Differential susceptibilities of the prosthetic heme of hemoglobin-based red cell substitutes: Implications in the design of safer agents

doi:10.1016/0006-2952(93)90621-3

Biochemical Pharmacology

Volume 46, Issue 12, 14 December 1993, Pages 2299-2305

https://doi.org/10.1016/0006-2952(93)90621-3 Get rights and content

Abstract

One approach to the development of an effective red cell substitute has been chemical modification of human hemoglobin to optimize oxygen transport and plasma half-life. Human hemoglobin A₀ and two of these modified hemoglobins, one prepared from the cross-linking of the α-chains at lysine residue 99 by bis(3,5-dibromosalicyl)fumarate (Hb-DBBF) and the other by acylation of lysine residue 82 of the β-chain by mono-(3,5-dibromosalicyl)fumarate (Hb-FMDA), were tested by HPLC for their susceptibility to oxidative damage caused by H₂O₂. Such oxidative insult may occur during ischemia and reperfusion of tissues after transfusion of red cell substitutes to patients with hypovolemic shock and trauma. Hb-DBBF was extremely susceptible to damage of its heme and protein moieties with stoichiometric amounts of H₂O₂, whereas Hb-FMDA was highly resistant, even at 10-fold molar excess and at an acidic pH of 4.7. Hemoglobin A₀ was of intermediate susceptibility, exhibiting alteration of heme and protein moieties at acidic but not neutral pH. Since the degradation of heme can release the potentially toxic agent iron, Hb-FMDA may be a more promising candidate than Hb-DBBF for development as a red cell substitute. A similar approach may be used to assess the susceptibility of other hemoglobin-based red cell substitutes to oxidative damage in order to determine the molecular basis of heme and protein alteration.

