Elsevier

Free Radical Biology and Medicine

Volume 53, Issue 6, 15 September 2012, Pages 1317-1326
Free Radical Biology and Medicine

Original Contribution
Haptoglobin alters oxygenation and oxidation of hemoglobin and decreases propagation of peroxide-induced oxidative reactions

https://doi.org/10.1016/j.freeradbiomed.2012.07.023Get rights and content

Abstract

We compared oxygenation and anaerobic oxidation reactions of a purified complex of human hemoglobin (Hb) and haptoglobin (Hb–Hp) to those of uncomplexed Hb. Under equilibrium conditions, Hb–Hp exhibited active-site heterogeneity and noncooperative, high-affinity O2 binding (n1/2=0.88, P1/2=0.33 mm Hg in inorganic phosphate buffer at pH 7 and 25 °C). Rapid-reaction kinetics also exhibited active-site heterogeneity, with a slower process of O2 dissociation and a faster process of CO binding relative to uncomplexed Hb. Deoxygenated Hb–Hp had significantly reduced absorption at the λmax of 430 nm relative to uncomplexed Hb, as occurs for isolated Hb subunits that lack T-state stabilization. Under comparable experimental conditions, the redox potential (E1/2) of Hb–Hp was found to be +54 mV, showing that it is much more easily oxidized than uncomplexed Hb (E1/2=+125 mV). The Nernst plots for Hb–Hp oxidation showed no cooperativity and slopes less than unity indicated active-site heterogeneity. The redox potential of Hb–Hp was unchanged by pH over the range of 6.4–8.3. Exposure of Hb–Hp to excess hydrogen peroxide (H2O2) produced ferryl heme, which was found to be more kinetically inert in the Hb–Hp complex than in uncomplexed Hb. The negative shift in the redox potential of Hb–Hp and its stabilized ferryl state may be central elements in the protection against Hb-induced oxidative damage afforded by formation of the Hb–Hp complex.

Highlights

► We examined the oxygenation and anaerobic oxidation reactions of Hb-Hp. ► The complex exhibited active-site heterogeneity and rapid ligand reaction kinetics. ► Redox potential of the complex was lower than uncomplexed Hb. ► The complex stabilizes the ferryl state and protects against oxidative damage.

Introduction

This paper addresses the mechanisms by which Hp helps protect against the oxidative cascade associated with cell-free Hb. Propagation of oxidative reactions by the so-called pseudoperoxidase activities of Hb both in vitro and in vivo has been the subject of many investigations in recent years [1]. These oxidative reactions are much more pronounced for cell-free Hb than for Hb within red blood cells, since antioxidant enzymes within red blood cells decrease the levels of superoxide ions (O2•−) and hydrogen peroxide (H2O2) and thereby minimize the extent of Hb-induced oxidative side reactions [2]. Consequently, Hb-induced oxidative damage is especially problematic in hemolytic anemias and when cell-free Hb is introduced as an O2 therapeutic in circulation, and presents both physiological and clinical challenges. Oxidation reactions of cell-free Hb solutions intended for therapeutic use after blood loss have been shown to compromise tissue O2-sensing mechanisms and lead to the induction of degradative cytotoxic pathways that can be detrimental to animals transfused with these solutions [3]. In addition, hemolysis of red blood cells in aged blood during storage and after transfusion and/or in hemolytic disorders has been associated with vaso-occlusion and vasculopathy as a result of the release of Hb into the circulation [2], [4].

H2O2-linked oxidative reactions of Hb have been studied extensively as models for the propagation of oxidative reactions by the pseudoperoxidase activities of Hb [5]. The reaction between H2O2 and ferrous Hb (Fe(II)Hb) is a two-electron process that results in the formation of ferryl Hb (Fe(IV)Hb) (reaction (1)). When H2O2 reacts with ferric Hb (Fe(III)Hb), a cation radical species (reaction (2)) is formed as a second reaction product [1].HbFe(II)+H2O2→O2−=Fe(IV)Hb+H2OHbFe(III)+H2O2→O2−=Fe(IV)Hb•+H2O

Because of their high redox potentials and hence thermodynamic ease of participation in redox reactions, both ferryl heme and protein radicals are capable of inducing a wide range of adverse oxidative reactions. Their formation in cell-free Hb extends the oxidative cascade to other biological molecules as well as fueling a self-destructive pathway that involves irreversible oxidation of key amino acids on the Hb molecule [1], [6].

Several diverse and complex physiological pathways are normally deployed in mammalian circulation to control and/or limit the oxidative reactions that occur when Hb is released from the protective environment of red blood cells. Hp, the focus of this paper, is one of several plasma proteins that orchestrate oxidative inactivation and clearance of free Hb and its oxidation by-products from circulating blood. Interaction of Hb with Hp sequesters the heme-containing subunits of Hb as αβ dimers within a Hb–Hp complex [7], [8].

