Ferryl Hb Research Articles

Impact of cold storage on the oxygenation and oxidation reactions of red blood cells.

Introduction: Electrostatic binding of deoxyhemoglobin (Hb) to cytoplasmic domain of band 3 anion transport protein occurs as part of the glycolytic regulation in red blood cells (RBCs). Hb oxidation intermediates not only impact RBC's oxygenation but also RBC's membrane through the interaction with band 3. It is not known however whether these critical pathways undergo changes during the storage of RBCs. Methods and Results: Oxygen parameters of fresh blood showed a sigmoidal and cooperative oxygen dissociation curve (ODC) for the first week of storage. This was followed by a large drop in oxygen affinity (P50) (from 30 to 20mmHg) which remained nearly unchanged with a slight elevation in Bohr coefficients and a significant drop in extracellular acidification rates (ECAR) at the 42-day storage. Oxidation of Hb increased with time as well as the formation of a highly reactive ferryl Hb under oxidative stress conditions. Ferryl Hb interacted avidly with RBC's membrane's band 3, but to lesser extent with old ghost RBCs. Discussion: The observed alterations in RBC's oxygen binding may have been affected by the alterations in band 3's integrity which are largely driven by the internal iron oxidation of Hb. Restoring oxygen homeostasis in stored blood may require therapeutic interventions that target changes in Hb oxidation and membrane changes.

Potential Antioxidative Activity of Homocysteine in Erythrocytes under Oxidative Stress.

Homocysteine is an amino acid containing a free sulfhydryl group, making it probably contribute to the antioxidative capacity in the body. We recently found that plasma total homocysteine (total-Hcy) concentration increased with time when whole blood samples were kept at room temperature. The present study was to elucidate how increased plasma total-Hcy is produced and explore the potential physiological role of homocysteine. Erythrocytes and leukocytes were separated and incubated in vitro; the amount of total-Hcy released by these two kinds of cells was then determined by HPLC-MS. The effects of homocysteine and methionine on reactive oxygen species (ROS) production, osmotic fragility, and methemoglobin formation in erythrocytes under oxidative stress were studied. The reducing activities of homocysteine and methionine were tested by ferryl hemoglobin (Hb) decay assay. As a result, it was discovered that erythrocytes metabolized methionine to homocysteine, which was then oxidized within the cells and released to the plasma. Homocysteine and its precursor methionine could significantly decrease Rosup-induced ROS production in erythrocytes and inhibit Rosup-induced erythrocyte's osmotic fragility increase and methemoglobin formation. Homocysteine (but not methionine) was demonstrated to enhance ferryl Hb reduction. In conclusion, erythrocytes metabolize methionine to homocysteine, which contributes to the antioxidative capability under oxidative stress and might be a supplementary protective factor for erythrocytes against ROS damage.

Caffeic acid: an antioxidant with novel antisickling properties.

It is well documented that caffeic acid (3,4‐dihydroxycinnamic acid) (CA) interacts with and inhibits the oxidative reactions of myoglobin (Mb) and hemoglobin (Hb), and this interaction underlies its antioxidative action in meat. Sickle cell hemoglobin (HbS) is known for its tendency to oxidize more readily than normal HbA in the presence of hydrogen peroxide (H2O2), which leads to a more persistent and highly oxidizing ferryl Hb (HbFe4+). We have investigated the effects of CA on HbS oxidation intermediates, specifically on the ferric/ferryl forms. At a low concentration of H2O2 (0.5‐fold over heme), we observed a fivefold reduction in the amount of HbFe4+ accumulated in a mixture of ferric and H2O2 solution. Higher levels of H2O2 (onefold and twofold over heme) led to a lesser threefold and twofold reduction in the content of HbFe4+, respectively, possibly due to the saturation of the binding sites on the Hb molecule. The most intriguing finding was that when 5‐molar excess CA over heme was used, and a considerable increase in the delay time of HbS polymerization to approximately 200 s was observed. This delay in polymerization of HbS is theoretically sufficient to avoid microcapillary blockage and prevent vasoconstrictions in vivo. Mass spectrometry analysis indicated that CA was more extensively covalently bonded to βCys93 than to βCys112 and αCys104. The dual antioxidant and antisickling properties of CA may be explored further to maximize its therapeutic potential in SCD.

