Release Of Hemin Research Articles

Hemin-induced platelet activation is regulated by the ACKR3 chemokine surface receptor and has implications for passivation of vulnerable atherosclerotic plaques.

In vulnerable atherosclerotic plaques, intraplaque hemorrhages (IPH) result in hemolysis of red blood cells and release of hemoglobin and free hemin. Hemin activates platelets and leads to thrombosis. Agonism of the inhibitory platelet receptor ACKR3 inhibits hemin-dependent platelet activation and thrombus formation. To characterize the effect of hemin and ACKR3 agonism on isolated human platelets, multi-color flow cytometry and classical experimental setup such as light transmission aggregometry and a flow chamber assay were used. Hemin induces platelet aggregation and ex vivo platelet-dependent thrombus formation on immobilized collagen under a low shear rate of 500 s-1, indicating that free hemin is a strong activator of platelet-dependent thrombosis. Recently, we described that ACKR3 is a prominent inhibitory receptor of platelet activation. Specific ACKR3 agonists but not conventional antiplatelet compounds such as COX-1 inhibitor (indometacin), ADP-receptor blocker (cangrelor), or PAR1 inhibitor (ML161) inhibit both hemin-dependent aggregation and thrombus formation. To further characterize the effect of hemin on platelet subpopulations, we established a multi-color flow cytometry assay. We found that hemin induces procoagulant (CD42bpos/PAC-1neg/AnnexinVpos), aggregatory (CD42bpos/PAC-1pos/AnnexinVneg), and inflammatory (CD42bpos/CXCR4pos/ACKR3pos/AnnexinVpos) platelet subpopulations. Treatment with ACKR3 agonists significantly decreased the formation of procoagulant and ACKR3pos platelets in response to hemin. We conclude that hemin is a strong activator for the formation of procoagulant platelets and thrombus formation which is dependent on the function of ACKR3. Activation of ACKR3 using specific agonists may offer a therapeutic strategy to regulate the vulnerability of atherosclerotic plaques in areas of IPH.

Self-amplified activatable nanoprodrugs for enhanced chemodynamic/chemo combination therapy

Chemodynamic therapy (CDT) has gained increasing attention by virtue of its high tumor specificity and low side effect. However, the low concentration of hydrogen peroxide (H2O2) in the tumor site suppresses the therapeutic efficacy of CDT. To improve the efficacy, introducing other kind of therapeutic modality is a feasible choice. Herein, we develop a self-amplified activatable nanomedicine (PCPTH NP) for chemodynamic/chemo combination therapy. PCPTH NP is composed of a H2O2-activatable amphiphilic prodrug PEG-PCPT and hemin. Upon addition of H2O2, the oxalate linkers within PCPTH NP are cleaved, which makes the simultaneous release of CPT and hemin. The released CPT can not only kill cancer cells but also upregulate the intracellular reactive oxygen species (ROS) level. The elevated ROS level may accelerate the release of drugs and enhance the CDT efficacy. PCPTH NP shows a H2O2 concentration dependent release profile, and can effectively catalyze H2O2 into hydroxyl radical (·OH) under acidic condition. Compared with PCPT NP without hemin, PCPTH NP has better anticancer efficacy both in vitro and in vivo with high biosafety. Thus, our study provides an effective approach to improve the CDT efficacy with high tumor specificity.

The interaction of perfluorooctane sulfonate with hemoproteins and its relevance to molecular toxicology

Perfluorooctane sulfonate (PFOS), a representative of perfluorinated compounds in industrial and commercial products, has posed a great threat to animals and humans via environmental exposure and dietary consumption. Herein, we investigated the effects of PFOS binding on the redox state and stability of two hemoproteins (hemoglobin (Hb) and myoglobin (Mb)). Fluorescence spectroscopy, circular dichroism and UV–vis absorption spectroscopy demonstrated that PFOS could induce the conformational changes of proteins along with the exposure of heme cavity and generation of hemichrome, which resulted in the increased release of free hemin. After that, free hemin liberated from hemoproteins led to reactive oxygen species formation, lipid peroxidation, cell membrane damage and loss of cell viability in vascular endothelial cells, while neither Hb nor Mb did show cytotoxicity. Chemical inhibitors of ferroptosis effectively mitigated hemin-caused toxicity, identifying the hemin-dependent ferroptotic cell death mechanisms. These data demonstrated that PFOS posed a potential threat of toxicity through a mechanism which involved its binding to hemoproteins, decreased oxygen transporting capacity, and increased hemin release. Altogether, our findings elucidate the binding mechanisms of PFOS with two hemoproteins, as well as possible risks on vascular endothelial cells, which would have important implications for the human and environmental toxicity of PFOS.

