Interaction of the chlorite-based drug WF10 and chlorite with hemoglobin, methemoglobin and ferryl hemoglobin

https://doi.org/10.1016/j.abb.2015.09.009Get rights and content

Highlights

  • The chlorite-containing drug WF10 converts oxyhemoglobin into methemoglobin.

  • WF10 (chlorite) inactivates methemoglobin.

  • Ferryl hemoglobin is converted by WF10 into methemoglobin.

  • Chlorite is the active principle of WF10 in all interconversions of hemoglobin forms.

  • Chlorite caused the same spectral changes applying a chlorite concentration as in WF10.

Abstract

The interaction of the chlorite-based drug solution WF10 with human oxyhemoglobin and oxidized hemoglobin forms was investigated monitoring the corresponding spectral changes in heme states. The chlorite component of WF10 converts oxyhemoglobin into methemoglobin with a rate of 35.4 M−1s−1. Methemoglobin is also formed upon the interaction of ferryl hemoglobin and WF10/chlorite. The rate of this interconversion depends on the oxidation state of ferryl hemoglobin. This rate is 114 M−1s−1, when ferryl hemoglobin was generated upon reaction of oxyhemoglobin and hydrogen peroxide. A considerable higher rate (6600 M−1s−1) is measured between the chlorite components of WF10 and ferryl hemoglobin after formation of the latter species from methemoglobin. WF10/chlorite inactivates also methemoglobin as evidenced by the continuous decrease of the Soret band and all other absorbances with a rate of 8.3 M−1s−1. In all interconversions, the chlorite component of WF10 was the active principle as shown in experiments applying pure chlorite at the same concentration as in WF10. Thus, WF10 is able to diminish efficiently the yield of cytotoxic hemoglobin species that might appear after excessive hemolysis of red blood cells under pathologic situations.

Introduction

Binding and transport of dioxygen is realised in our organism by the tetrameric hemoglobin in red blood cells and the monomeric myoglobin in muscle tissue. In each subunit of both proteins, a protoporphyrin IX group is present with a central iron ion that can exist in several redox forms. In both proteins, only heme iron in the ferrous state (Fe2+) is able to bind dioxygen.

Therefore, it is highly important to maintain these hemes in the reduced state. In erythrocytes, this is mainly achieved by methemoglobin reductase converting methemoglobin, which contains ferric (Fe3+) iron, to ferrous hemoglobin. The spontaneous formation of methemoglobin occurs usually at low rate not exciting a methemoglobin yield higher than 1% [1], [2]. Enhanced methemoglobin levels are reported for some gene-associated disorders affecting hemoglobin homeostasis in red blood cells, and after intoxication by redox-active agents like nitrite [3].

Several strong disease scenarios are known to be accompanied by massive intravascular hemolysis [4]. Free hemoglobin is much more prone to oxidation than hemoglobin in red blood cells [4]. Especially nitric oxide converts hemoglobin into methemoglobin with a high rate that reduces the bioavailability of nitric oxide [5], [6], [7]. Ferric iron-protoporphyrin IX, also known as hemin, is easily released from methemoglobin [8], [9]. The hydrophobic hemin inserts into lipoproteins, cell membranes and hydrophobic areas of proteins like albumin [10]. In the presence of hydrogen peroxide hemoglobin, methemoglobin as well as hemin are oxidized to products containing iron in the highly reactive oxo-ferryl state (Fe4+ with an adjacent oxygen atom) and bearing in some cases an additional radical moiety [11]. Oxo-ferryl heme species are potent oxidants towards numerous biological substrates [12].

In plasma, the cytoprotective proteins haptoglobin and hemopexin form stable complexes with free hemoglobin and free hemin, respectively. These complexes are removed from circulating blood via receptor-mediated uptake by macrophages and hepatocytes [10], [13]. Under strong stress situations in combination with excessive hemolysis, these protecting systems are fast exhausted. Then free heme is not efficiently removed from plasma and becomes toxic. Damage of kidney glomeruli, liver toxicity, promotion of oxidative processes in lipoproteins and cell membranes as well as hemolysis of further red blood cells belong to these toxic effects [4], [9], [10], [14], [15]. Under these conditions, heme is also known to act as a damage-associated molecular pattern via toll-like receptor 4 signalling in macrophages [16] and endothelial cells [17].

