Kinetic method for assaying the halogenating activity ...

Free Radical Research, 2015; Early Online: 1–13 © 2015 Informa UK, Ltd. ISSN 1071-5762 print/ISSN 1029-2470 online DOI: 10.3109/10715762.2015.1017478

ORIGINAL ARTICLE

Kinetic method for assaying the halogenating activity of myeloperoxidase based on reaction of celestine blue B with taurine halogenamines A. V. Sokolov1,2,3, V. A. Kostevich1,2, S. O. Kozlov1,3, I. S. Donskyi1, I. I. Vlasova2, A. O. Rudenko1, E. T. Zakharova1, V. B. Vasilyev1,3 & O. M. Panasenko2,4

Free Radic Res Downloaded from informahealthcare.com by 89.223.101.14 on 03/20/15 For personal use only.

1Department of Molecular Genetics, Research Institute of Experimental Medicine, Saint-Petersburg, Russia, 2Department of Biophysics, Research Institute of Physico-Chemical Medicine, Moscow, Russia, 3Department of Fundamental Problems of Medicine and Medical Technology, Saint-Petersburg State University, Saint-Petersburg, Russia, and 4The Russian National Research Medical University named after N.I. Pirogov, Moscow, Russia

Abstract Myeloperoxidase (MPO) is a challenging molecular target which, if put under control, may allow regulating the development of inflammatory reactions associated with oxidative/halogenative stress. In this paper, a new kinetic method for assaying the halogenating activity of MPO is described. The method is based on measuring the rate of iodide-catalyzed oxidation of celestine blue B (CB) by oxygen and taurine N-chloramine (bromamine). The latter is produced in a reaction of taurine with HOCl (HOBr). CB is not a substrate for the peroxidase activity of MPO and does not react with hydrogen peroxide and superoxide anion radical. Taurine N-chloramine (bromamine) reacts with CB in molar ratio of 1:2. Using the new method, we studied the dependence of MPO activity on concentration of substrates and inhibitors. The specificity of MPO inhibition by non-proteolyzed ceruloplasmin is characterized. The inhibition of taurine N-chloramine production by neutrophils and HL-60 cells in the presence of MPO-affecting substances is demonstrated. The new method allows determining the kinetic parameters of MPO halogenating activity and studying its inhibition by various substances, as well as screening for potential inhibitors of the enzyme. Keywords: myeloperoxidase, halogenating activity, celestine blue B, taurine, HOCl, ceruloplasmin Abbreviations: CB, celestine blue B; CP, ceruloplasmin; MCD, monochlorodimedone; MPO, myeloperoxidase; PMA, phorbol-12-myristate-13-acetate; Tau, taurine; TMB, 3,3’,5,5′-tetramethylbenzidine; TNB, 5-thio-2-nitrobenzoic acid

Introduction Myeloperoxidase (MPO, EC 1.7.1.11, donor:H2O2 oxidoreductase) is the enzyme of azurophilic granules of neutrophils, comprising approximately 5% dry weight of the cell. Each monomer of the dimeric MPO consists of the light and the heavy polypeptide chains and a heme covalently bound to the latter. Interaction of H2O2 with MPO heme leads to formation of Compound I, a highly reactive state of MPO heme, which catalyzes the twoelectron oxidation of either chloride or bromide in the socalled “halogenating cycle” with formation of hypochlorous acid (HOCl) or hypobromous acid (HOBr), respectively (Figure 1, 1,2). Their anions are termed “hypochlorite (OClˉ)” and “hypobromite (OBrˉ).” Since pKa of HOCl and HOBr lies within the physiological range of pH, hereinafter, we use the terms “hypochlorite” and “hypobromite” in reference to the mixtures of molecular and ionic forms of the acids which are simultaneously present in the medium at physiological pH (HOCl/OClˉ and HOBr/ OBrˉ). Like halogenides, thiocyanate (SCNˉ) is known to be easily oxidized in the halogenating cycle. Some

substances, which are electron donors, including aromatic compounds, are oxidized by Compound I in the peroxidase cycle of MPO via formation of Compound II at the intermediate stage (Figure 1, 3,3′). Compound II, which is inactive toward halogenides, is also produced when Compound I reacts with an excess of H2O2 (Figure 1, 1′). Formation of hypochlorite, hypobromite, and hypothiocyanite in halogenating cycle underlies the antimicrobial activity of MPO. Along with other mechanisms of innate immunity, it provides the defense of a host organism against pathogenic bacteria. On the other hand, the oxidants produced by MPO can induce an oxidative/halogenative stress that usually accompanies inflammatory diseases [1]. MPO is currently considered as a target for new antiphlogistic drugs, though the list of substances put in a claim for regulators of the enzyme’s activity being relatively short [2,3]. The number of methods used for the measurements of MPO activity is strongly limited by the peculiarities of MPO catalytic cycle and by an extremely high reactivity of MPO-generated oxidants. Data analysis is often difficult and the results are ambiguous.