Amphiphilic Dendrimer Doping Enhanced pH-Sensitivity of Liposomal Vesicle for Effective Co-delivery toward Synergistic Ferroptosis-Apoptosis Therapy of Hepatocellular Carcinoma.

Ferroptosis, characterized by the accumulation of reactive oxygen species and lipid peroxides, has emerged as an attractive strategy to reverse drug resistance. Of particular interest is the ferroptosis-apoptosis combination therapy for cancer treatment. Herein, a nanoplatform is reported for effective co-delivery of the anticancer drug sorafenib (S) and the ferroptosis inducer hemin (H), toward synergistic ferroptosis-apoptosis therapy of advanced hepatocellular carcinoma (HCC) as a proof-of-concept study. Liposome is an excellent delivery system; however, it is not sufficiently responsive to the acidic tumor microenvironment (TME) for tumor-targeted drug delivery. The pH-sensitive vesicles are therefore developed (SH-AD-L) by incorporating amphiphilic dendrimers (AD) into liposomes for controlled and pH-stimulated release of sorafeniband hemin in the acidic TME, thanks to the protonation of numerous amine functionalities in AD. Importantly, SH-AD-L not only blocked glutathione synthesis to disrupt the antioxidant system, but also increased intracellular Fe2+ and ·OH concentrations to amplify oxidative stress, both of which contribute to enhanced ferroptosis. Remarkably, high levels of ·OH also augmented sorafenib-mediated apoptosis in tumor cells. This study demonstrates the efficacy of ferroptosis-apoptosis combination therapy, as well as the promise of the AD-doped TME-responsive vesicles for drug delivery in combination therapy to treat advanced HCC.

Analysis of ridges and grooves shape in grating coupler for optimization of integrated optics sensor structures.

The paper presents a theoretical analysis of a sensor structure based on a planar waveguide and grating coupler designed to determine selected physical properties of blood – hemoglobin concentration and oxidation level. In particular analysis were focused on optimization of selected geometrical properties of grating coupler (shape of ridges and grooves) to obtain maximum efficiency of uncoupling of light from the sensor structure. The analysis were carried out for three type of ridges and grooves shape in grating coupler: rectangular, triangular and sinusoidal. Full Text: PDF ReferencesI. . Singh, A.Weston, A. Kundur, G. Dobie, Haematology Case Studies with Blood Cell Morphology and Pathophysiology; Elsevier: Amsterdam, The Netherlands, (2017). DirectLink P. Jarolim, M. Lahav, SC. Liu, J. Palek, "Effect of hemoglobin oxidation products on the stability of red cell membrane skeletons and the associations of skeletal proteins: correlation with a release of hemin", Blood 76, 10 (1990). CrossRef E. Beutler, J. Waalen, "The definition of anemia: what is the lower limit of normal of the blood hemoglobin concentration?", Blood 107, 5 (2006). CrossRef M. Kiroriwal, P. Singal M. Sharma, A. Singal, "Hemoglobin sensor based on external gold-coated photonic crystal fiber", Optics & Laser Technology 149, 107817 (2022). CrossRef A. A. Boiarski, J. R. Busch, B. S. Bhullar, R. W. Ridgway, V. E. Wood, "Integrated optic sensor with macro-flow cell", Proc. SPIE Integrated Optics and Microstructures 1793 (1993). CrossRef L. Cheng, S. Mao, Z. Li, Y. Han and H. Y. Fu, "Grating Couplers on Silicon Photonics: Design Principles, Emerging Trends and Practical Issues", Micromachines 11, 666 (2020). CrossRef P. Struk, "Design of an Integrated Optics Sensor Structure Based on Diamond Waveguide for Hemoglobin Property Detection", Materials 12, 175 (2019). CrossRef P. Struk, "Numerical analysis of integrated photonics structures for hemoglobin sensor application", Phot. Lett. Poland 12, 2 (2020). CrossRef P.V. Lambeck, "Integrated optical sensors for the chemical domain", Meas. Sci. Technol. 17, (2006). CrossRef W. Lukosz, "Integrated optical chemical and direct biochemical sensors", Sens. Actuators B Chem 29 (1995). CrossRef P. Struk, T. Pustelny, K. Gołaszewska,E. Kaminska, M.A. Borysiewicz, M. Ekielski, A. Piotrowska, "Hybrid photonics structures with grating and prism couplers based on ZnO waveguides", Opto-Electron. Rev. 21, (2013). CrossRef P. Struk, "Design of an integrated optics sensor structure for hemoglobin property detection", Proc. SPIE 11204, (2019). CrossRef OptiFDTD Technical Background and Tutorials - Finite Difference Time Domain Photonics Simulation Software, Optiwave Systems Inc. (2008). DirectLink K. Yee, "Cutoff Frequencies of Eccentric Waveguides", IEEE Transactions 14, 3 (1966). CrossRef