Oxidative degradation perturbs physico-chemical properties of hemoglobin in cigarette smokers: a threat to different biomolecules

Context Cigarette smokers develop structural modification in hemoglobin (Hb) and this modification enable Hb to undergo higher rate of auto-oxidation, leading to generation of further intracellular ROS. Objective In this study, we exhibited the possible cause and consequences of Hb modification in cigarette smokers. Methods Twenty-two smokers and 16 nonsmokers, aged 25 to 35 years, having a smoking history of 7–10 years were recruited in this study. Carbonyl content, ferryl form, peroxidase-like and esterase-like activities of Hb were assayed. Free iron release by Hb, erythrocyte membrane-bound Hb and plasma Hb were also measured along with assessment of important biomolecular degradations by Hb. Results and discussion Increase in carbonyl content in Hb indicates its oxidative degradation. Increase in ferryl Hb formation, peroxidase-like activity and decrease in esterase like activity of Hb along with increased release of nonheme iron (from Hb) clearly indicates alteration in physico-chemical properties of Hb in smokers. Moreover, increase in erythrocyte membrane-bound Hb and plasma-free Hb provide further evidences for higher rate of Hb oxidation in smokers’ erythrocyte. The rates of protein, lipid, sugar and DNA degradation were noticed to be higher by smokers’ Hb; and were further attenuated by desferrioxamine as well as mannitol. Conclusion We conclude that in cigarette smokers, there is oxidative degradation of Hb and the degradation causes alteration in its physico-chemical properties, which in turn may degrade different biomolecules in its close vicinity by releasing more iron and production of more superoxide as well as hydroxyl radical.

Ferrous hemoglobin and hemoglobin-based oxygen carriers acting as a peroxidase can inhibit oxidative damage to endothelial cells caused by hydrogen peroxide.

Oxidative damage caused by the ferryl hemoglobin is one of the major clinical adverse reactions of hemoglobin-based oxygen carriers (HBOCs), while the production of reactive oxygen species in a pathological state can oxidize hemoglobin (HbFe2+ ) to ferryl Hb, which can then enter the pseudoperoxidase cycle, making hemoglobin highly toxic. In this study, we found that ferrous hemoglobin and polymerized porcine hemoglobin (one of the HBOCs) have the peroxidase activity different from the pseudoperoxidase activity of ferric hemoglobin. Ferrous hemoglobin can catalyze the reaction of tyrosine (Tyr) with hydrogen peroxide. In addition, the results also indicated that ferrous hemoglobin and pPolyHb have a strong inhibitory effect on the pseudoperoxidase activity of ferric hemoglobin. Therefore, hydrogen peroxide was consumed in a large amount, which greatly prevented hemoglobin from becoming oxidized and entering the pseudoperoxidase cycle, thus inhibiting ferryl Hb toxicity. We further cultured human umbilical vein endothelial cells and monitored cell morphology, viability, cell cycle, apoptosis, lactate dehydrogenase (LDH) release, and malondialdehydes (MDAs) formation when incubated with H2 O2 , Tyr, and HbFe2+ . HbFe2+ and pPolyHb reduced cell cycle arrest, apoptosis, LDH release, and MDA formation. These results showed that reducing oxidative damage induced by H2 O2 and converted hemoglobin from a molecule that is toxic to one that inhibits oxidative damage, suggesting a new strategy for development of a safer HBOCs.