Correspondence: Alexey V. Sokolov, Research Institute of Experimental Medicine, ul. Pavlova 12, Saint-Petersburg 197376, Russia. Fax: (812)234–9489. E-mail: [email protected] (Received date: 29 September 2014; Accepted date: 6 February 2015; Published online: 19 March 2015)


2 A. V. Sokolov et al.

Figure 1. Principal reactions catalyzed by MPO and methods of their registration. Upon interaction with H2O2 (1), MPO active site transforms into a highly reactive Compound I, and utilization of H2O2 can be registered using electrochemical sensor. Then a two-electron oxidation of Cl by Compound I (2) generates HOCl with simultaneous recovery of the native state of MPO, thus completing the cycle of halogenation. In the presence of peroxidase substrates (RH), sequential one-electron transfer of oxidative equivalents from MPO active site to these substances results in the reduction of Compound I to the native enzyme via formation of Compound II and with generation of the reaction products which are usually colored (R ), and thus peroxidase cycle is complete (3,3′). Being inactive with halogenides, Compound II is also formed when Compound I is exposed to the excess of H2O2 (1′). HOCl produced in halogenating cycle is assayed by measuring a decrease of MCD or NADPH absorbance (4). Chloramine Tau (Tau-Cl), which is a stable product of reaction between taurine (Tau) and HOCl, causes bleaching of TNB (6). It can also oxidize TMB in the presence of I (7) as a catalyst (8). Compounds oxidized by MPO in peroxidase cycle are underlined. Arrows indicate increasing (↑) or decreasing (↓) absorption of target compounds.

×

On account that hydrogen peroxide is a substrate of MPO, measuring its decrease using electrochemical sensors (hydrogen peroxide electrode) seems to be the most adequate assay for MPO activity [3]. However, since H2O2 can be consumed both in the peroxidase and in the halogenating cycles, H2O2 loss in a complex medium containing peroxidase substrates accounts for the total MPO activity. Besides, additional tests are necessary to ensure that oxidants produced by MPO do not affect the stability of H2O2 enzymatic sensors. Another problem is the necessity of specific media for electrochemical measurements. Components of a medium may affect MPO activity. Spectral assays of enzymatic activity are more available and less sophisticated. The measurements of MPO peroxidase activity using spectrophotometer (oxidation of aromatic chromogenic substrates–guaiacol, o-dianisidine, 3,3′,5,5′-tetramethylbenzidine, etc.) or spectrofluorimeter (oxidation of tyrosine) are easy to perform [4–6]. However, the optimum conditions for peroxidase reaction do not coincide with those for halogenating activity of MPO, and peroxidase substrates can modulate the oxidation of halogenides [7]. As shown in our recent study [8], tyrosine, being a substrate of MPO peroxidase reaction, can enhance the enzyme’s chlorinating activity favoring the transition from Compound II to the native MPO and, consequently, to Compound I (Figure 1, 3′ and 1). By now, not much is known about the physiological role of MPO peroxidase activity, whereas the enzyme’s halogenating activity was demonstrated to be responsible for its antimicrobial function and at the same time it can induce non-specific damage of biological molecules, causing pathologic phenomena [1].

Depending on the oxidant that reacts with a chromophore or a fluorophore, spectral assays of MPO halogenating activity may be conventionally classified into two groups, that is, assays in which compounds are oxidized by hypochlorite (Figure 1, 4) and assays based on halogenated amines of taurine (Tau) (Figure 1, 5–8). Halogenated amines of Tau are long-living oxidants formed upon the reaction of Tau with hypochlorite or hypobromite (Figure 1, 5) [9]. Secreted together with MPO during the respiratory burst of neutrophils, Tau is likely to accumulate and transfer MPO oxidative equivalents in the form of Tau halogenamines, thus providing the most effective realization of antimicrobial properties of the enzyme [10,11]. Methods based on oxidation of compounds by hypochlorite include those in which monochlorodimedone (MCD) [12], ascorbic acid [13], or NADPH [14] is used (Figure 1, 4). MCD and ascorbic acid were demonstrated to be substrates of MPO peroxidase cycle, so these reagents cannot be used to measure MPO halogenating activity [15,16]. NADPH is difficult to work with because of the reagent’s instability. Besides, oxidation of all compounds listed above is monitored by measuring the absorbance in UV region, which prevents their use for studying the effects of compounds containing phenolic or heterocyclic groups (including proteins) on MPO activity. Currently available assays for MPO halogenating activity based on Tau halogenamines allow to solely measure the final concentration of halogenites after the arrest of the MPO-catalyzed reaction by sodium azide and catalase. Adding catalase is essential for the elimination of residual H2O2, because color compounds used to quantify Tau halogenamines (such as 3,3′,5,5′-tetramethylbenzidine


