Peroxidase activity of hemoglobin-haptoglobin complexes: covalent aggregation and oxidative stress in plasma and macrophages

Abstract

As a hemoprotein, hemoglobin (Hb) can, in the presence of H(2)O(2), act as a peroxidase. In red blood cells, this activity is regulated by the reducing environment. For stroma-free Hb this regulation is lost, and the potential for Hb to become a peroxidase is high and further increased by inflammatory cells generating superoxide. The latter can be converted into H(2)O(2) and feed Hb peroxidase activity. Haptoglobins (Hp) bind with extracellular Hb and reportedly weaken Hb peroxidase activity. Here we demonstrate that: (i) Hb peroxidase activity is retained upon binding with Hp; (ii) in the presence of H(2)O(2), Hb-Hp peroxidase complexes undergo covalent cross-linking; (iii) peroxidase activity of Hb-Hp complexes and aggregates consumes reductants such as ascorbate and nitric oxide; (iv) cross-linked Hb-Hp aggregates are taken up by macrophages at rates exceeding those for noncovalently cross-linked Hb-Hp complexes; (v) the engulfed Hb-Hp aggregates activate superoxide production and induce intracellular oxidative stress (deplete endogenous glutathione and stimulate lipid peroxidation); (vi) Hb-Hp aggregates cause cytotoxicity to macrophages; and (vii) Hb-Hp aggregates are present in septic plasma. Overall, our data suggest that under conditions of severe inflammation and oxidative stress, peroxidase activity of Hb-Hp covalent aggregates may cause macrophage dysfunction and microvascular vasoconstriction, which are commonly seen in severe sepsis and hemolytic diseases.

FIGURE 1.

H₂O₂-dependent oxidation of small molecular mass peroxidase substrates by free Hb and Hb·Hp complexes monitored by EPR and by absorbance at 265 nm. A, typical EPR spectrum (inset) and time course of ascorbate radical EPR signals. B, lifespan of EPR signals from ascorbate radicals generated by Hb·Hp complexes in the presence of H₂O₂. The reaction conditions were: 50 mm sodium phosphate buffer (pH 7.4), 100 μm DTPA, 2.5 μm Hb, 20 μm ascorbate, 100 μm H₂O₂, and Hp as indicated (means ± S.D. from three independent experiments; *, p < 0.05 versus Hb). C, ascorbate oxidation by Hb·Hp complexes. Inset, time course of characteristic ascorbate absorbance at 265 nm. The reaction conditions were: 50 mm sodium phosphate buffer (pH 7.4), 100 μm DTPA, 1.25 μm Hb, 20 μm ascorbate, 100 μm H₂O₂, and Hp as indicated. Line a, Hb; line b, Hb·Hp −1:0.15; line c, Hb·Hp −1:0.8; line d, Hb·Hp-1:1 (means ± S.D. from three independent experiments; *, p < 0.05 versus Hb). D, representative native PAGE stained for peroxidase activities of free Hb and its complexes with Hp 1-1, Hp 2-2 assessed by West Pico chemiluminescence substrate. E, averaged results of densitometric analysis of peroxidase activities of Hb complexes with Hp 1-1 and Hp 2-2 normalized to the activity of free Hb (*, p < 0.05 versus Hb). F, Lineweaver-Burk plots for peroxidase activity of Hb assayed by Amplex Red oxidation in the presence and absence of Hp. Hb (0.25 μm) was preincubated for 10 min with Hp at a ratio of 1:1. The peroxidase activity was assessed by fluorescence of resorufin in PBS (pH 7.4) containing DTPA (100 μm), H₂O₂ (50 μm), and Amplex Red in the range of 5–150 μm. Line 1, Hb; line 2, Hb·Hp.

FIGURE 2.

