Peroxidase Activity of Hemoglobin·Haptoglobin Complexes

Abstract

As a hemoprotein, Hb, 2 in the presence of oxidizing equivalents such as H 2 O 2 , can act as a peroxidase with very high oxidizing potential (1). In red blood cells, this potentially dangerous activity is strictly regulated by the reducing environment and the lack of oxidizing equivalents. The inadvertently appearing ferric forms of Hb are short-lived, and the hemoprotein is effectively converted into ferro-Hb (deoxyHb) by metHb reductase (2). Normally, less than 2% of total Hb exists in the form of MetHb because the rate of Hb reduction is far greater than its oxidation (2). For stroma-free Hb, however, this intracellular regulation is lost, and the likelihood for Hb to act as a peroxidase is high. This possibility is markedly increased in the course of severe inflammation (e.g. in sepsis) by the generation of superoxide radicals by immune cells. The latter can be spontaneously or catalytically (by extracellular superoxide dismutase) converted into H 2 O 2 , a fuel for Hb peroxidase activity. In line with this, several clinical and experimental investigations have established that lethality in sepsis is increased in the setting of hemolysis (3-5).Circulating haptoglobin (Hp) provides an important endogenous defense against the toxic effects of Hb (6, 7). The major biological function of this abundant plasma protein is binding and recycling of stroma-free Hb via the macrophage CD163 receptor-mediated pathway (8, 9). It has been proposed that Hp possesses antioxidant activity and diminishes oxidative stress induced by stroma-free Hb (10 -12). The antioxidant action of Hp toward Hb has been associated, at least in part, with weakening of its peroxidase activity (10) or preventing oxidation and cross-linking of Hb (7).Previous work has demonstrated that peroxidase activity of different hemoproteins, including Hb, can induce protein self-oxidation leading to covalent cross-linking and aggregation (13-16). Whether these hetero-oligomeric covalent aggregates retain the peroxidase activity is unknown. If the aggregates retain peroxidase activity, they may continue to be a source of oxidative stress both in circulation as well as in phagocytizing cells involved in their clearance such as macrophages.In the current work, we determined the extent to which: (i) Hb peroxidase activity is decreased by binding with Hp; (ii) peroxidase activity of Hb⅐Hp complexes initiates cross-linking into covalent hetero-oligomers; (iii) peroxidase activity of Hb⅐Hp complexes and aggregates utilizes nitric oxide (NO ⅐ ); (iv) Hb⅐Hp aggregates are taken up by macrophages as compared with noncovalent Hb⅐Hp complexes; and (v) the engulfed Hb⅐Hp aggregates induce oxidative stress and cytotoxicity. Here, we report that Hb⅐Hp complexes and aggregates are potent peroxidases capable of inducing oxidative stress in both plasma and macrophages. We further demonstrate the presence of Hb⅐Hp aggregates in septic plasma.

Dysbiosis by neutralizing commensal mediated inhibition of pathobionts

“…Among many other components, blood contains serum, hemin and hemoglobin24 but the hemolytic capacity of pathobionts can also increase their concentration25. It is already described that hemoglobin and hemin have a peroxidase activity172627. P. intermedia and P. gingivalis also generate layers of haems with catalytic activity to degrade the H 2 O 2 using hemin and hemoglobin28.…”

“…It can modulate the composition of oral biofilms by inhibiting or enhancing the growth of certain oral species or by promoting biofilm formation and inhibiting the adhesion of certain species16. Additionally, serum and blood components can have peroxidase activity17. This is of significance since these could interact with the H 2 O 2 produced by commensal bacteria to suppress the outgrowth of pathobionts.…”

Preferential Silent Survival of Intracellular Bacteria in Hemoglobin-Primed Macrophages

“…Redox-active Hb has been reported to aggregate and induce cytotoxicity in macrophages [33]. Since we observed a higher intracellular bacterial count in Hb-primed macrophages (fig.…”

Cell-Free Plasma Hemoglobin and Male Gender Are Risk Factors for Acute Kidney Injury in Low Risk Children Undergoing Cardiopulmonary Bypass

“…PHb’s role in oxidative stress lies, partly, in its peroxidase activity [27] which is influenced by reducing agents such as NO. In the presence of oxidizing agents, Hp bound hemoglobin also has peroxidase activity [27, 28]. Supporting this, PHb correlated with increased lipid peroxidation after CPB in both adults and children [11, 20].…”