(TMB), dihydrorhodamine [17], and 5-thio-2-nitrobenzoic acid (TNB) [18]) may be oxidized in the peroxidase cycle of MPO (Figure 1, underlined). TNB is the most widely used chromophore for the detection of Tau halogenamines accumulation in solutions. Bleaching of TNB depends on pH of a solution, which is a disadvantage of the method. At pH below 8.0 TNB loses color, which impedes detection of MPO activity at acidic pH required for the enzyme to display most of its chlorinating activity. Moreover, thiol-containing compounds are able to restore the color of TNB making the results of measurements unreliable. Iodide was used to catalyze the oxidation of TMB and dihydrorhodamine by halogenated amines of Tau and provided considerable specificity for the detection of MPO halogenating activity. Chlorinated amines interact with Iˉ, which results in formation of ICl. Interaction of ICl with a compound results in the release of iodide which participates in the next catalytic cycle (Figure 1, 7,8). Neither TNB nor TMB can be used to assay the kinetics of MPO halogenating activity, since both substances are substrates of the enzyme’s peroxidase cycle. It is worth noticing that measurements of the end-point amounts of Tau halogenamines produced in the reaction mixture provide no evidence whether the halogenating reaction has reached its plateau, and for that reason kinetic methods should be more informative [15]. Elaboration of a new assay for the kinetic measurements of MPO-induced production of halogenated amines of Tau seems to be topical. First, to develop a kinetic method we had to choose a compound beyond the large spectrum of substrates of MPO peroxidase reaction. Second, absorbance spectrum of a chromogenic compound should lie in the visible region and should be stable at pH between 5.0 and 8.0. Here we describe a kinetic method for assaying the halogenating activity of MPO, based on measuring the rate of iodide-catalyzed celestine blue B (CB; CI 51050) bleaching in the presence of oxygen either by Tau N-bromamine. The experimental conditions and CB absorption coefficient are provided for the measurements of MPO activity at pH between 5.2 and 7.4. Results obtained by the new method were compared with those provided by routine assays of MPO halogenating activity. Kinetic parameters of MPO halogenating activity were determined. The effects of synthetic inhibitors, scavengers of hypohalogenites, as well as the effect of ceruloplasmin (CP), the physiological inhibitor of MPO, on the enzyme’s halogenating activity were studied. The possibility to register production of reactive halogen species by neutrophils and HL-60 cells was demonstrated. Materials and methods Chemicals The following chemicals were used: UNOsphere Q and UNOsphere S, Bio-Gel A-1.5m fine (“Bio-Rad”, USA);

Assay for halogenating activity of MPO 3

NaCl, NaBr, NaSCN, and KI (“Merck”, Germany); NaN3, catalase from bovine liver, Coomassie R-250, 2-mercaptoethanol, ammonium persulfate, and Tris (“Serva”, Germany); KO2, NADPH, NaOCl, albumin from human serum, 6-aminocaproic acid, 4-aminobenzoic acid hydrazide, CB, dapsone, 5,5′-dithiobis(2-nitrobenzoic acid), glycine, L-cysteine, L-methionine, L-tyrosine, MCD, neomycin trisulfate, plasmin, phorbol-12-myristate-13-acetate (PMA), sodium dodecyl sulfate (SDS), salicylhydroxamic acid, Tau, TMB, triethanolamine, phenylmethylsulfonyl fluoride, phenyl-agarose, Sephacryl S-200 HR, xanthine, and xanthine oxidase from bovine milk (“Sigma”, USA); DEAE-Toyopearl 650-M (“Toyosoda”, Japan); acrylamide, N,N′-methylene-bis-acrylamide, and N,N,N′N′tetramethylethylenediamine (“Laboratory MEDIGEN”, Russia); and fibrinogen from human plasma (“OlvexDiagnosticum”, Russia). TNB was obtained after hydrolysis of 2 mM 5, 5′-dithiobis(2-nitrobenzoic acid): 1 M NaOH was added dropwise to achieve pH: 12.0. After 2 min, 0.2 M NaH2PO4 was added dropwise till pH was decreased to 8.0. Water was added to double the total volume of solution, after which the obtained 2 mM TNB was stored in the dark at 4°C. 0.05 M citric acid and 0.1 M Na2HPO4, hereinafter referred to as “sodium-phosphate-citrate buffer” (pH indicated), were used to prepare buffer solutions with pH ranging from 5.0 to 7.6. All solutions were prepared using apyrogenic deionized water with resistivity 18.2 MW·cm. Peptide RPYLKVNPR was obtained by solid-phase synthesis at the Institute of Highly Purified Biopreparations (Saint-Petersburg, Russia). The purity of the peptide was 99.5% as judged by high-performance liquid chromatography (HPLC) and amino acid analysis. Heparin and neomycin were immobilized on BrCN-activated agarose. Protein purification MPO from human leukocytes was purified by successive chromatography on heparin-Sepharose, phenyl-agarose, and Sephacryl S-200 HR. A430/A280 (RZ) of purified MPO was 0.85, which is indicative of the enzyme’s homogeneity [19]. Stable at storage monomeric CP which is stable for storage was purified from human blood plasma by chromatography on UNOsphere Q and neomycin-agarose. Purified CP contained more than 95% of non-proteolyzed protein and had A610/A280  0.050 [20]. Proteolyzed CP was obtained by limited plasmin hydrolysis of intact CP for 1 h at 37°C (CP:plasmin  1:500, w/w). Hydrolysis was stopped by adding 6-aminocaproic acid to the final concentration of 10 mM. About 90% of the prepared proteolyzed CP were fragments with Mr 116 and 19 kDa as judged by SDS-polyacrylamide gel electrophoresis (PAGE). Homogeneous transferrin was isolated from human plasma after the protein’s saturation with Fe3 and successive chromatography on DEAE-Toyopearl, UNOsphere S, and Sephacryl S-200 HR.