Substitutions in the β subunits of sickle-cell hemoglobin improve oxidative stability and increase the delay time of sickle-cell fiber formation

After reacting with hydrogen peroxide (H2O2), sickle-cell hemoglobin (HbS, βE6V) remains longer in a highly oxidizing ferryl form (HbFe4+=O) and induces irreversible oxidation of “hot-spot” amino acids, including βCys-93. To control the damaging ferryl heme, here we constructed three HbS variants. The first contained a redox-active Tyr in β subunits (F41Y), a substitution present in Hb Mequon; the second contained the Asp (K82D) found in the β cleft of Hb Providence; and the third had both of these β substitutions. Both the single Tyr-41 and Asp-82 constructs lowered the oxygen affinity of HbS but had little or no effects on autoxidation or heme loss kinetics. In the presence of H2O2, both rHbS βF41Y and βF41Y/K82D enhanced ferryl Hb reduction by providing a pathway for electrons to reduce the heme via the Tyr-41 side chain. MS analysis of βCys-93 revealed moderate inhibition of thiol oxidation in the HbS single F41Y variant and dramatic 3- to 8-fold inhibition of cysteic acid formation in rHbS βK82D and βF41Y/K82D, respectively. Under hypoxia, βK82D and βF41Y/K82D HbS substitutions increased the delay time by ∼250 and 600 s before the onset of polymerization compared with the rHbS control and rHbS βF41Y, respectively. Moreover, at 60 °C, rHbS βK82D exhibited superior structural stability. Asp-82 also enhanced the function of Tyr as a redox-active amino acid in the rHbS βF41Y/K82D variant. We conclude that the βK82D and βF41Y substitutions add significant resistance to oxidative stress and anti-sickling properties to HbS and therefore could be potential genome-editing targets.

Oxidized hemoglobin forms contribute to NLRP3 inflammasome-driven IL-1β production upon intravascular hemolysis

Damage associated molecular patterns (DAMPs) are released form red blood cells (RBCs) during intravascular hemolysis (IVH). Extracellular heme, with its pro-oxidant, pro-inflammatory and cytotoxic effects, is sensed by innate immune cells through pattern recognition receptors such as toll-like receptor 4 and nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3), while free availability of heme is strictly controlled. Here we investigated the involvement of different hemoglobin (Hb) forms in hemolysis-associated inflammatory responses.We found that after IVH most of the extracellular heme molecules are localized in oxidized Hb forms. IVH was associated with caspase-1 activation and formation of mature IL-1β in plasma and in the liver of C57BL/6 mice. We showed that ferrylHb (FHb) induces active IL-1β production in LPS-primed macrophages in vitro and triggered intraperitoneal recruitment of neutrophils and monocytes, caspase-1 activation and active IL-1β formation in the liver of C57BL/6 mice. NLRP3 deficiency provided a survival advantage upon IVH, without influencing the extent of RBC lysis or the accumulation of oxidized Hb forms. However, both hemolysis-induced and FHb-induced pro-inflammatory responses were largely attenuated in Nlrp3−/− mice.Taken together, FHb is a potent trigger of NLRP3 activation and production of IL-1β in vitro and in vivo, suggesting that FHb may contribute to hemolysis-induced inflammation. Identification of RBC-derived DAMPs might allow us to develop new therapeutic approaches for hemolytic diseases.

The Reaction of Oxy Hemoglobin with Nitrite: Mechanism, Antioxidant-Modulated Effect, and Implications for Blood Substitute Evaluation.

The autocatalytic reaction between nitrite and the oxy form of globins involves free radicals. For myoglobin (Mb), an initial binding of nitrite to the iron-coordinated oxygen molecule was proposed; the resulting ferrous-peroxynitrate species was not detected, but its decay product, the high-valent ferryl form, was demonstrated in stopped-flow experiments. Reported here are the stopped flow spectra recorded upon mixing oxy Hb (native, as well as chemically-derivatized in the form of several candidates of blood substitutes) with a supraphysiological concentration of nitrite. The data may be fitted to a simple kinetic model involving a transient met-aqua form, in contrast to the ferryl detected in the case of Mb in a similar reaction sequence. These data are in line with a previous observation of a transient accumulation of ferryl Hb under auto-catalytic conditions at much lower concentrations of nitrite (Grubina, R. et al. J. Biol. Chem. 2007, 282, 12916). The simple model for fitting the stopped-flow data leaves a small part of the absorbance changes unaccounted for, unless a fourth species is invoked displaying features similar to the oxy and tentatively assigned as ferrous-peroxynitrate. Density functional theory (DFT) calculations support this latter assignment. The reaction allows for differentiating between the reactivities of various chemically modified hemoglobins, including candidates for blood substitutes. Polymerization of hemoglobin slows the nitrite-induced oxidation, in sharp contrast to oxidative-stress type reactions which are generally accelerated, not inhibited. Sheep hemoglobin is found to be distinctly more resistant to reaction with nitrite compared to bovine Hb, at large nitrite concentrations (stopped-flow experiments directly observing the oxy + nitrite reaction) as well as under auto-catalytic conditions. Copolymerization of Hb with bovine serum albumin (BSA) using glutaraldehyde leads to a distinct increase of the lag time compared to native Hb as well as to any other form of derivatization examined in the present study. The Hb-BSA copolymer also displays a slower initial reaction with nitrite under stopped-flow conditions, compared to native Hb.