Hp is composed of α (9 kDa) and β (33 kDa) subunits and in humans exists in two allelic forms, Hp1 and Hp2, which differ only in their respective α chains. The α1-chain in Hp1 carries one sulfhydryl group, while α2 in Hp2 is longer and carries two sulfhydryl groups [9]. Polymeric structures with variable numbers of the Hp subunits are found in vivo. In human sera, Hp molecules comprise two α1β units (Hp 1-1), a variable number of α2β units (Hp1-2), or two α1β units and a variable number of α2β units (Hp 2-2).

The binding between Hp and Hb in the Hb–Hp complex is among the strongest noncovalent interactions known in biological systems [9]. The Hb bound to Hp is recognized by CD163 receptors on macrophages and this CD 163–Hb–Hp complex is then internalized and the released heme is subsequently degraded by the heme oxygenase system within macrophages [10]. Recent in vitro and in vivo studies have shed some light on the role of Hp in the CD163–Hb clearance pathways [11], [12], but the precise molecular mechanism of Hp protection against Hb-induced oxidative toxicity has yet to be determined.

We anticipated that knowledge of the redox properties of the heme-containing subunits within the Hb–Hp complex would provide further insight into the mechanisms underlying the protective action of Hp. Accordingly, we characterized the anaerobic oxidation processes of a purified Hb–Hp complex relative to those of uncomplexed Hb, using a spectroelectrochemical method developed by our laboratories. We also report the ligand-binding equilibria and kinetics of Hb and of the purified Hb–Hp complex under similar buffer conditions, and compare the pseudoperoxidase reactions of these proteins induced by their interactions with H2O2.

The results obtained led to a fascinating picture that may explain, for the first time, the molecular basis of Hp action in protecting against Hb-induced oxidative toxicity. The formation of the Hb–Hp complex appears to limit the propagation of Hb-derived radicals, resulting in a less reactive form of cell-free Hb. As detailed in this paper, spectroelectrochemical studies with uncomplexed Hb and the Hb–Hp complex show a shift of the E1/2 value for Hb–Hp complex system by 70 mV in the negative direction, indicating stabilization of the Fe(III) state in the complex compared to free Hb. The ligand-binding experiments show elevated ligand affinity and chain-based heterogeneity for the Hb–Hp complex, whereas the reaction with H2O2 demonstrates kinetic stabilization of the Fe(IV) state. Taken together, the elevated O2 affinity, increased ease of oxidation, and kinetic inertness of the ferryl Hb form may be central elements in the protection against Hb-induced oxidative damage afforded by formation of the Hb–Hp complex.

Section snippets

Materials

Highly purified Hp solution was a kind gift from Bio Products Laboratory (BPL), Hertfordshire, England. The isolation and fractionation of this protein from human plasma were done as previously reported [13]. Typical size-exclusion HPLC separation profiles of Hp samples used in this study shows the following molecular weight distribution: 60% with 2 αβ (dimer, Hp 1-1), 21% with 3 αβ (trimer, mostly Hp 1-2), and 19% larger forms (polymer, mostly Hp 2-2). Adult human Hb (Hb) was prepared by

Ligand-binding equilibria and kinetics for Hb and Hb–Hp

Fig. 1 shows representative Hill plots for O2 binding to the purified Hb–Hp complex and for uncomplexed Hb. Hb–Hp showed significantly increased O2-binding affinity relative to Hb (log P1/2=–0.48 instead of 0.95, respectively; P1/2=0.33 instead of 9.05 mm Hg, respectively) when measured in 200 mM phosphate, pH 7.0 at 25 °C. Cooperativity of O2 binding, indicated by Hill plots with slopes >1 (n1/2=2.8 for uncomplexed Hb), was absent in studies with the Hb–Hp complex. Instead, significant

Discussion

The pseudoperoxidase activity of cell-free Hb can produce ferryl heme and ferryl heme-based radicals that can participate in several damaging reactions in living systems. The propagation of radical reactions associated with ferryl heme formation is due to their high redox potentials. In order to reduce the adverse effects of the ferryl Hb/ferryl Hb-based radicals, two strategies can therefore be hypothesized: (1) blocking the formation of ferryl Hb/ferryl Hb-based radicals, and (2)

Acknowledgments

This work was supported by CBER’s MODSCI Funding (A.I.A.). A.L.C. thanks the National Science Foundation (CHE 0809466) and Duke University for financial support and the Santa Fe Institute for hospitality during the period this manuscript was written. We thank Dr. Jin Baek (CBER/FDA) for technical advice and suggestions for the immunoblot experiments.

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