4 A. V. Sokolov et al. Spectrophotometry


Absorbance and the rate of enzymatic reactions were registered on a spectrophotometer SF-2000-02 “OKB Spectr” (Saint-Petersburg, Russia). Concentrations of the compounds used in the study were determined using the following extinction coefficients: dimeric MPO – e430  178 000 M 1  cm 1 [21], CP – e610  10 000 M 1  cm 1 [22], H2O2 – e240  43.6 M 1  cm 1 [23], ClOˉ – e292  350 M 1  cm 1 [24], BrOˉ – e329  345 M 1  cm 1 [25], NADPH – e339  6 200 M–1  cm–1 [14], oxidized TMB – e645  30 231 M–1  cm–1 [18], TNB – e412  14 150 M–1  cm–1 [26], and MCD – e290  17 700 M–1  cm–1 [15]. Screening the dyes applicable for a kinetic assay of halogenating activity Water (or 50% water–AcCN, v/v) solutions of dyes (0.05 mM, acridine orange, acridine yellow, alizarin red S, alizarin yellow R, auramine O, auramine OO, aurine, azocarmine G, benzopurpurin, benzyl orange, bromochlorophenol blue, bromocresol green, bromocresol purple, bromophenol blue, bromothymol blue, carmine, CB, chlorophenol red, Congo red, cresol purple, cresol red, crystal violet, curcumin, eriochrome black T, eriochrome red B, Evan’s blue, fuchsine, indigo carmine, Janus green B, malachite green, methylene green, methyl blue, methyl orange, methyl red, methyl violet, methyl yellow, naphthyl red, neutral red, Nile blue, phenol red, Ponceau S, pyronin G, rhodamine B, safranin T, tartrazine, thiazine red, tropaeolin O, tropaeolin OO, tropaeolin OOO, trypaflavine, trypan blue, thionine, thymol blue, victoria blue B, and xylene cyanol) were tested for change in color after adding NaOCl, Tau N-chloramine, and Tau N-bromamine (5–50 mM). The dyes thus selected were tested for susceptibility to oxidation by 5 nM MPO and/or 50 mM H2O2. Nonoxidized dyes were selected and tested for stability against bleaching upon addition of 1 mg of KO2 (per 10 ml of 0.05 mM dye) and of the system containing 50 mM xanthine and 0.05 U/ml of xanthine oxidase in 50 mM K-phosphate buffer, pH: 7.4. Studying the mechanisms of celestine blue B interaction with reactive halogen species The starting CB and the product of its reaction with NaOCl, KI, and Tau were purified by reverse-phase HPLC on a Luna C18 column (water–AcCN gradient 0→60% was used for elution). Electrospray ionization mass spectra and tandem mass spectrometry (MS/MS) spectra of purified substances were obtained on a Bruker Maxis 4G Q/TOF. To determine rate constants for the reaction of CB with NaOCl (in the presence and absence of Tau and KI), the stepwise injection analysis (SWIA) was used [27]. Briefly, the core device for carrying out SWIA was flow injection analyzer PIAKON-30-1 (Rosanalit, Saint-Petersburg, Russia). It was supplemented with a bidirectional peristaltic pump ensuring the reverse flow, a six-port titanium

valve, a 50-mm optical Z-flow cell, and connecting tubes (PTFE, i.d. 0.5 mm). A tungsten lamp and a USB650 UV–VIS fiber optical CCD detector (OceanOptics, USA) were fitted to the flow system. Mixtures of 15–60 mM CB and 7.5–30 mM NaOCl (with and without Tau and KI) were injected into the optical cell, and the rate of A650 decrease was registered. The data obtained were used to calculate rate constants. Detection of MPO chlorinating activity Chlorinating activity of MPO was measured using TNB. MPO at different concentrations was added to a mixture of 100 mM NaCl, 10 mM Tau, and 1–5 nM MPO in Naphosphate-citrate buffers, at pH: 5.8 and 7.4. Reaction was triggered by H2O2 (the final concentration, 50 mM). It was stopped 5 min later by catalase (20 mg/ml). 0.2 M Naphosphate buffer (pH: 8.0) was added to neutralize the medium when the enzyme’s activity was measured at pH: 5.8. Test tubes with mixtures were placed on ice and 5 min later 50 mM TNB was added. A412 was measured 5 min after sample incubation in the dark, and the rate of hypochlorite production was calculated. Chlorinating activity of MPO was also assayed using MCD or NADPH in a mixture consisting of 100 mM NaCl, 100 mM MCD, or NADPH and 2–20 nM MPO in Na-phosphate-citrate buffers, at pH: 5.8 and 7.4. Reaction was triggered by H2O2 (the final concentration, 50 mM). Reaction rate (ΔA290/min or ΔA339/min) was measured and the rate of hypochlorite production was calculated. Assaying the H2O2 utilization rate The rate of H2O2 utilization in MPO-catalyzed reactions was determined using planar sensors (kindly granted by “RUSENS”, Russia; www.rusens.com), the active principle of which is nanofilm of Prussian blue [28]. The facility contained a microcell (300 mL) with planar sensor, a magnetic stirrer for continuous mixing, and a potentiostat P-8 “Elins” (Chernogolovka, Russia) for recording the current. The latter is proportional to H2O2 concentration. After the injection of H2O2 (50 mM) into the microcell containing 100 mM KCl, 10 mM K-phosphate buffer, pH: 5.8 and 7.4, and 10 mM Tau, we registered the initial current. Then the MPO-catalyzed reaction was triggered by MPO addition (to the final concentration of 1–5 nM) and subsequent changes of the current were recorded. The rate of H2O2 loss was calculated from the linear part of the kinetic curve. Development of the CB-based method The best experimental conditions for CB-based assay of halogenating activity of MPO were selected from a large number of combinations when 200 mM of CB was added to a variety of solutions containing Na-phosphate-citrate buffers at pH: 5.0–7.8, NaCl (0–200 mM), NaBr (0–5 mM), H2O2 (0–150 mM), KI (0–50 mM), Tau (0–10 mM), and MPO (0–10 nM). Reaction was triggered by adding H2O2