The chlorite-based immunomodulatory drug solution WF10 and the more diluted form Oxoferin are applied to dampen strong inflammatory states and to improve wound healing processes [18], [19], [20], [21]. However, the mechanism of action of these drugs remains puzzling. WF10 is a ten-fold diluted sterile, pyrogen-free, aqueous solution of the drug substance OXO-K993 (NUVO Research Inc., Mississauga, Canada), also referred as tetrachlorodecaoxygen. WF10 contains 4.25% chlorite (62.9 mM), 1.9% chloride (53 mM), 1.5% chlorate (18 mM), 0.7% sulfate (7.3 mM) and sodium ions as cationic species in an aqueous solution [19].

Recently, it has been found that chlorite reacts readily with the oxo-ferryl states compound I and compound II of the heme proteins myeloperoxidase and lactoperoxidase [22], which are both involved in defence against foreign microorganisms [23], [24]. Similar reactions were observed for horseradish peroxidase [25]. Chlorite also inactivates the resulting ferric state of myeloperoxidase and lactoperoxidase by iron release from heme [22], but not in horseradish peroxidase [25]. Chlorite and WF10 are able to oxidize hemoglobin to methemoglobin [26], [27], [28], [29]. Although a chlorite-induced release of iron from methemoglobin has been mentioned [30], no systematic investigations are known about effects of chlorite and WF10 on oxidized forms of hemoglobin.

We present here a comprehensive investigation of the reactions of chlorite and WF10 with hemoglobin, methemoglobin and ferryl hemoglobin. This work is directed to provide a novel mechanism for the action of WF10 under strong inflammatory conditions that are characterised by the presence of excess free oxidized hemoglobin forms and enhanced levels of hydrogen peroxide. Mechanistic details of reactions of chlorite and WF10 with different forms of hemoglobin are given.

Section snippets

Chemicals

Hemoglobin A0 (ferrous stabilized human), phosphate buffered saline (PBS), hydrogen peroxide solution (30% (w/w) in H2O), potassium ferricyanide (III), potassium cyanide, sodium l-ascorbate, uric acid as well as Sephadex® G-25 were purchased from Sigma–Aldrich, Taufkirchen, Germany. A 10 mM hydrogen peroxide solution was freshly prepared from a stock solution (H2O2, 30%, stored at 4 °C) with Millipore water. H2O2 concentrations were determined using ε240 = 43.6 M−1cm−1 as well as ε230 = 74 M−1cm

Interaction of WF10 with ferrous hemoglobin

Effects of WF10 on oxyhemoglobin were investigated in the first series of experiments. The position of the Soret band maximum at 414 nm and absorbance maxima at 541 nm and 576 nm remained unchanged in PBS-diluted ferrous oxyhemoglobin over at least 20 min at 37 °C (Fig. 1A). The addition of diluted WF10 (1:200, with a final chlorite concentration of 315 μM) caused a gradual shift of the Soret band maximum to 405/406 nm (Fig. 1B) and a decrease of the absorbances at wavelengths higher than

Discussion

The WF10 component chlorite has been shown to react in three different ways with hemoglobin and oxidized heme forms. It oxidizes hemoglobin to methemoglobin as demonstrated by the shift of the absorbance maximum from 414 nm to 406 nm. Secondly, chlorite destroys methemoglobin as evidenced by the continuous decrease of the Soret band around 406 nm and all other absorbance values. And thirdly, it reduces ferryl hemoglobin to methemoglobin as shown by the corresponding shift of the Soret band

Acknowledgement

The authors gratefully acknowledge the financial support provided by the Sächsische Aufbaubank from a funding of the European Regional Development Fund (ERDF) (SAB Project Nr: 100116526) and the German Federal Ministry of Education and Research (BMBF 1315883).

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