Detection of heme-nitrosyl complexes and protein-derived (Tyr^•) radicals (peroxidase intermediates) by EPR spectroscopy. A, typical low temperature (77 K) EPR spectra of Hb·Hp complexes and aggregates (insets) and magnitudes of EPR signals of heme-nitrosylated Hb (bar a), Hb·Hp complexes (bar b), Hb aggregates (bar c), and Hb·Hp aggregates (bar d). To form nitrosyl complexes, 40 μm Angeli's salt was added to the solution containing 10 μm Hb, and incubation was continued for 30 min, after which the samples were frozen (means ± S.D. from 3–7 independent experiments; *, p < 0.05 versus Hb; **, p < 0.05 versus Hb·Hp complexes). B, H₂O₂-dependent formation of protein-derived (Tyr^•) radicals (peroxidase intermediates) by Hb (bar a), Hb·Hp complexes (bar b), Hb aggregates (bar c), and Hb·Hp aggregates (bar d). Shown are typical low temperature (77 K) EPR spectra of Hb·Hp complexes and aggregates (insets) (measured at g = 2.005) and magnitudes of the respective protein-derived (Tyr) radicals (means ± S.D. from three to six independent experiments; *, p < 0.05 versus Hb). C, power saturation curves for protein-derived radicals of Hb and Hb·Hp complexes and aggregates. Hb aggregates and Hb·Hp aggregates were obtained by incubating Hb (10 μm) and Hb·Hp in the presence of H₂O₂ (50 μm added four times with a 15-min interval). 200 μm ascorbate was added to the formed aggregates and incubated for 15 min to reduce all protein immobilized radicals, and the residual ascorbate was removed by ascorbate oxidase (1 unit/100 μl; 5 min). 100 μm H₂O₂ was added to Hb, Hb aggregates, Hb·Hp complexes, and Hb·Hp aggregates, then samples were frozen for 20 s, and EPR spectra of protein-derived radicals were measured. Catalase (10 μg/ml) was added to stop the reaction.

FIGURE 3.

PAGE of H₂O₂-induced covalent aggregation of Hb and Hp. SDS-PAGE (12% acrylamide) in the presence of DTT (40 μm) stained with silver (panel a) and Western blots using anti-Hb antibody (panel b) and anti-Hp antibody (panel c). The arrows and brackets indicate positions of protein aggregates containing both Hb and Hp that were formed upon the addition of H₂O₂. Monomeric form of Hb (marked by a double-headed arrow) disappeared almost completely after incubation of Hb with H₂O₂ as a result of its oligomerization. Incubation system contained Hb (1 μm) and Hp (equal weight amounts of Hp 1-1 and Hp 2-2 at a ratio of 1:1) in the presence or absence of H₂O₂ (50 μm) in 50 mm phosphate buffer (pH 7.4). MW, molecular mass.

FIGURE 4.

Inhibition of Hb·Hp peroxidase-reactive intermediates and hetero-oligomerization and immuno-spin trapping of H₂O₂-induced protein-immobilized radicals as evidenced by PAGE. A–C, protection against H₂O₂-induced Hb·Hp aggregation by Amplex Red, PAPANONOate, and ascorbate as evidenced by SDS-DTT-PAGE (7.5% acrylamide). The arrows and brackets indicate positions of protein aggregates containing both Hb and Hp formed upon the addition of H₂O₂. Staining was performed using SilverSNAP stain kit (A), anti-Hb antibody (B and C, panels a) and anti-Hp antibody (B and C, panels b). D, detection of protein-immobilized radical intermediates using antibody against a spin trap DMPO after SDS-PAGE (7.5% acrylamide) followed by Western blot analysis. The arrows and brackets indicate positions of protein aggregates reacting with DMPO. Staining was performed using anti-DMPO antibody (panel a), anti-Hb antibody (panel b), and anti-Hp antibody (panel c). E, inhibition of Hb/Hpt hetero-oligomerization by DMPO as evidenced by SDS-DTT-PAGE (7.5% acrylamide) stained with silver. The arrow indicates the position of covalent protein aggregates formed upon the addition of H₂O₂. The incubation system contained Hb (1 μm) and Hp1–1 in the presence or absence of H₂O₂ (50 μm) and the indicated concentrations of Amplex Red, PAPANONOate, ascorbate, and DMPO in 50 mm phosphate buffer (pH 7.4). MW, molecular mass.

FIGURE 5.

Comparison of the peroxidase activity of free Hb, Hb·Hp complexes, and aggregates. A, assessment of peroxidase activity of free Hb, Hb·Hp complexes, and Hb·Hp aggregates after native PAGE with subsequent Western blotting using West Pico chemiluminescence substrate. Panel a, representative gel stained with West Pico for peroxidase activity. Panel b, representative gel stained with SilverSNAP stain kit for detection of protein. Panel c, averaged peroxidase activity assessments based on the densitometry of bands (normalized to the amount of Hb) (*, p < 0.05 versus Hb). B, HPLC separation of Hb·Hp complexes and Hb·Hp aggregates and assessments of peroxidase activity in the fractions. Panel a, HPLC chromatogram of Hb·Hp complexes and Hb·Hp aggregates separated on BioSep-SEC-S4000 column; insets show native PAGE of two fractions corresponding to Hb·Hp complexes and Hb·Hp aggregates. Aggregation of Hb·Hp was induced by incubation of Hb and Hp with H₂O₂ (H₂O₂/Hb ratio 15:1, 60 min). Panels b and c, peroxidase activity of Hb·Hp complexes and Hb·Hp aggregates measured by fluorescence of Amplex Red oxidation product, resorufin (panel b) and by EPR spectroscopy of ascorbate radical signals (presented as lifespan of ascorbate radical signals) (panel c) (*, p < 0.05 versus Hb·Hp complexes). C, H₂O₂-dependent peroxidase activity of Hb·Hp aggregates revealed on SDS-PAGE. The incubation system contained Hb (1 μm) and Hp (equal weight amounts of Hp 1-1 and Hp 2-2 at a ratio of 1:1) in the presence or absence of H₂O₂ (25 μm) in 50 mm phosphate buffer (pH 7.4). After PAGE in 2.5% acrylamide, 0.5% agarose, zymography was performed using West Pico chemiluminescence substrate. The arrows and brackets indicate positions of Hb·Hp aggregates.

