Haptoglobin: the hemoglobin detoxifier in plasma

doi:10.1016/j.tibtech.2012.10.003

Trends in Biotechnology

Volume 31, Issue 1, January 2013, Pages 2-3

https://doi.org/10.1016/j.tibtech.2012.10.003 Get rights and content

Hemoglobin (Hb) is one of the most studied proteins. However, oxidative toxicity associated with free Hb in circulation and its contribution to inflammation and complications of transfusion have only recently become active areas of research. New insights into the protective mechanisms of haptoglobin (Hp), a plasma protein, and a timely resolution of the crystal structure of the Hb–Hp complex made it possible to definitively link the functional and structural interplay between the two proteins. Here, we summarize current knowledge of the interactions between Hb and Hp under oxidative stress conditions, and how Hb's own damaging radicals are harnessed by complex formation. Potential therapeutic benefits of using Hp for inactivation and clearance of free Hb under a number of clinical settings are considered.

Section snippets

Extracellular Hb is toxic

Hemolysis and transfusion of old blood or artificial blood can result in varying quantities of free Hb in circulation, which can induce radical-generating reactions that are life threatening in critically ill patients [1]. The latter scenario provided both scientific and industrial communities with a unique opportunity to closely examine the toxicity associated with free Hb in animals and humans 2, 3. However, much of the research was focused on Hb-mediated nitric oxide (NO) depletion and

Hp short-circuits Hb's radicals

Biological systems express molecular chaperone proteins such as Hp, which bind and accelerate the clearance of free Hb through the macrophage CD163 [1]. Recent experiments reveal that Hp freezes the higher oxidation state of Hb (Fe^IV) and short-circuits subsequent radical-generating steps after exposure to oxidants such as H₂O₂ 7, 8. Ferryl Hb stabilization is accompanied by a significant increase in the concentration of ferryl protein radical on the penultimate tyrosine 145 of the Hb β

Hp shields the Hb oxidative ‘hot spot’

The crystal structure of the porcine dimeric Hb–Hp complex was recently determined at 2.9 Å [9]. The Hp molecules dimerize through β-strand swapping between two complement control protein (CCP) domains forming a novel fusion CCP domain structure. The Hp serine protease domain engages in extensive interactions with both α and β subunits of Hb. A striking finding is that those amino acids that are susceptible to oxidation are protected against damaging radicals by the tight Hp and Hb interactions (

Targets for therapeutic opportunities

Hp was shown to effectively suppress Hb oxidative toxicity in dogs and guinea pigs and, more recently, in reversing toxicity associated with the infusion of old red blood cells in guinea pigs 5, 12. The unraveling of the structural–functional aspects of Hp and the ‘rediscovery’ of these therapeutic potentials led to the development of several strategies to treat complications of hemolysis in multiple chronic disease states, specifically, the detoxification of extracellular Hb in sickle cell

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Cited by (66)

