for free Hb and PEG-LEH dispersions using stopped-flow UV-visible spectroscopy, while vasoactivity was assessed in rat aortic ring strips using both endogenous and exogenous sources of NO. It was observed that PEG-LEH dispersions had lower O2 release and NO dioxygenation rate constants compared with acellular Hbs. However, no difference was observed in the CO association rate constants between free Hb and PEG-LEH dispersions. Furthermore, it was observed that Hb encapsulation inside vesicles prevented Hb dependent inhibition of NO-mediated vasodilation. In addition, the magnitude of the vasoconstrictive effects of Hb and PEG-LEH dispersions correlated with their respective rates of NO dioxygenation and O2 release. Overall, this study emphasizes the pivotal role Hb encapsulation plays in regulating gaseous ligand binding/release kinetics and the vasoactivity of Hb.     

The reaction between nitrite and oxyhemoglobin: a mechanistic study

The nitrite anion (NO(-)(2)) has recently received much attention as an endogenous nitric oxide source that has the potential to be supplemented for therapeutic benefit. One major mechanism of nitrite reduction is the direct reaction between this anion and the ferrous heme group of deoxygenated hemoglobin. However, the reaction of nitrite with oxyhemoglobin (oxyHb) is well established and generates nitrate and methemoglobin (metHb). Several mechanisms have been proposed that involve the intermediacy of protein-free radicals, ferryl heme, nitrogen dioxide (NO(2)), and hydrogen peroxide (H(2)O(2)) in an autocatalytic free radical chain reaction, which could potentially limit the usefulness of nitrite therapy. In this study we show that none of the previously published mechanisms is sufficient to fully explain the kinetics of the reaction of nitrite with oxyHb. Based on experimental data and kinetic simulation, we have modified previous models for this reaction mechanism and show that the new model proposed here is consistent with experimental data. The important feature of this model is that, whereas previously both H(2)O(2) and NO(2) were thought to be integral to both the initiation and propagation steps, H(2)O(2) now only plays a role as an initiator species, and NO(2) only plays a role as an autocatalytic propagatory species. The consequences of uncoupling the roles of H(2)O(2) and NO(2) in the reaction mechanism for the in vivo reactivity of nitrite are discussed.     

Low oxygen affinity derivatives of human hemoglobin by fixation of polycarboxylic dextran to the oxyform

Solutions of modified adult human hemoglobin (Hb) have potential applications as physiological oxygen carriers. The chemical modification that has been the most studied during the last few years is the cross-linking of the protein between its two αβ dimers, in order, first, to hamper their diffusion through the kidney and therefore increase the plasma persistence of Hb, and second, to decrease its oxygen affinity. However, despite the cross-linking, the vascular retention time is only increased by a factor of three, and a supplementary modification of cross-linked Hb is needed in order to further improve its in vivo half-life. The Hb derivatives described in this paper were obtained by the covalent fixation of benzene tetracarboxylate-substituted dextran onto oxyHb. The resulting conjugates all exhibited a higher P₅₀ than native Hb. The experiments carried out in the presence of inositolhexaphosphate showed that the allosteric sites of Hb molecules were occupied by the polymeric reagent. The important decrease in the Bohr effect and the lack of the Cl^? effect on the oxygen-binding properties proved that the Val 1α residue was also substituted. Finally, the ability of some conjugates to unload as much O₂ as blood, together with their other properties, make them quite promising candidates as red cell substitutes. © 1993 John Wiley & Sons, Inc.     

Erythrocyte nitric oxide transport reduced by a submembrane cytoskeletal barrier

Encapsulation of hemoglobin (Hb) within red blood cells (RBCs) preserves nitric oxide (NO) bioactivity. With encapsulation, millimolar concentrations of Hb quench only a fraction of NO bioactivity, whereas mere micromolar concentrations of cell-free Hb completely quench NO bioactivity. A submembrane cytoskeletal barrier has been hypothesized to account for the lowered quenching of NO bioactivity. In order to substantiate this hypothesis, here, the underlying submembrane cytoskeletal barrier was physically reduced and the rate of NO entry into the modified RBC measured. The submembrane cytoskeletal barrier of normal and depleted RBCs was characterized using atomic force microscopy and the lipid to protein ratio measured. The reduction in the submembrane cytoskeletal barrier resulted in an increase in the rate of NO entry. We suggest that the underlying submembrane cytoskeleton may be a key component of RBC mediated regulation of NO bioavailability.     