Photoelectrochemical Detection of Exosomal miRNAs by Combining Target-Programmed Controllable Signal Quenching Engineering.

MicroRNAs extracted from exosomes (exosomal miRNAs) have recently emerged as promising biomarkers for early prognosis and diagnosis. Thus, the development of an effective approach for exosomal miRNA monitoring has triggered extensive attention. Herein, a sensitive photoelectrochemical (PEC) biosensing platform is demonstrated for exosomal miRNA assay via the target miRNA-powered λ-exonuclease for the amplification strategy. The metal-organic framework (MOF)-decorated WO3 nanoflakes heterostructure is constructed and implemented as the photoelectrode. Also, a target exosomal miRNA-activatable programmed release nanocarrier was fabricated, which is responsible for signal control. Hemin that acted as the electron acceptor was prior entrapped into the programmed control release nanocarriers. Once the target exosomal miRNAs-21 was introduced, the as-prepared programmed release nanocarriers were initiated to trigger the release of hemin, which enabled the quenching of the photocurrent. Under the optimized conditions, the level of exosomal miRNAs-21 could be accurately tracked ranging from 1 fM to 0.1 μM with a low detection limit of 0.5 fM. The discoveries illustrate the possibility for the rapid and efficient diagnosis and prognosis prediction of diseases based on the detection of exosomal miRNAs-21 and would provide feasible approaches for the fabrication of an efficient platform for clinical applications.

Effect of high hydrostatic pressure on the oxidation of washed muscle with added chicken hemoglobin

The role of chicken hemoglobin in lipid oxidation of washed chicken muscle exposed to high hydrostatic pressure (0, 200, 400 and 600 MPa) was examined. The observed decrease in redness was higher with elevated pressures (5.0 vs 3.7). During storage, redness decreased in samples exposed to 400 and 600 MPa. This decrease was concomitant with the progression of oxidation and the 2.5 fold decrease of soluble heme. An additional experiment was conducted to examine the effect of the hemoglobin mode of addition into pressurized muscle. The exposure at 600 MPa led to washed muscle oxidation even in the absence of hemoglobin, thus indicating that pressure treatments triggered lipid oxidation. However, the presence of native or pressurized hemoglobin into pressurized washed muscle caused more hexanal than the pressurized control without hemoglobin. Overall, results suggest that membrane disruption and the release of hemin are crucial for the onset of oxidation.

Hemin inhibits the large conductance potassium channel in brain mitochondria: A putative novel mechanism of neurodegeneration