Kinetic inequivalence between α and β subunits of ligand dissociation from ferrous nitrosylated human haptoglobin:hemoglobin complexes. A comparison with O<inf>2</inf> and CO dissociation
2021, Journal of Inorganic Biochemistry
Haptoglobin (Hp) counterbalances the adverse effects of extra-erythrocytic hemoglobin (Hb) by trapping the αβ dimers of Hb in the bloodstream. In turn, the Hp:Hb complexes display Hb-like reactivity. Here, the kinetics of NO dissociation from ferrous nitrosylated Hp:Hb complexes (i.e., Hp1–1:Hb(II)-NO and Hp2–2:Hb(II)-NO, respectively) are reported at pH 7.0 and 20.0 °C. NO dissociation from Hp:Hb(II)-NO complexes has been followed by replacing NO with CO. Denitrosylation kinetics of Hp1–1:Hb(II)-NO and Hp2–2:Hb(II)-NO are biphasic, the relative amplitude of the fast and slow phase being 0.495 ± 0.015 and 0.485 ± 0.025, respectively. Values of k_off(NO)1 and k_off(NO)2 (i.e., (6.4 ± 0.8) × 10⁻⁵ s⁻¹ and (3.6 ± 0.6) × 10⁻⁵ s⁻¹ for Hp1–1:Hb(II)-NO and (5.8 ± 0.8) × 10⁻⁵ s⁻¹ and (3.1 ± 0.6) × 10⁻⁵ s⁻¹ for Hp2–2:Hb(II)-NO) are unaffected by allosteric effectors and correspond to those reported for the α and β subunits of tetrameric Hb(II)-NO and isolated α(II)-NO and β(II)-NO chains, respectively. This highlights the view that the conformation of the Hb α₁β₁ and α₂β₂ dimers matches that of the Hb high affinity conformation. Moreover, the observed functional heterogeneity reflects the variation of energy barriers for the ligand detachment and exit pathway(s) associated to the different structural arrangement of the two subunits in the nitrosylated R-state. Noteworthy, the extent of the inequivalence of α and β chains is closely similar for the O₂, NO and CO dissociation in the R-state, suggesting that it is solely determined by the structural difference between the two subunits.
βCysteine 93 in human hemoglobin: a gateway to oxidative stability in health and disease
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βcysteine 93 residue plays a key role in oxygen (O₂)-linked conformational changes in the hemoglobin (Hb) molecule. This solvent accessible residue is also a target for binding of thiol reagents that can remotely alter O₂ affinity, cooperativity, and Hb's sensitivity to changes in pH. In recent years, βCys93 was assigned a new physiological role in the transport of nitric oxide (NO) through a process of S-nitrosylation as red blood cells (RBCs) travel from lungs to tissues. βCys93 is readily and irreversibly oxidized in the presence of a mild oxidant to cysteic acid, which causes destabilization of Hb resulting in improper protein folding and the loss of heme. Under these oxidative conditions, ferryl heme (HbFe⁴⁺), a higher oxidation state of Hb is formed together with its protein radical (^.HbFe⁴⁺). This radical migrates to βCys93 and interacts with other “hotspot” amino acids that are highly susceptible to oxidative modifications. Oxidized βCys93 may therefore be used as a biomarker of oxidative stress, reflecting the deterioration of Hb within RBCs intended for transfusion or RBCs from patients with hemoglobinopathies. Site specific mutation of a redox active amino acid(s) to reduce the ferryl heme or direct chemical modifications that can shield βCys93 have been proposed to improve oxidative resistance of Hb and may offer a protective therapeutic strategy.
This mini-review summarizes the role of βcysteine 93 in oxidative stability of hemoglobin in human blood. βCys93 has been recognized as an end point for radicals originating from heme during oxidative stress and therefore it may be an important biological marker of oxidative stability of hemoglobin within red blood cells intended for transfusion or in hemoglobinopathies.
Haptoglobin: From hemoglobin scavenging to human health
2020, Molecular Aspects of Medicine
Citation Excerpt :
Binding of Hb to Hp does not appear to inhibit completely the oxidative properties of the heme group and consequently radicals are still formed within the Hp:Hb complex (Buehler et al., 2009; Vallelian et al., 2015). However, Hb residues prone to oxidation are located at the Hp:Hb interface and the protective role of Hp may be achieved through shielding and stabilizing radicals on Hb (Andersen et al., 2012; Banerjee et al., 2012; Alayash et al., 2013; Cooper et al., 2013). In particular, Hb residues Tyr42α and Cys93β representing a way for radical's migration from α1Hb to β2Hb (or from α2Hb to β1Hb) are located in close proximity in tetrameric Hb (Pimenova et al., 2010).
Haptoglobin (Hp) belongs to the family of acute-phase plasma proteins and represents the most important plasma detoxifier of hemoglobin (Hb). The basic Hp molecule is a tetrameric protein built by two α/β dimers. Each Hp α/β dimer is encoded by a single gene and is synthesized as a single polypeptide. Following post-translational protease-dependent cleavage of the Hp polypeptide, the α and β chains are linked by disulfide bridge(s) to generate the mature Hp protein. As human Hp gene is characterized by two common Hp1 and Hp2 alleles, three major genotypes can result (i.e., Hp1-1, Hp2-1, and Hp2-2). Hp regulates Hb clearance from circulation by the macrophage-specific receptor CD163, thus preventing Hb-mediated severe consequences for health. Indeed, the antioxidant and Hb binding properties of Hp as well as its ability to stimulate cells of the monocyte/macrophage lineage and to modulate the helper T-cell type 1 and type 2 balance significantly associate with a variety of pathogenic disorders (e.g., infectious diseases, diabetes, cardiovascular diseases, and cancer). Alternative functions of the variants Hp1 and Hp2 have been reported, particularly in the susceptibility and protection against infectious (e.g., pulmonary tuberculosis, HIV, and malaria) and non-infectious (e.g., diabetes, cardiovascular diseases and obesity) diseases. Both high and low levels of Hp are indicative of clinical conditions: Hp plasma levels increase during infections, inflammation, and various malignant diseases, and decrease during malnutrition, hemolysis, hepatic disease, allergic reactions, and seizure disorders. Of note, the Hp:Hb complexes display heme-based reactivity; in fact, they bind several ferrous and ferric ligands, including O₂, CO, and NO, and display (pseudo-)enzymatic properties (e.g., NO and peroxynitrite detoxification). Here, genetic, biochemical, biomedical, and biotechnological aspects of Hp are reviewed.