Mechanisms involved in hemoglobin-mediated oxidation of lipids in washed fish muscle and inhibitory effects of phospholipase A2.

Hemoglobin (Hb) is a lipid oxidation promoter in fish muscle. Phospholipase A2 (PLA2; EC 3.1.1.4) is linked to an increased resistance to lipid oxidation of frozen-thawed cod fillets via an unknown mechanism. The present study aimed to investigate the mechanism of Hb-mediated lipid oxidation with a focus on ferryl Hb and methemoglobin (metHb), the pro-oxidative Hb species, and to examine how porcine pancreatic PLA2 inhibits Hb-mediated lipid oxidation in washed cod muscle (WCM). Lipid hydroperoxides (LOOHs) and thiobarbituric acid reactive substances (TBARS) were measured as primary and secondary lipid oxidation products, respectively. The formation of metHb and ferryl Hb was also monitored. Ferryl Hb and metHb formed during the Hb-mediated lipid oxidation. PLA2 inhibited the formation of LOOHs and TBARS and suppressed the formation of metHb and ferryl Hb. WCM was pre-oxidized by hemin to increase the amount of LOOHs. PLA2 promoted the depletion of LOOHs in the pre-oxidized WCM with limited TBARS formation at the expense of the heme moiety of Hb. The results of the present study suggest that ferryl Hb may play a role in Hb-mediated lipid oxidation and that PLA2 from pig pancreas may work together with Hb as a novel antioxidant with an ability to remove pre-formed LOOHs from a lipid substrate. © 2017 Society of Chemical Industry.

Bioinspired Polydopamine-Coated Hemoglobin as Potential Oxygen Carrier with Antioxidant Properties.

Oxidative side reaction is one of the major factors hindering the development of hemoglobin-based oxygen carriers (HBOCs). To avoid the oxidative toxicity, we designed and synthesized polydopamine-coated hemoglobin (Hb-PDA) nanoparticles via simple one-step assemblage without any toxic reagent. Hb-PDA nanoparticles showed oxidative protection of Hb by inhibiting the generation of methemoglobin (MetHb) and ferryl (Fe IV) Hb, as well as excellent antioxidant properties by scavenging free radicals and reactive oxygen species (ROS). Interestingly, the scavenging rate of Hb-PDA nanoparticles for ABTS+ radical is at most 89%, while for DPPH radical it reaches 49%. In addition, Hb-PDA efficiently reduced the intracellular H2O2-induced ROS generation. Moreover, Hb-PDA nanoparticles exhibited high oxygen affinity, low effect on blood constituents, and low cytotoxicity. The results indicate that polydopamine-coated hemoglobin might be a promising approach for constructing novel oxygen carriers with the capacity to reduce oxidative side reaction.

Antioxidants Attenuate Oxidative Stress-Induced Hidden Blood Loss in Rats.