Intracerebral hemorrhage (ICH) is a pathological condition that accompanies certain neurological diseases like hemorrhagic stroke or brain trauma. Its effects are severely destructive to the brain and can be fatal. There is an entire spectrum of harmful factors which are associated with the pathogenesis of ICH. One of them is a massive release of hemin from the decomposed erythrocytes. It has been previously shown, that hemin can inhibit the large-conductance Ca2+-regulated potassium channel in the plasma membrane. However, it remained unclear whether this phenomenon applies also to the mitochondrial large-conductance Ca2+-regulated potassium channel. The aim of the present study was to determine the impact of hemin on the activity of the large conductance Ca2+-regulated potassium channel in the brain mitochondria (mitoBKCa). In order to do so, we have used a patch-clamp technique and shown that hemin inhibits mitoBKCa in human astrocytoma U-87 MG cell line mitochondria. Since opening of the mitochondrial potassium channels is known to be cytoprotective, we have elucidated whether hemin can attenuate some of the beneficiary effects of potassium channel opening. We have studied the effect of hemin on reactive oxygen species synthesis, and mild mitochondrial uncoupling in isolated rat brain mitochondria. Taken together, our data show that hemin inhibits mitoBKCa and partially abolishes some of the cytoprotective properties of potassium channel opening. Considering the role of the mitoBKCa in cytoprotection, it can be presumed that its inhibition by hemin may be a novel mechanism contributing to the severity of the ICH symptoms. However, the validity of the presented results shall be further verified in an experimental model of ICH.

MicroRNA‐301b reduces PPARγ expression in transgenic sickle mice and in hemin‐treated human pulmonary artery endothelial cells (1089.22)

Sickle cell disease (SCD) is characterized by chronic intravascular hemolysis resulting in the release of hemin into the blood. Our preliminary microarray data indicate that PPARγ expression is significantly reduced in hemin‐treated human pulmonary artery endothelial cells (HPAECs). To examine post‐transcriptional mechanisms of PPARγ regulation in pulmonary hypertension in SCD (SCD‐PH), Townes knockin SCD mice aged 12‐weeks were gavaged daily with vehicle or rosiglitazone (RSG, 10 mg/kg/day) for 10 d. PPARγ was decreased and ET‐1 expression was increased in sickle mice (SS) compared to littermate control mice (AA). Using an in vitro model that mimics acute extracellular hemin release, HPAECs were treated with DMSO or hemin (5 μM) for 72 h ± RSG (10 μM) for the final 24 h. Similarly, hemin reduced PPARγ and increased ET‐1 expression in HPAECs. MicroRNA (miR‐301b), which negatively regulates PPARγ, was increased in SCD mice and in hemin‐treated HPAECs. In contrast, PPARγ activation attenuates increases in miR‐301b levels in SS mice and in hemin‐induced HPAECs. These findings suggest that hemolysis in SCD increases miR‐301b to reduce PPARγ and increase ET‐1 levels. These results suggest novel pathways for SCD‐PH pathogenesis and therapy.Grant Funding Source: Supported by AHA‐SDG 13SDG14150004,CEB F16788‐00, Atlanta VA 1I01BX001910, NIH HL074518, DK102167

Hemolysis and free hemoglobin revisited: exploring hemoglobin and hemin scavengers as a novel class of therapeutic proteins

AbstractHemolysis occurs in many hematologic and nonhematologic diseases. Extracellular hemoglobin (Hb) has been found to trigger specific pathophysiologies that are associated with adverse clinical outcomes in patients with hemolysis, such as acute and chronic vascular disease, inflammation, thrombosis, and renal impairment. Among the molecular characteristics of extracellular Hb, translocation of the molecule into the extravascular space, oxidative and nitric oxide reactions, hemin release, and molecular signaling effects of hemin appear to be the most critical. Limited clinical experience with a plasma-derived haptoglobin (Hp) product in Japan and more recent preclinical animal studies suggest that the natural Hb and the hemin-scavenger proteins Hp and hemopexin have a strong potential to neutralize the adverse physiologic effects of Hb and hemin. This includes conditions that are as diverse as RBC transfusion, sickle cell disease, sepsis, and extracorporeal circulation. This perspective reviews the principal mechanisms of Hb and hemin toxicity in different disease states, updates how the natural scavengers efficiently control these toxic moieties, and explores critical issues in the development of human plasma–derived Hp and hemopexin as therapeutics for patients with excessive intravascular hemolysis.

BINDING TO CHICKPEA (CICER ARIETINUM L.) PA2 ALBUMIN ENHANCES HEMIN-DEPENDENT OXIDATIVE REACTIONS

In an assay for antioxidant activity, chickpea albumin (PA2), human and bovine serum albumins increased hemin-dependent oxidative degradation of β-carotene in a concentration-dependent manner up to saturation at 1.6 µM PA2. On the contrary, while human and bovine serum albumins decreased oxidative degradation of hemin in the presence of H2O2, PA2 albumin had a stimulatory effect on this phenomenon. The prooxidant effect of PA2 might have deleterious effects in the gut because it may enhance oxidative tissue damage as a result of release of hemin from heme-containing foods such as red meats.