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These results are consistent with our previous observations that confirm that H2O2 induces extensive decomposition of β-globin chains of Hb (14). Other internal oxidative changes (initiated by the ferryl heme) that can be monitored by RP-HPLC are heme and the AHPs that result from covalent attachment to the protein (40, 41). As shown in Fig. 5, changes in both heme and AHPs occur as a function of pH and H2O2 concentration.
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.
Redox properties of human hemoglobin in complex with fractionated dimeric and polymeric human haptoglobin
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A number of specific oxidative changes have been reported to occur when the protein is reacted with excess H2O2 in vitro. First, the heme vinyl groups may become modified and covalently linked to the protein [63]. Second, extensive globin chain cross-links and irreversible modifications of several amino acids occur primarily in the “hot spots” of β subunits.
Haptoglobin (Hp) is an abundant and conserved plasma glycoprotein, which binds acellular adult hemoglobin (Hb) dimers with high affinity and facilitates their rapid clearance from circulation after hemolysis. Humans possess three main phenotypes of Hp, designated Hp 1-1, Hp 2-1, and Hp 2-2. These variants exhibit diverse structural configurations and have been reported to be functionally nonequivalent. We have investigated the functional and redox properties of Hb–Hp complexes prepared using commercially fractionated Hp and found that all forms exhibit similar behavior. The rate of Hb dimer binding to Hp occurs with bimolecular rate constants of ~0.9 μM⁻¹ s⁻¹, irrespective of the type of Hp assayed. Although Hp binding does accelerate the observed rate of HbO₂ autoxidation by dissociating Hb tetramers into dimers, the rate observed for these bound dimers is three- to fourfold slower than that of Hb dimers free in solution. Co-incubation of ferric Hb with any form of Hp inhibits heme loss to below detectable levels. Intrinsic redox potentials (E_1/2) of the ferric/ferrous pair of each Hb–Hp complex are similar, varying from +54 to +59 mV (vs NHE), and are essentially the same as reported by us previously for Hb–Hp complexes prepared from unfractionated Hp. All Hb–Hp complexes generate similar high amounts of ferryl Hb after exposure to hydrogen peroxide. Electron paramagnetic resonance data indicate that the yields of protein-based radicals during this process are approximately 4 to 5% and are unaffected by the variant of Hp assayed. These data indicate that the Hp fractions examined are equivalent to one another with respect to Hb binding and associated stability and redox properties and that this result should be taken into account in the design of phenotype-specific Hp therapeutics aimed at countering Hb-mediated vascular disease.
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The precise role of Hp in the CD163-mediated Hb clearance pathway remains therefore elusive Exposure of Hb to H2O2 induces structural modifications, which include altered heme protein product formation, extensive cross-linking of α-globin chains, and irreversible oxidative modifications of specific amino acids within the CD163 binding region of the Hb β-globin chain.8,9 These structural alterations result in reduced CD163 binding, and severely impair endocytosis of oxidized Hb by CD163.10
Detoxification and clearance of extracellular hemoglobin (Hb) have been attributed to its removal by the CD163 scavenger receptor pathway. However, even low-level hydrogen peroxide (H₂O₂) exposure irreversibly modifies Hb and severely impairs Hb endocytosis by CD163. We show here that when Hb is bound to the high-affinity Hb scavenger protein haptoglobin (Hp), the complex protects Hb from structural modification by preventing α-globin cross-links and oxidations of amino acids in critical regions of the β-globin chain (eg, Trp15, Cys93, and Cys112). As a result of this structural stabilization, H₂O₂-exposed Hb-Hp binds to CD163 with the same affinity as nonoxidized complex. Endocytosis and lysosomal translocation of oxidized Hb-Hp by CD163-expressing cells were found to be as efficient as with nonoxidized complex. Hp complex formation did not alter Hb's ability to consume added H₂O₂ by redox cycling, suggesting that within the complex the oxidative radical burden is shifted to Hp. We provide structural and functional evidence that Hp protects Hb when oxidatively challenged with H₂O₂ preserving CD163-mediated Hb clearance under oxidative stress conditions. In addition, our data provide in vivo evidence that unbound Hb is oxidatively modified within extravascular compartments consistent with our in vitro findings.
The reaction of hydrogen peroxide with hemoglobin induces extensive α-globin crosslinking and impairs the interaction of hemoglobin with endogenous scavenger pathways
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Cell-free hemoglobin (Hb) enhances the oxidation-related toxicity associated with inflammation, ischemia, and hemolytic disorders. Hb is highly vulnerable to oxidative damage, and irreversible structural changes involving iron/heme oxidation, heme-adduct products, and amino acid oxidation have been reported. Specific structural features of Hb, such as unconstrained α−chains and molecular size, determine the efficiency of interactions between the endogenous Hb scavengers haptoglobin (Hp) and CD163. Using HPLC, mass spectrometry, and Western blotting, we show that H₂O₂-mediated Hb oxidation results in the formation of covalently stabilized globin multimers, with prominent intramolecular crosslinking between α−globin chains. These structural alterations are associated with reduced Hp binding, reduced CD163 interaction, and severely impaired endocytosis of oxidized Hb by the Hp-CD163 pathway. As a result, when exposed to oxidized Hb, CD163-positive HEK293 cells and human macrophages do not increase hemeoxygenase-1 (HO-1) expression, the physiological anti-oxidative macrophage response to Hb exposure. Failed Hb clearance, inadequate HO-1 expression, and the subsequent accumulation of oxidatively damaged Hb species might thus contribute to pathologies related to oxidative stress.
Enzyme-inorganic nanoporous materials: Stabilization of proteins intercalated in α-zirconium(IV) phosphate by a denaturant
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Improving the stability of proteins and enzymes at solid–liquid interfaces is challenging and it has important implications in biocatalysis and biosensors. Here, we report that moderate concentrations of urea stabilize heme proteins, such as met-hemoglobin (Hb) or met-myoglobin (Mb) bound to α-zirconium phosphate (α-ZrP). The half-life of Hb intercalated in the galleries of α-ZrP, for example, increased from 127 h (no urea) to >235 h (2 M urea). The peroxidase-like activity of Hb intercalated in α-ZrP was also improved by the addition of urea, and the product yields increased from 44% (no urea) to 79% (2 M urea). These improvements are not limited to Hb, and similar results also observed with Mb. Urea also inhibited the dithionite reduction of the ferric form of the intercalated heme proteins to their corresponding ferrous forms but not the oxidation of the ferric form to the corresponding high valent iron-oxo species. These observations open new possibilities to control the properties of immobilized proteins.
Structural basis of peroxide-mediated changes in human hemoglobin: A novel oxidative pathway
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Citation Excerpt :
In Fig. 2F, a new heme peak (400 nm) eluted at a retention time similar to intact α-and β-globin chains, further indicating the presence of AHP products. These results are consistent with the previous observation that H2O2 induces extensive decomposition of β-globin chains (24). The loss of intact globin chains within HbA0 is readily seen in the CD spectra of HbA0 treated with increasing H2O2 concentrations.
Hydrogen peroxide (H₂O₂) triggers a redox cycle between ferric and ferryl hemoglobin (Hb) leading to the formation of a transient protein radical and a covalent hemeprotein cross-link. Addition of H₂O₂ to highly purified human hemoglobin (HbA₀) induced structural changes that primarily resided within β subunits followed by the internalization of the heme moiety within α subunits. These modifications were observed when an equal molar concentration of H₂O₂ was added to HbA₀ yet became more abundant with greater concentrations of H₂O₂. Mass spectrometric and amino acid analysis revealed for the first time that βCys-93 and βCys-112 were oxidized extensively and irreversibly to cysteic acid when HbA₀ was treated with H₂O₂. Oxidation of further amino acids in HbA₀ exclusive to the β-globin chain included modification of βTrp-15 to oxyindolyl and kynureninyl products as well as βMet-55 to methionine sulfoxide. These findings may therefore explain the premature collapse of the β subunits as a result of the H₂O₂ attack. Analysis of a tryptic digest of the main reversed phase-high pressure liquid chromatography fraction revealed two α-peptide fragments (α128 - α139) and a heme moiety with the loss of iron, cross-linked between αSer-138 and the porphyrin ring. The novel oxidative pathway of HbA₀ modification detailed here may explain the diverse oxidative, toxic, and potentially immunogenic effects associated with the release of hemoglobin from red blood cells during hemolytic diseases and/or when cell-free Hb is used as a blood substitute.

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Differential susceptibilities of the prosthetic heme of hemoglobin-based red cell substitutes: Implications in the design of safer agents

Abstract

J Biol Chem

J Biochem Biophys Methods

Biophys J

J Biol Chem

Biochem Biophys Res Commun

J Biol Chem

Arch Biochem Biophys

J Biol Chem

J Biol Chem

J Biol Chem

Comp Biochem Physiol

FEBS Lett

Biochem Pharmacol

Am Heart J

Arch Biochem Biophys

Blood substitutes; Minireview

Hemoglobin-Based Red Cell Substitutes

Equilibrium oxygen binding to human hemoglobin cross-linked between the α chains by bis(3,5-dibromosalicyl)fumarate

J Biol Chem