Objective:Hidden blood loss (HBL), commonly seen after total knee or hip arthroplasty, causes postoperative anemia even after reinfusion or blood transfusion based on the visible blood loss volume. Recent studies demonstrated that oxidative stress might be involved in HBL. However, whether the antioxidants proanthocyanidin (PA) or hydrogen water (HW) can ameliorate HBL remains poorly understood. The aim of this study was to evaluate the effects of PA and HW on HBL.Materials and Methods:A rat HBL model was established through administration of linoleic acid with or without treatment with PA or HW. The levels of hemoglobin (Hb), red blood cell (RBC) count, superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-PX) activity, malondialdehyde (MDA), and ferryl Hb were measured.Results:RBC and Hb values as well as the activity of SOD and GSHPX were reduced after administration of linoleic acid, which was ameliorated by treatment with PA or HW. In addition, the quantity of MDA was significantly decreased with the administration of PA or HW.Conclusion:PA and HW could ameliorate HBL in a rat model by reducing oxidative stress, suggesting that they might be used as a novel therapeutic approach in the prophylaxis or treatment of HBL in clinics.

Determination of extinction coefficients of human hemoglobin in various redox states

The role of hemoglobin (Hb) redox forms in tissue and organ toxicities remain ambiguous despite the well-documented contribution of Hb redox reactivity to cellular and subcellular oxidative changes. Moreover, several recent studies, in which Hb toxicity were investigated, have shown conflicting outcomes. Uncertainties over the potential role of these species may in part be due to the protein preparation method of choice, the use of published extinction coefficients and the lack of suitable controls for Hb oxidation and heme loss. Highly purified and well characterized redox forms of human Hb were used in this study and the extinction coefficients of each Hb species (ferrous/oxy, ferric/met and ferryl) were determined. A new set of equations were established to improve accuracy in determining the transient ferryl Hb species. Additionally, heme concentrations in solutions and in human plasma were determined using a novel reversed phase HPLC method in conjugation with our photometric measurements. The use of more accurate redox-specific extinction coefficients and method calculations will be an invaluable tool for both in vitro and in vivo experiments aimed at determining the role of Hb-mediated vascular pathology in hemolytic anemias and when Hb is used as oxygen therapeutics.

Arachidonic acid causes hidden blood loss–like red blood cell damage through oxidative stress reactions

BackgroundHidden blood loss (HBL) often occurs in the prosthetic replacement for joint, but the mechanism is still not clear. Materials and methodsThis study tried to establish an animal model of HBL by injecting arachidonic acid (AA) into the Sprague-Dawley rats. Different concentrations of AA were injected into the tail veins of the rats, and blood samples were collected before and after administration at 24, 48, and 72 h. A complete blood count was obtained by to find the hemoglobin (Hb) and red blood cell (RBC) count changes. The glutathione peroxidase (GSH-PX) and total superoxide dismutase (T-SOD) activities and hydrogen peroxide (H2O2) levels were detected. The morphological changes of erythrocyte were observed under a polarizing microscope. The absorbance values of the blood samples were tested to determine the presence of ferryl Hb. ResultsHBL occurred in the experimental groups when the concentration of AA reached 10 mmol/L; Hb and RBC values decreased sharply at 24- and 48-h postinjection. This was followed by reduced activities of GSH-PX and T-SOD and decreased levels of H2O2. Moreover, the pathologic changes of red cell morphology mainly presented as pleomorphic RBC morphology, including cell rupture. The absorbance values of the blood samples were in accordance with ferryl Hb features. RBC and Hb values were relatively stable at 72 h. The GSH-PX and T-SOD activities and H2O2 levels gradually increased up to a balanced state. ConclusionsThe study concluded that high concentrations of AA can induce oxidative stress reactions in the body, causing acute injury of RBCs, which is closely related to HBL.