Mitochondrial and Microsomal Ferric b5 Cytochromes Exhibit Divergent Conformational Plasticity in the Context of a Common Fold

Native-state hydrogen-deuterium exchange (HDX) monitored by NMR spectroscopy has been used to compare conformational plasticity in ferric rat liver outer mitochondrial membrane cytochrome b5 (rOM b5) and ferric bovine liver microsomal cytochrome b5 (bMc b5). Analysis of the data indicated that rOM b5 is the less conformationally flexible protein on the time scale probed by the HDX experiments. The data also suggest a likely contributor to the much higher kinetic barrier for the release of hemin from OM b5s in comparison to Mc b5s, a characteristic that may be to a large extent the source of their divergent functional properties. Specifically, the data indicate that conformational mobility within helices alpha4 and alpha5, which flank the loop harboring axial ligand His63, is considerably more restricted in rOM b5 than in bMc b5. The lower conformational flexibility of alpha4 and alpha5 in rOM b5 can reasonably be attributed to more extensive hydrophobic packing in that region of the protein, arising from two conserved side chain packing motifs in OM cytochrome b5s [Altuve, A., Wang, L., Benson, D. R., and Rivera, M. (2004) Biochem. Biophys. Res. Commun. 314, 602-609].

Methemoglobin contributes to the growth of human tumor cells

Methemoglobin (metHb) has been reported to be present in areas surrounding solid tumors. The effects of human metHb on the growth of one human hepatocellular carcinoma cell line and one human glioma cell line that simply replicate in Ham's nutrient mixture F12 (F12) were investigated. MetHb, depending on its concentration, stimulated or inhibited the in vitro growth of both cancer cell lines. The stimulatory or inhibitory effect was due to the release of hemin from metHb, which was recognized by its characteristic light absorption spectrum. The possibility of metHb or hemin acting initially through a 3′, 5′-cyclic guanosine monophosphate- (cGMP-) or prostaglandin E 2- (PGE 2-) mediated pathway to enhance cell growth was excluded. Ferric iron derived from the catabolic degradation of hemin increased cell growth, whereas biliverdin (Bv) and its reduction product, bilirubin (Br), decreased cell growth. Hemoglobin oxidized to metHb in conditions found in tumors showing neovascularization and hemorrhage may contribute significantly to increased proliferation of cancerous cells.

Melatonin protects human red blood cells from oxidative hemolysis: new insights into the radical-scavenging activity.

Antioxidant activity of melatonin in human erythrocytes, exposed to oxidative stress by cumene hydroperoxide (cumOOH), was investigated. CumOOH at 300 microM progressively oxidized a 1% suspension of red blood cells (RBCs), leading to 100% hemolysis in 180 min. Malondialdehyde and protein carbonyls in the membrane showed a progressive increase, as a result of the oxidative damage to membrane lipids and proteins, reaching peak values after 30 and 40 min, respectively. The membrane antioxidant vitamin E and the cytosolic reduced glutathione (GSH) were totally depleted in 20 min. As a consequence of the irreversible oxidative damage to hemoglobin (Hb), hemin accumulated into the RBC membrane during 40 min. Sodium dodecyl sulfate (SDS) gel electrophoresis of membrane proteins showed a progressive loss of the cytoskeleton proteins and formation of low molecular weight bands and protein aggregates, with an increment of the intensity of the Hb band. Melatonin at 50 microM strongly enhanced the RBC resistance to oxidative lysis, leading to a 100% hemolysis in 330 min. Melatonin had no effect on the membrane lipid peroxidation, nor prevented the consumption of glutathione (GSH) or vitamin E. However, it completely inhibited the formation of membrane protein carbonyls for 20 min and hemin precipitation for 10 min. The electrophoretic pattern provided further evidence that melatonin delayed modifications to the membrane proteins and to Hb. In addition, RBCs incubated for 15 min with 300 microM cumOOH in the presence of 50 microM melatonin were less susceptible, when submitted to osmotic lysis, than cells incubated in its absence. Extraction and high-performance liquid chromatography (HPLC) analysis showed a much more rapid consumption of melatonin during the first 10 min of incubation, then melatonin slowly decreased up to 30 min and remained stable thereafter. Equilibrium partition experiments showed that 15% of the melatonin in the incubation mixture was recovered in the RBC cytosol, and no melatonin was extracted from RBC membrane. However, 35% of the added melatonin was consumed during RBC oxidation. Hydroxyl radical trapping agents, such as dimethylsulfoxide or mannitol, added into the assay in a 1,000 times molar excess, did not vary melatonin consumption, suggesting that hydroxyl radicals were not involved in the indole consumption. Our results indicate that melatonin is actively taken up into erythrocytes under oxidative stress, and is consumed in the defence of the cell, delaying Hb denaturation and release of hemin. RBCs are highly exposed to oxygen and can be a site for radical formation, under pathological conditions, which results in their destruction. A protective role of melatonin should be explored in hemolytic diseases.