or MPO, and then ΔA650/min was monitored to calculate the rate of hypohalogenite formation using coefficient 15 200 M 1  cm 1 on account that 1 mole of oxidant reacts with two moles of CB (see Results). Turnover number for MPO was calculated as the ratio between reaction rate and concentration of MPO. To determine IC50 for different compounds, a graph reflecting the reaction rate dependence on logarithm of molar concentration of a compound was plotted. Km and kcat for chloride, bromide, and H2O2 were calculated by Hanes–Woolf linearization. When MPO was inhibited by CP, Ki was calculated as [CP]/(kcat/kcat′-1). Water solution of CB (4 mM, 0.45 mm filtered) was stored in vitrum nigro. Its maximum absorbance changed neither upon five-day storage at 4°C nor during the five hours of experiments run at room temperature. Tau N-chloramine production by neutrophils and HL-60 cells Cells HL-60 were cultured in suspension culture flacons in RPMI-1640 medium with 10% fetal calf serum, 1% L-glutamine, 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid–NaOH (pH: 7.4), 50 U/ml of penicillin, and 100 mM streptomycin in humidified air (5% CO2 at 37°С). Suspension of 1.5  106 cells per ml was diluted 1:10 with the same medium containing 1.5% dimethylsulfoxide to trigger differentiation, and then cultured for five days. Differentiated cells were twice washed with Hanks’ solution without Ca2 and Mg2. Neutrophils were isolated from freshly taken blood of healthy donors using sodium citrate as anticoagulant [29]. Cells were isolated after sedimentation of erythrocytes by Ficoll– Hypaque (r  1.078 g/ml) centrifugation, followed by hypotonic lysis of red blood cells and washing the neutrophils twice with Hanks’ solution containing neither Ca2 nor Mg2. Finally, neutrophils or differentiated HL-60 cells (5  105 and 106 cells per ml) were resuspended in 10 mM phosphate buffer at pH: 7.4, containing 140 mM NaCl, 10 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2, 1 mg/ ml glucose, and 20 mM Tau. Effect of dapsone (4 mM), 4-aminobenzoic acid hydrazide (4 mM), and CP (2 mM) on reactive halogen species production by cells (5  105 and 106 cells per ml) activated by PMA (100 nM) was studied. Cells in 24-well plates containing various supplements were incubated in a thermal shaker (37°C) for 60 min. Reaction was stopped by adding catalase (2 nM) and placement of 24-well plates on ice for at least 10 min. Then cells were pelleted by centrifugation (10 min, 5000 g, 4°C). The supernatant (200 ml) was mixed with 50 ml of 0.5 mM CB; 0.5 mM CB and 25 mM KI; 0.5 mM TMB; 0.5 mM TMB and 25 mM KI or 0.5 mM TNB in 96-well plates. A650 (CB and TMB) and A412 (TNB) were registered on a plate reader (CLARIOstar, BMG LABTECH, Germany). Tau N-chloramine production by cells was calculated using calibration plots reflecting dependence between A650 (A412 in case of TNB) and NaOCl (2–40 mM) added in 96-well plates with 10 mM phosphate buffer at pH: 7.4, containing 140 mM NaCl, 10 mM KCl, 1 mM

CaCl2, 0.5 mM MgCl2, 1 mg/ml glucose, 20 mM Tau and 100 mM CB (with/without 5 mM KI), 100 mM TMB (with/ without 5 mM KI), or 100 mM TNB. Statistical analysis Experiments were repeated three times (n  3) and the mean values were calculated as Хm  (1/n)ΣXi, where Хi is a value of each following sample. The standard error was expressed as S*/n, where S* 