Storability of porcine blood in forensics: How far should we go?
2020, Forensic Science International
Previous studies on the storability of porcine blood for bloodstain pattern analysis (BPA) focused on abattoir blood only and did not include measurements of viscoelasticity. Although known to provoke echinocyte formation, EDTA is widely used for BPA issues. We compared ageing samples taken from live pigs with abattoir blood and detected considerable differences in hematocrit (HCT), total protein and shear viscosity that even worsened with time. Upon storage, high shear viscosity continuously increased, resulting in a partial loss of the typical shear thinning property of blood. Furthermore, we explored CPDA-1, the gold standard in preserving red blood cells (RBCs), for storage of forensic samples. We found it to be a superior choice for anticoagulation, as the rise of high shear viscosity was attenuated compared to EDTA. When performing oscillation measurements, we found a sudden change of viscoelasticity of blood after 22 days, providing a cut-off for storage time. To highlight the importance of hematological and hemorheological changes upon cold storage, we performed simple drip pattern experiments. These tests revealed a tendency to smaller stain diameters and higher numbers of satellite spatter. While this contradicts expectations from elevated viscosity values, we associate this trend to microscopic inhomogeneities due to storage. We recommend CPDA-1 for prolonged storage of BPA samples and suggest the use of comprehensive test protocols including viscoelasticity for determination of the maximum shelf life of pig blood.
Ligand-dependent inequivalence of the α and β subunits of ferric human hemoglobin bound to haptoglobin
2020, Journal of Inorganic Biochemistry
Citation Excerpt :
Since plasmatic free Hb indeed brings about the formation of free radicals, thus inducing the destruction of cellular constituents and extracellular macromolecules, living organisms need an efficient mechanism for extraerythrocytic Hb scavenging [5–7]. The molecular chaperone haptoglobin (Hp) binds αβ dimers of plasma Hb leading to a very stable non-covalent complex, which (i) allows the removal of Hb via the reticuloendothelial system and the CD163 receptor-mediated endocytosis in hepatocytes, Kupffer cells, and tissue macrophages, and (ii) impairs heme oxidation and release [4,8–11]. Further, the exposure of the Hp:Hb complexes to hydrogen peroxide induces the formation of ferryl metal centers, which are more kinetically inert than those of Hb; this plays a relevant role in the protection against Hb-mediated oxidative damage [12].
Haptoglobin (Hp) prevents extra-erythrocytic hemoglobin- (Hb-)mediated damage. Hp binds αβ dimers of Hb, displaying heme-based reactivity. Here, kinetics and thermodynamics of cyanide, thiocyanate and imidazole binding to ferric human Hb (Hb(III)), Hb(III) dimers complexed with the human Hp phenotypes 1-1 and 2-2 (Hp1-1:Hb(III) and Hp2-2:Hb(III), respectively), and α(III) and β(III) chains are reported and analyzed in parallel with fluoride and azide binding properties (at pH 7.0 and 20.0 °C). Cyanide and fluoride bind to Hb(III), Hp1-1:Hb(III), Hp2-2:Hb(III), α(III), and β(III) with a simple behavior. In contrast, azide, thiocyanate and imidazole binding to Hb(III), Hp1-1:Hb(III) and Hp2-2:Hb(III) follows a two-step process, whereas ligand binding to α(III) and β(III) chains follows a simple behavior. However, azide, thiocyanate and imidazole binding to Hb(III), Hp1-1:Hb(III) and Hp2-2:Hb(III) is characterized by a simple equilibrium, reflecting the compensation of kinetic parameters. The fast and the slow step of azide, thiocyanate and imidazole binding to Hb(III), Hp1-1:Hb(III) and Hp2-2:Hb(III) mirror the ligand binding properties of the β(III) and α(III) chains, respectively. Values of kinetic and thermodynamic parameters for binding of ferric ligands to Hp1-1:Hb(III) and Hp2-2:Hb(III) match very well with those obtained for ligation of Hb(III) and α(III) and β(III) chains, confirming the ligand-dependent kinetic inequivalence of α(III) and β(III) subunits. However, a variation between tetrameric Hb(III) on one side and Hp1-1:Hb(III), Hp2-2:Hb(III), α(III), and β(III) on the other side for the rate-limiting step (likely referable to the dissociation of heme-coordinated H₂O from the heme-Fe(III) atom) suggests a structural change(s) upon dimers to tetramer assembly in Hb(III).
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Trends in Biotechnology

LetterHaptoglobin: the hemoglobin detoxifier in plasma

Section snippets

Extracellular Hb is toxic

Hp short-circuits Hb's radicals

Hp shields the Hb oxidative ‘hot spot’

Targets for therapeutic opportunities

J. Biol. Chem.

Am. J. Pathol.

Free Radic. Biol. Med.

Blood aging, safety, and transfusion: capturing the “radical” menace

Antioxid. Redox Signal.

Oxygen therapeutics: can we tame haemoglobin?

Nat. Rev. Drug Discov.

Hemoglobin-based oxygen carriers: current status and future directions

Anesthesiology

Letter
Haptoglobin: the hemoglobin detoxifier in plasma