The spontaneous hemin release from Lumbricus terrestris hemoglobin

The slow, spontaneous release of hemin from earthworm, Lumbricus terrestris, hemoglobin has been studied under mild conditions in the presence of excess apomyoglobin. This important protein is surprisingly unstable. The reaction is best described as hemin released from the globin into water, followed by quick engulfment by apomyoglobin. The energetics of this reaction are compared with those of other types of hemoglobins. Anomalously low activation energy and enthalpy are counterbalanced by a negative entropy. These values reflect significant low frequency protein motion and dynamics of earthworm hemoglobin and may also indicate an open structure distal to the heme. This is also supported by the infrared spectrum of the carbonyl hemoprotein, which indicates several types of distal interactions with the bound CO. The reported low heme to polypeptide ratio for this protein may be due to facile heme and hemin release by the circulating protein.

Free Radical and Oxidative Damage in Human Blood Cells.

Free radicals and oxidative damage play important roles in aging and many degenerative disorders such as cancer, cardiovascular diseases, and Alzheimer disease. Antioxidants can alleviate some of the harmful effects of oxidative damage. In this report, we describe that we have been using human red blood cells (RBCs) as a model system to delineate the effects of oxidative damage on human cells, particularly on glucose-6-phosphate dehydrogenase (G6PD)-deficient human RBCs. By using a monolayer technique, we found that oxidative denaturation of hemoglobin leads to the release of hemin into the RBC membrane and the released hemin is capable of oxidizing membrane proteins via a thiyl radical intermediate as detected by the electron spin resonance technique. By using a Laser Viscodiffractometer (Vidometer) to measure RBC deformability, we found that the deformability of G6PD-deficient RBCs was drastically reduced by hydroxyl radicals. Perhaps as a consequence of enhanced susceptibility to oxidative stress, G6PD-deficient individuals have lower antioxidant levels, particularly vitamin C, than normal individuals. Interestingly, we have also found that RBC deformability could be affected by two environmental pollutants, namely, platinum and palladium, which can enhance hydroxyl radical formation in the presence of hydrogen peroxide and ferrous ion (Fenton reaction).

Effect of hemoglobin oxidation products on the stability of red cell membrane skeletons and the associations of skeletal proteins: correlation with a release of hemin

Oxidative injury to hemoglobin (Hb) leads to formation of methemoglobin (MetHb), reversible hemichromes (rHCRs), and irreversible hemichromes (iHCRs). iHCRs precipitate and form Heinz bodies that attach to the red cell membrane causing injury that leads to hemolysis. The molecular mechanisms of this membrane damage have not been fully elucidated. We have studied the effect of Hb oxidation products on the mechanical stability of red cell membrane skeletons and the associations of membrane skeletal proteins. Hb and MetHb stabilized the isolated membrane skeletons, whereas further oxidation to rHCRs abolished this stabilizing effect. Crude iHCRs prepared by phenylhydrazine oxidation of Hb destabilized membrane skeletons by decreasing formation of the spectrin-protein 4.1-actin complex, the effect similar to that of pure hemin. Whereas virtually no hemin was released from Hb and MetHb, high concentrations of hemin were released from crude iHCR preparations. After removal of this hemin fraction by Dowex resin, the iHCRs lost their destabilizing effect. We conclude that as the oxidation of Hb proceeds, the stabilizing effect of Hb on the membrane skeleton is gradually lost and the deleterious effect increases. The destabilization of the red cell membrane skeleton in the presence of crude iHCR is caused by release of hemin, which lowers the stability of membrane skeleton by weakening the spectrin-protein 4.1-actin interaction.