∑ ( X X i

(n1)

m

)2

,

and the confidence interval was calculated as Xm(S*/ n1/2)tn-1,1-a/2, for which t was found in the table of values under condition that is stated in our experiments a  0.05. Results Screening the dyes for applicability in halogenating activity kinetic assay Having tested more than 50 dyes for bleaching in reaction with NaOCl, Tau N-chloramine, and Tau N-bromamine, we chose celestine blue B (CB; CI 51050) routinely used in microscopy, since it was the only compound that remained stable in the presence of H2O2, MPO–H2O2, MPO–H2O2–NaSCN, KO2, and xanthine/xanthine oxidase (Figure 2A). Celestine blue B oxidation by reactive halogen species Water solutions of CB have a characteristic absorption spectrum with the maximum at 650 nm (Figure 2B). Upon adding either hypochlorite or hypobromite, absorbance at 650 nm decreased with simultaneous increase of a less intensive absorption peak at 520 nm, which can be attributed to the pink product of CB oxidation (Figure 2B). The isosbestic point between these two spectra was at 540 nm. Plotting the dependence of ΔA650 on concentration of NaOCl allowed us to calculate a coefficient (Figure 2C), which, in turn, allows estimating the amount of HOCl in solution, on account that 2 moles of CB are oxidized by 1 mole of HOCl. The coefficient was equal to 15 200 M 1  cm 1. Performing the reaction at various pH values we found out that the dependence of ΔA650 on NaOCl concentration was not pH sensitive (Figure 2C). It can be concluded that MPO halogenating activity (HOCl production) can be assayed in the broad range of pH (from 5.0 to 7.4) if CB is used in reaction. Furthermore, no clearcut distinctions in the effect of various reactive halogen species on the absorbance of CB at 650 nm were observed at different pH values. The results were similar to that presented in Figure 2B, 2C when CB in solution was treated with hypobromite, Tau N-chloramine, or Tau



Figure 2. Reactivity of CB toward reactive oxygen and halogen species. (A) A650 of CB solution (50 mM) measured 30 min after adding 50 mM H2O2, NaOCl, NaOBr, Tau N-chloramine, or Tau N-bromamine, KO2 (0.1 mg/ml), a mixture containing 50 mM xanthine and 0.05 U/ ml xanthine oxidase (X/XO) in 50 mM K-phosphate buffer at pH: 7.4; and a mixture containing 5 nM MPO, 50 mM H2O2, 5 mM NaCl, NaBr, or NaSCN, in 50 mM Na-phosphate buffer at pH: 5.5. (B) Absorption spectra of CB (200 mM) at different NaOCl concentrations (0–90.9 mM). Arrows indicate the trend of absorbance changes at 520 and 650 nm; optical pathway, 5 mm. (C) Dependence of A650 on concentration of NaOCl added to CB solution (200 mM) (calibration curve). Data of five independent measurements in Na-phosphate-citrate buffer (рН: 5.0, 5.6, 6.2, 6.8, and 7.4) are summarized. (D) Dependence of A650 on concentration of NaOBr, Tau N-chloramine, or Tau N-bromamine added to CB solution (200 mM) (calibration curve) in Na-phosphate-citrate buffer (рН: 5.6).


N-bromamine (Figure 2D). It can be concluded that all the studied compounds react with CB in a similar manner. In the presence of Tau, the rate of CB bleaching by hypochlorite (hypobromite) slowed down, but adding 5 mM KI brought the rate of CB oxidation back to the level when only hypochlorite (or hypobromite) was added (Figure 3A). Using SWIA we calculated the rate constants for reaction between CB and NaOCl both in the absence and presence of Tau and KI. Those were 25300  700 M 1  s 1 and 119000  2000 M 1  s 1, respectively. Obviously, when Tau and KI are added to the reaction mixture, NaOCl-induced oxidation of CB is accelerated several times. Nitrogen bubbling through CB solution before adding NaOCl completely prevented CB oxidation. However, the dye was rapidly oxidized when oxygen access was restored (Figure 3B). This experiment unequivocally demonstrated that CB oxidation by NaOCl is an oxygen-dependent process. Next, we employed MS analysis to identify the product of CB oxidation (Figure 3C). Mass spectrum of CB showed a peak of 328.1317 Da, which corresponds to its calculated mass (С17H18N3O4, precision 1.7 ppm). The product of CB oxidation has m/z of 362.1346 Da (Figure 3C, 1). Measurement of MS/MS spectrum allowed us to suggest that the increment results from addition of two hydroxyls to the aromatic ring of CB (С17H20N3O6, precision 0.9 ppm, Figure 3C, Table I).


Tentative scheme of CB oxidation by reactive halogen species and oxygen is presented in Figure 3D. 1 mole of HOCl is reduced by 2 moles of CB to water and chloride: H  HOCl  2eˉ  Clˉ  H2O (Figure 3D, reaction 1). The cation radical formed in the aromatic ring of CB is oxidized by dissolved oxygen to an unstable epoxide (Figure 3D, reaction 2) that is hydrolyzed to glycol in water solution (Figure 3D, reaction 3). We can speculate that adding KI to the reaction mixture resulted in formation of ICl: HOCl  Iˉ  H  ICl  H2O. This product accelerates oxidation of 2 moles of CB, and the overall reaction is accelerated. As a result, chloride and iodide are formed: ICl  2eˉ  Iˉ  Clˉ. Regenerated iodide serves as a catalyst for subsequent reactions. Application of CB for measurements of MPO halogenating activity The fundamental requirement for CB to be used as a dye for measuring the MPO halogenating activity is its stability against oxidation in the MPO peroxidase cycle. Indeed, in the absence of chloride or bromide in a mixture of 5 nM MPO, 200 mM CB, 500 mM Tau, 5 mM KI, and 50 mM H2O2 in Na-phosphate-citrate buffer (pH: 5.8 and 7.0), CB absorbance did not change. However, when chloride or bromide was added to the reaction mixture, the rate of CB bleaching was linearly dependent on MPO concentration (Figure 4A). At pH: 5.8, the enzyme turnover numbers