FIGURE 6.

Uptake of Hb·Hp complexes and aggregates by THP-1 cells. A, CD163 expression in differentiated and nondifferentiated THP-1 cells. In THP-1 cells treated with PMA and dexamethasone (Dex), flow cytometry analysis showed an increase in surface expression of CD163. Expression of CD163 was detected by flow cytometry using phycoerythrin (PE)-conjugated antibody. Inset, typical histogram of nondifferentiated (gray) or differentiated (unfilled) THP-1 cells (means ± S.D. from three independent experiments; ***, p < 0.001 versus nondifferentiated cells). B, uptake of Hb, Hb·Hp complexes and Hb·Hp aggregates by THP-1 cells as evidenced by flow cytometry. Note that the uptake of fluorescently labeled Hb·Hp aggregates was higher than that of Hb·Hp complexes. Inset, shown is a typical histogram of differentiated THP-1 cells incubated with Hb·Hp complexes (filled purple) or Hb·Hp aggregates (green unfilled) (means ± S.D. from four independent experiments; *, p < 0.05; **, p < 0.01 versus Hb; ##, p < 0.01 versus Hb·Hp). C, confocal microscopy of THP-1 cells incubated with fluorescently labeled Hb, Hb·Hp complexes, or Hb·Hp aggregates. Inset, shown is a typical photomicrograph of THP-1 cells with engulfed Hb·Hp aggregates (green). Blue, staining of nuclei with Hoechst 33342. The images were obtained using an Olympus Fluoview 1000 confocal microscope with DP25 digital camera. Magnification was 600×, and the aperture was 150 μm. For quantification, three fields were randomly chosen (150 cells in each field) (means ± S.D. from three independent experiments; ***, p < 0.001 versus Hb; ###, p < 0.001 versus Hb·Hp complexes.) D, detection of Hb·Hp aggregates in THP-1 cells by SDS-PAGE. Lane i, naïve THP-1 cells; lane ii, THP-1 cells incubated with Hb·Hp aggregates.

FIGURE 7.

Uptake of Hb·Hp aggregates by THP-1 cells is accompanied by increased production of superoxide radicals and oxidative stress. A, superoxide generation by THP-1 cells incubated with free Hb, Hb aggregates, Hb·Hp complexes, or aggregates measured by flow cytometry with dihydroethidium. Inset, shown is a typical histogram of ethidium fluorescence response from control (filled) and Hb·Hp aggregate-loaded (unfilled) THP-1 cells (means ± S.D. from three independent experiments; *, p < 0.05 versus control). B, intracellular GSH levels in THP-1 cells incubated with free Hb, Hb aggregates, Hb·Hp complexes, or aggregates (means ± S.D. from three independent experiments; *, p < 0.05 versus control). C, Western blot analysis of 4-HNE protein adducts in THP-1 cells treated with Hb·Hp aggregates (panel a). Densitometric analysis of anti-4-HNE antibody-positive bands (normalized to amount of actin) (panel b) (*, p < 0.05 versus control). D, cytotoxicity of Hb, Hb·Hp complexes and Hb·Hp aggregates in differentiated THP-1 cells (means ± S.D. from three independent experiments; **, p < 0.001 versus control). MW, molecular mass.

FIGURE 8.

Detection of Hb·Hp aggregates in septic plasma. A, gels after native PAGE (7.5% acrylamide) were stained with West Pico chemiluminescence substrate for peroxidase activity (panel a), Western blot analyses with anti-Hb (panel b), and anti-Hp antibodies (panel c). B, Western blot analyses with anti-Hb (panel a) and anti-Hp (panel b) antibodies after SDS-DTT-PAGE (7.5% acrylamide). The arrows and brackets indicate the positions of high molecular mass aggregates.