Effect of Hemoglobin Oxidation Products on the Stability of Red Cell Membrane Skeletons and the Associations of Skeletal Proteins: Correlation With a Release of Hemin

Oxidative injury to hemoglobin (Hb) leads to formation of methemoglobin (MetHb), reversible hemichromes (rHCRs), and irreversible hemichromes (iHCRs). iHCRs precipitate and form Heinz bodies that attach to the red cell membrane causing injury that leads to hemolysis. The molecular mechanisms of this membrane damage have not been fully elucidated. We have studied the effect of Hb oxidation products on the mechanical stability of red cell membrane skeletons and the associations of membrane skeletal proteins. Hb and MetHb stabilized the isolated membrane skeletons, whereas further oxidation to rHCRs abolished this stabilizing effect. Crude iHCRs prepared by phenylhydrazine oxidation of Hb destabilized membrane skeletons by decreasing formation of the spectrin-protein 4.1-actin complex, the effect similar to that of pure hemin. Whereas virtually no hemin was released from Hb and MetHb, high concentrations of hemin were released from crude iHCR preparations. After removal of this hemin fraction by Dowex resin, the iHCRs lost their destabilizing effect. We conclude that as the oxidation of Hb proceeds, the stabilizing effect of Hb on the membrane skeleton is gradually lost and the deleterious effect increases. The destabilization of the red cell membrane skeleton in the presence of crude iHCR is caused by release of hemin, which lowers the stability of membrane skeleton by weakening the spectrin-protein 4.1-actin interaction.

Association of hemin with protein 4.1 as compared to spectrin and actin

The interaction of hemin with protein 4.1 isolated from red cell membrane cytoskeleton has been studied. Spectrophotometric titration has shown one strong binding site and additional lower affinity sites for hemin. From fluorescence quenching data an association binding constant of 1.3 · 10 7 M −1 has been calculated for the primary site. The conformation of cytoskeletal proteins after hemin binding was followed by the use of far UV circular dichroism and compared to that of the serum hemin trap, albumin. The secondary structure of albumin was unchanged in the presence of high hemin concentrations. Both spectrin and actin lost their conformation upon hemin binding in a ligand-concentration and time-dependent manner. Unlike spectrin and actin, the secondary structure of protein 4.1 was unaffected by hemin binding to the primary site, but, at higher hemin concentrations, some reduction in the ellipticity of protein 4.1 appeared. The findings of this study suggest that protein 4.1 may serve as the cytoskeletal temporary sink for small amounts of membrane-intercalated hemin similarly to the function of albumin in the serum. However, an increased release of hemin under pathological conditions may cause hemin association with the cytoskeletal proteins and as a result the cell membrane is expected to be distorted.

Detection of Hemin Release During Hemoglobin S Denaturation

Sickle hemoglobin is relatively unstable upon oxidation or mechanical shaking. During denaturation, it generates oxygen radicals and hemichromes and ultimately precipitates in the form of micro-Heinz bodies. It is not clear, however, whether the degradation product hemin, which is a potent hemolytic agent and a potential perturbant to protein-protein interactions in the red cell membrane skeleton, is also generated during sickle hemoglobin denaturation. By specific absorption of hemin with Dowex AG 1-X8 anion-exchange resin at high-ionic strength conditions, we now separate hemin for quantitation from the bulk hemoglobin and its derivatives. We demonstrate that upon mechanical shaking oxyhemoglobin S denatures much faster than oxyhemoglobin A and that a considerably higher level of hemin is detected in the shaken hemoglobin S as compared with hemoglobin A. By using the same method to measure the hemin content in the hemolysate of fresh red cells from patients with sickle cell disease, we detect a three- to fivefold increase in the hemin content in these patients (0.4 to 0.75 mumol/L) as compared with normal individuals (0.1 to 0.15 mumol/L). These data suggest that the instability of sickle oxyhemoglobin leads to increased intracellular precipitation of hemoglobin and the release of hemin, which may play a role in the membrane lesion of sickle red cells.