Figure 3. CB oxidation by reactive halogen species. (A) Time dependence of A650 changes in CB solution (200 mM) upon adding NaOCl or NaOBr (50 mM) in the presence of Tau (2 mM) with/without 5 mM KI. (B) Time dependence of A650 changes in CB solution (200 mM) upon adding NaOCl (50 mM): (1) nitrogen bubbling through CB solution before adding NaOCl, (2) after providing oxygen access. (C) Mass spectra (1, 2) and MS/MS spectra (3, 4) of the initial CB (2, 4) and oxidized CB (1, 3). (D) Tentative scheme of reactions of CB with HOCl (ICl) (reaction 1) with subsequent oxidation by oxygen (reaction 2) and hydrolysis of unstable epoxide in water solution (reaction 3).

8 A. V. Sokolov et al. Table I. Analysis of MS/MS spectra of CB glycol. Formula

Precision, Δm, ppm

362.1346 345.1086 327.0988 316.0715 301.1179

C17H20 N3O6 C17H17 N2O6 C17H15 N2O5 C15H12 N2O6 C16H17 N2O4

0.3 1.4 3.7 8.1 1.2

271.1073 261.1237 243.1118 231.1138 215.1166

C15H15 N2O3 C14H17 N2O3 C14H15 N2O2 C13H15N2O2 C13H15N2O

1.1 1.2 3.9 4.5 6.2


Ion mass, Da

Neutral loss – NH3 Н2О and NH3 NH3 and C2H4 H2O and CHNO (O  C  NH)

obtained for chloride and bromide were 94.7  1.4 s 1 and 67.5  1.1 s 1, respectively. These values are close to those presented in the literature [14,30,31]. Concentration optimum for KI as the catalyst was 5 mM (Figure 4B). Increasing Tau concentrations from 0.5 to 4.0 mM did not cause any noticeable changes in the reaction rate (Figure 4C).

Therefore, in the system studied, MPO was the enzyme producing Tau N-halogenamines, and concentrations of KI and Tau chosen for experiments provided an efficient transfer of oxidative equivalents to CB. The dependence of MPO activity on H2O2 concentration had extreme character (Figure 4D). That dependence was described earlier [32]. Its extreme character results from the enzyme’s transition at concentrations of H2O2 exceeding 100 mM to Compound II which cannot accomplish oxidation of halogenides (Figure 1, 1′). When 100 mM tyrosine, which is a peroxidase substrate, was added to MPO in the presence of 100 mM H2O2 and 100 mM NaCl at pH: 7.4, the turnover number increased from 2.5 s 1 to 8 s 1. Plots illustrating the dependence of MPO activity on chloride or bromide concentration give evidence of a stronger affinity of the enzyme for bromide (Figure 4E). Observation of pH dependence of MPO activity is in good agreement with the data of other authors who showed that the optimum of MPO activity with chloride lies in the range of pH: 5.6–6.2, while that for reaction with bromide is between pH of 5.0 and 6.2 [14,30,31]. It should be

Figure 4. Dependence of reaction rate (A) and turnover number (B-F) on concentrations of MPO (A), KI (B), Tau (C), H2O2 (D), NaBr, or NaCl (E), and on pH of Na-phosphate-citrate buffer (F) in the course of MPO-catalyzed formation of Tau N-chloramines (1) and Tau N-bromamines (2). Unless specified otherwise, reaction mixture contained 200 mM CB, 5 nM MPO, 5 mM KI, 500 mM Tau, 50 mM H2O2, 100 mM NaCl (1), or 4 mM NaBr (2), and Na-phosphate-citrate buffer at pH: 5.8.


noticed that at neutral pH, MPO displays higher activity in reaction with bromide (Figure 4F). To verify the reliability of data obtained by the new method, we compared those with the results obtained by routine methods at pH: 5.8 and 7.4. Besides, H2O2 utilization in the presence of MPO was measured by electrochemical sensor, which was an independent method (Table II). This comparative study provided very close values of enzyme turnover number for all methods in which Tau was used.


Effect of various compounds on halogenating activity of MPO Table III summarizes the inhibitory effects (the half maximum inhibitory concentration: IC50) of various compounds on MPO activity estimated using the proposed CB assay. Among those compounds were synthetic inhibitors (NaN3, 4-aminobenzoic acid hydrazide, dapsone, and salicylhydroxamic acid), scavengers of H2O2 (catalase) and of hypochlorite (methionine and cysteine), a substrate of halogenating cycle (SCNˉ), CP which is the physiological inhibitor of MPO, and synthetic peptide RPYLKVNPR having the sequence of amino acid stretch (883–892) between domains 5 and 6 of CP. IC50 of synthetic inhibitors lies in the range 4–55 mM (Table III). Virtually the same effect was observed for scavengers of hypochlorite, that is, cysteine and methionine. Inhibitory effect of thiocyanate is likely to result from inability of a product formed in the reaction between hypothiocyanate and Tau to oxidize CB. Indeed, CB absorbance did not change in the presence of 50–200 mM NaSCN in a mixture of 5 nM MPO, 200 mM CB, 500 mM Tau, 5 mM KI, and 50 mM H2O2 in Na-phosphate-citrate buffer (pH: 5.8 and 7.0). The strongest inhibition of MPO was achieved when either non-proteolyzed CP or the peptide structurally identical to the stretch of amino acids 883–892 linking domains 5 and 6 in CP were used. In contrast, inhibitory effect of proteolyzed CP was very low and similar to that of some plasma proteins, namely albumin, fibrinogen, and transferrin (Figure 5). Hence, our data on CP effect on kinetics of MPO chlorinating and brominating activity allow regarding CP as a non-competitive inhibitor of MPO, which causes no changes in Km for chloride, bromide, and H2O2 (Table IV).