Hemoglobin S Oxidation Promotes Plasma-Derived Microparticle Membrane Alterations and Toxicity

Polymerization of sickle cell hemoglobin S (HbS) is recognized as a key event in the pathophysiology of sickle cell disease (SCD). Repeated HbS polymerization promotes an altered red blood cell (RBC) membrane, hemolysis, and microparticle (MP) formation, which have been shown to play significant roles in the interaction of RBCs with vascular endothelium and progression of vaso-occlusive events. Circulating RBC-derived MPs are elevated in SCD patients and they release a significant portion of their contents including oxidized HbS and heme to the cells of the vasculature. We have recently reported that free HbS oxidizes faster, remains locked in a highly oxidizing form (ferryl) longer, and loses heme faster than normal HbA (Kassa et al., J Biol Chem 290: 27939, 2015). The contributions of HbS higher oxidation states (ferric and ferryl heme) to MP formation, membrane alterations, and heme loss are poorly defined in SCD. RBC-derived MPs (ranging in size between 100-300 nm in diameter) generated by sheer stress or isolated by ultracentrifugation from the plasma (circulating) of SCD patients (N=6), ethnically matched control subjects (N=5), humanized transgenic sickle mice (Townes-SS, N=4), and control wild-type mice (Townes-AA, N=4) were identified by flow cytometry using CD235a glycophorin antibody and annexin V for externalized phosphatidylserine (PS). Time courses of Hb oxidation, obtained during 30 hour incubations of mouse or human MPs were biphasic. The initial levels of oxidized (ferric) Hb (30 to 45%) were slightly reduced within the first ~10 hours, likely due to the presence of RBC residual reductive enzymes within MPs. This was followed by a second phase in which Hb oxidation (ferric Hb) increased linearly and uncontrollably to 65 to 75% of total Hb. SCD MP's contained highly reactive ferryl Hb intermediates, carbonylated membrane proteins, and phosphorylated band 3 proteins. Quantitative proteomic analysis indicated a higher level of protein oxidation in MPs derived from SCD mice and patients. Five-fold higher levels of irreversibly oxidized βCys93 oxidation were found in untreated versus hydroxyurea-treated SCD patients. Intriguingly, HbS β subunits from SCD MPs were ubiquitinated and MPs isolated from untreated SCD patients had 25-fold higher ubiquitination levels than hydroxyurea-treated SCD patients that were comparable to normal controls. MP ubiquitination levels were correlated with HbS and an overall increase in MP oxidative stress, and inversely correlated with HbF. Compared to respective control MPs, incubation of either mice or human SCD MPs with human endothelial cells (HUVEC) activated apoptotic pathways and impacted cellular bioenergetic parameters by lowering mitochondrial oxygen consumption rates to a greater degree in a manner that was correlated with the redox state of Hb iron within MPs. Human endothelial cells incubated with SCD MPs showed greater intracellular reactive oxygen species production and heme oxygenase-1 induction. In summary, Hb transformation to higher oxidation forms is markedly increased in MPs generated from SCD mice and patients, which when incubated with endothelial cells, lead to mitochondrial dysfunction and apoptotic cell death. These mechanistic analyses of RBC-derived SCD microparticles suggest potential anti-oxidative reducing modalities that may interrupt MP heme-mediated pathophysiology in patients with SCD. DisclosuresBelcher:Cydan/Imara: Research Funding; CSL-Behring: Research Funding. Vercellotti:CSL-Behring: Research Funding; Imara: Research Funding.

Haemoglobin in pelvic fluid rescues Fallopian tube epithelial cells from reactive oxygen species stress and apoptosis.

Fallopian tube fimbrial epithelium is considered to be the major site of origin of ovarian high-grade serous carcinoma, with p53 loss being the earliest and universal change. We previously reported that reactive oxygen species (ROS) in the ovulatory follicular fluids (FFs) are mutagenic and cytotoxic to fimbrial epithelial cells, which are bathed in the peritoneal fluid mixed with FFs. Here, we observed that ferryl haemoglobin (Hb), which was abundantly present in ovulatory FFs and pelvic peritoneal fluids, could rescue p53-deficient immortalized fimbrial epithelial (FE25) cells and oviduct epithelial cells from Trp53-null mice from lethal ovulatory ROS stress. Ferryl Hb and FF containing high Hb levels protected FE25 cells from apoptosis, mainly by consuming extracellular ROS and reducing NADPH oxidase-mediated cell death. The remaining extracellular ROS could still induce DNA double-strand breaks in the fimbrial epithelial cells. Our study revealed that ferryl Hb in peritoneal fluid rescued ROS-stressed, DNA-damaged fimbrial epithelial cells from death, and suggested that peritoneal blood from various sources may contribute to the ovulation-induced transformation of Fallopian tube epithelium. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Differential heme release from various hemoglobin redox states and the upregulation of cellular heme oxygenase-1.