Regulation of vascular function by haemoglobin

A critical element in the ability of endothelial NO to function in the vasculature is preventing its reaction with erythrocytic Hb (haemoglobin). Emerging concepts suggest that the biophysical and rheological properties of the red blood cell are important in meeting this criterion. It has been recognized for some time that cell-free Hb may react with endothelial NO and that this may underlie the problems with Hb-based blood substitutes. More recent data extend these concepts to haemolytic diseases, including sickle cell disease, and have also identified novel therapeutic strategies to prevent interactions of cell-free Hb with NO. In this overview we have hypothesized that production of high concentrations of NO can overcome the diffusional barriers presented by the red cell and result in formation of S-nitrosohaemoglobin. By doing so, it is hypothesized that Hb may mediate the vasodilatory potential of NO and contribute to the hypotensive responses observed in acute inflammatory diseases, including sepsis.     

Nitric oxide effect on the hemoglobin-oxygen affinity.

The biological roles of nitric oxide (NO)-hemoglobin (Hb) derivatives are obscure. It is proposed that NO can function as an allosteric regulator of hemoglobin oxygen-binding properties. We aimed to estimate the effects of NO donors and NO-synthase substrate (L-arginine) on hemoglobin-oxygen affinity (HOA) in experiments in vitro with the various ratios between NO formed and Hb and various oxygen pressures. HOA index (p50), blood pH, plasma and red blood cell (RBC) concentrations of nitrite/nitrate and methemoglobin amounts were measured after the experiments. In our experiments, blood incubation with NO donors (glyceryltrinitrate, molsidomine, sodium nitroprusside, S-nitrosocysteine) or NO-synthase substrate (L-arginine) did not change HOA even at NO:Hb ratio of 1:1. At the same time our results showed that oxygenated blood incubation with S-nitrosocysteine induced an oxyhemoglobin dissociation curve shift leftwards. This indicates a leading role of met-Hb in a modification of Hb oxygen-binding properties. However other NO-modified forms of hemoglobin (S-nitroso- and nitrosylhemoglobin) also may be involved in the regulation of HOA. The results obtained indicate that nitric oxide can be the allosteric effector of hemoglobin, increasing or decreasing its oxygen affinity - possibly, through the generation of different NO-Hb derivatives.     

Low micromolar intravascular cell-free hemoglobin concentration affects vascular NO bioavailability in sickle cell disease: a computational analysis

In sickle cell disease, the changes in RBC morphology destabilize the red blood cell (RBC) membrane and lead to hemolysis. Several experimental and clinical studies have associated intravascular hemolysis with pulmonary hypertension in sickle cell disease. Cell-free hemoglobin (Hb) from intravascular hemolysis has high affinity for nitrixc oxide (NO) and can affect the NO bioavailability in the sickle cell disease, which may eventually lead to pulmonary hypertension. To study the effects of intravascular hemolysis related cell-free Hb concentrations on NO bioavailability, we developed a two-dimensional mathematical model of NO biotransport in 50-μm arteriole under steady-state sickle cell disease conditions. We analyzed the effects of flow-dependent NO production and axial and radial transport of NO, a recently reported much lower NO-RBC reaction rate constant, and cell-free layer thickness on NO biotransport. Our results show that the presence of cell-free Hb concentrations as low as 0.5 μM results in an approximately three- to sevenfold reduction in the predicted smooth muscle cell NO concentrations compared with those under physiological conditions. In addition, increasing the diffusional resistance for NO in vascular lumen from cell-free layer or reducing NO-RBC reaction rate did not improve the NO bioavailability at the smooth muscle cell layer significantly for cell-free Hb concentrations ≥1 μM. These results suggest that lower NO bioavailability due to low micromolar cell-free Hb can disturb NO homeostasis and cause insufficient bioavailability at the smooth muscle cell layer. Our results supports the hypothesis that hemolysis-associated reduction in NO bioavailability may play a role in the development of pathophysiological complications like pulmonary hypertension in sickle cell disease that are observed in several clinical and experimental studies.