Despite advances in our understanding of the oxidative pathways mediated by free hemoglobin (Hb), the precise contribution of its highly reactive redox forms to tissue and organ toxicities remains ambiguous. Heme, a key degradation byproduct of Hb oxidation, has recently been recognized as a damage‐associated molecular pattern (DAMP) molecule, able to trigger inflammatory responses. Equally damaging is the interaction of the highly redox active forms of Hb with other biological molecules. We determined the kinetics of heme loss from individual Hb redox states—ferrous (Fe2+), ferric (Fe3+), and ferryl (Fe4+)—using two different heme receptor proteins: hemopexin (Hxp), a naturally occurring heme scavenger in plasma, and a double mutant (H64Y/V86F), apomyoglobin (ApoMb), which avidly binds heme released from Hb. We show for the first time that ferric Hb (Fe3+) loses heme at rates substantially higher than that of ferryl Hb (Fe4+). This was also supported by a higher expression of heme oxygenase‐1 (HO‐1) when ferric Hb was added to cultured lung alveolar epithelial cells (E10). The reported cytotoxicity of Hb may therefore be attributed to a combination of accelerated heme loss from the ferric form and protein radical formation associated with ferryl Hb. Targeted therapeutic interventions can therefore be designed to curb specific oxidative pathways of Hb in hemolytic anemias and when Hb is used as an oxygen‐carrying therapeutic.

Oxidized Ferric and Ferryl Forms of Hemoglobin Trigger Mitochondrial Dysfunction and Injury in Alveolar Type I Cells.

Lung alveoli are lined by alveolar type (AT) 1 cells and cuboidal AT2 cells. The AT1 cells are likely to be exposed to cell-free hemoglobin (Hb) in multiple lung diseases; however, the role of Hb redox (reduction-oxidation) reactions and their precise contributions to AT1 cell injury are not well understood. Using mouse lung epithelial cells (E10) as an AT1 cell model, we demonstrate here that higher Hb oxidation states, ferric Hb (HbFe(3+)) and ferryl Hb (HbFe(4+)) and subsequent heme loss play a central role in the genesis of injury. Exposures to HbFe(2+) and HbFe(3+) for 24 hours induced expression of heme oxygenase (HO)-1 protein in E10 cells and HO-1 translocation in the purified mitochondrial fractions. Both of these effects were intensified with increasing oxidation states of Hb. Next, we examined the effects of Hb oxidation and free heme on mitochondrial bioenergetic function by measuring changes in the mitochondrial transmembrane potential and oxygen consumption rate. In contrast to HbFe(2+), HbFe(3+) reduced basal oxygen consumption rate, indicating compromised mitochondrial activity. However, HbFe(4+) exposure not only induced early expression of HO-1 but also caused mitochondrial dysfunction within 12 hours when compared with HbFe(2+) and HbFe(3+). Exposure to HbFe(4+) for 24 hours also caused mitochondrial depolarization in E10 cells. The deleterious effects of HbFe(3+) and HbFe(4+) were reversed by the addition of scavenger proteins, haptoglobin and hemopexin. Collectively, these data establish, for the first time, a central role for cell-free Hb in lung epithelial injury, and that these effects are mediated through the redox transition of Hb to higher oxidation states.

Sickle Cell Hemoglobin in the Ferryl State Promotes βCys-93 Oxidation and Mitochondrial Dysfunction in Epithelial Lung Cells (E10)

Polymerization of intraerythrocytic deoxyhemoglobin S (HbS) is the primary molecular event that leads to hemolytic anemia in sickle cell disease (SCD). We reasoned that HbS may contribute to the complex pathophysiology of SCD in part due to its pseudoperoxidase activity. We compared oxidation reactions and the turnover of oxidation intermediates of purified human HbS and HbA. Hydrogen peroxide (H2O2) drives a catalytic cycle that includes the following three distinct steps: 1) initial oxidation of ferrous (oxy) to ferryl Hb; 2) autoreduction of the ferryl intermediate to ferric (metHb); and 3) reaction of metHb with an additional H2O2 molecule to regenerate the ferryl intermediate. Ferrous and ferric forms of both proteins underwent initial oxidation to the ferryl heme in the presence of H2O2 at equal rates. However, the rate of autoreduction of ferryl to the ferric form was slower in the HbS solutions. Using quantitative mass spectrometry and the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide, we found more irreversibly oxidized βCys-93in HbS than in HbA. Incubation of the ferric or ferryl HbS with cultured lung epithelial cells (E10) induced a drop in mitochondrial oxygen consumption rate and impairment of cellular bioenergetics that was related to the redox state of the iron. Ferryl HbS induced a substantial drop in the mitochondrial transmembrane potential and increases in cytosolic heme oxygenase (HO-1) expression and mitochondrial colocalization in E10 cells. Thus, highly oxidizing ferryl Hb and heme, the product of oxidation, may be central to the evolution of vasculopathy in SCD and may suggest therapeutic modalities that interrupt heme-mediated inflammation.

Nitrite attenuated hypochlorous acid-mediated heme degradation in hemoglobin

Hypochlorous acid (HOCl) is elevated in many inflammatory diseases and causes the accumulation of free iron. Through the Fenton reaction, free iron has the ability to generate free radicals and subsequently is toxic. Recent studies have demonstrated that HOCl participates in heme destruction of hemoglobin (Hb) and free iron release. In this study, it was showed that nitrite (NO2−) could prevent HOCl-mediated Hb heme destruction and free iron release. Also, NO2− prevented HOCl-mediated loss of Hb peroxidase activity. After the NO2−/HOCl treatment, Tyr 42 in α-chain was found to be nitrated in Hb, attenuating the electron transferring abilities of phenolic compounds. The protective effects of NO2− on HOCl-induced heme destruction were attributed to its reduction of ferryl Hb and/or direct scavenging of HOCl. Therefore, NO2− could show protective effects in some inflammatory diseases by preventing HOCl-mediated heme destruction of hemoproteins and free iron release.

Dissection of the radical reactions linked to fetal hemoglobin reveals enhanced pseudoperoxidase activity.

In the presence of excess hydrogen peroxide (H2O2), ferrous (Fe+2) human hemoglobin (Hb) (α2β2) undergoes a rapid conversion to a higher oxidation ferryl state (Fe+4) which rapidly autoreduces back to the ferric form (Fe+3) as H2O2 is consumed in the reaction. In the presence of additional H2O2 the ferric state can form both ferryl Hb and an associated protein radical in a pseudoperoxidative cycle that results in the loss of radicals and heme degradation. We examined whether adult HbA (β2α2) exhibits a different pseudoenzymatic activity than fetal Hb (γ2α2) due to the switch of γ to β subunits. Rapid mixing of the ferric forms of both proteins with excess H2O2 resulted in biphasic kinetic time courses that can be assigned to γ/β and α, respectively. Although there was a 1.5 fold increase in the fast reacting γ /β subunits the slower reacting phases (attributed to α subunits of both proteins) were essentially the same. However, the rate constant for the auto-reduction of ferryl back to ferric for both proteins was found to be 76% higher for HbF than HbA and in the presence of the mild reducing agent, ascorbate there was a 3-fold higher reduction rate in ferryl HbF as opposed to ferryl HbA. Using quantitative mass spectrometry in the presence of H2O2 we found oxidized γ/β Cys93, to be more abundantly present in HbA than HbF, whereas higher levels of nitrated β Tyr35 containing peptides were found in HbA samples treated with nitrite. The extraordinary stability of HbF reported here may explain the evolutionary advantage this protein may confer onto co-inherited hemoglobinopathies and can also be utilized in the engineering of oxidatively stable Hb-based oxygen carriers.