Table III. IC50 values calculated for various inhibitors of MPO chlorinating activity in Na-phosphate-citrate buffer at pH: 6.0, containing 200 mM CB, 100 mM NaCl, 50 mM H2O2, 2 mM Tau, 5 mM KI, and 5 nM MPO. Compound

IC50, mM

NaN3 Salicylhydroxamic acid Dapsone 4-Aminobenzoic acid hydrazide Catalase Methionine Cysteine NaSCN CP (intact) Peptide RPYLKVNPR

55  9 21  8 7  2 4  1 0.001  0.0005 43  13 27  6 25  2 0.35  0.09 0.58  0.11

Tau N-chloramine registered upon activation of neutrophils and differentiated HL-60 cells Effect of PMA on Tau N-chloramine production by neutrophils and differentiated HL-60 cells was studied in experiments with CB, TMB, and TNB. Figure 6A presents evidence that the three targets showed similar results in Tau N-chloramine production by both types of MPOcontaining cells. CB can be used to study the effects of anti-inflammatory agents and of MPO inhibitors on Tau N-chloramine production by neutrophils and differentiated HL-60 cells. In our experiments, dapsone, 4-aminobenzoic acid hydrazide, and CP diminished production of Tau N-chloramine by PMA-activated neutrophils and differentiated HL-60 cells (Figure 6B). Discussion Kinetic measurements of MPO halogenating activity is an important issue in every study concerning the MPO catalytic mechanisms and inhibitor analysis. In this study, we used CB to obtain a new method of measuring the MPO halogenating activity both in enzyme solutions and in suspensions of MPO-containing cells. High sensitivity and specificity of the assay were provided by adding Tau and KI to the reaction mixture. Dependences of the MPO halogenating activity on concentration of the reagents presented in Figure 4 allow suggesting the optimal composition of the reaction mixture for measuring the

Table II. MPO chlorinating activity assayed at pH: 5.8 and 7.4 using various methods. Target for HOCl/Tau N-chloramine (reaction mixture contained 100 mM NaCl and 50 mM H2O2 in Na-phosphate-citrate buffer) 200 mM CB, 2 mM Tau, 5 mM KI, and 1–10 nM MPO 50 mM TNB, 10 mM Tau, and 1–5 nM MPO 100 mM monochlorodimedone and 2–20 nM MPO 100 mM NADPH and 2–20 nM MPO 100 mM 3,3′,5,5′-tetramethylbenzidine, 10 mM Tau, 50 mM KI, and 1–5 nM MPO H2O2 utilization measured with electrochemical sensor, 10 mM Tau, and 1–5 nM MPO* * 100 mM KCl in potassium-phosphate buffer.

Turnover, s 1 (pH 5.8)

(pH 7.4)

94.7  1.4 88.1  2.8 72.3  2.9 79.2  3.0 81.8  2.5 89.9  5.2

5.5  0.2 3.2  0.5 1.7  0.2 2.7  0.3 3.8  0.4 4.9  0.5



Figure 5. MPO turnover as detected from Tau N-chloramine formation in the presence of proteins (2 mM). Reaction mixture contained 200 mM CB, 5 nM MPO, 5 mM KI, 2 mM Tau, 50 mM H2O2, 100 mM NaCl, and Na-phosphate-citrate buffer at pH: 6.2. Control – without proteins. TF, transferrin; FI, fibrinogen; HSA, human serum albumin; CP, ceruloplasmin; CPpr, proteolyzed CP.

activity, that is, 5 mM KI, 2 mM Tau, 50 mM H2O2, and 200 mM CB. Usage of KI to catalyze CB oxidation is authorized by the fact that the rate constant for the reaction of iodide with Tau N-halogenamines is somewhat higher than that of iodide oxidation in the halogenating cycle of MPO. Control experiments showed that oxidation of iodide in halogenating cycle [33] does not occur before its concentration exceeds 1 mM (Km about 2.5 mM). The results obtained in this study demonstrate a number of advantages of the new method. Firstly, our kinetic method allows online monitoring of MPO activity. In contrast, methods with TMB and TNB require the reaction arrest before adding these reagents. This advantage comes from the fact that CB, contrary to TMB and TNB, is not a substrate of the peroxidase cycle of MPO. For instance, in NaCl- or NaBr-free medium CB was not oxidized by MPO and H2O2 (Figures 2A, 4E). Secondly, the maximum of CB absorption spectrum lies in the visible region and is not changed within a broad range of pH. This advantage allows visual control of the reaction and facilitates studying the dependence of MPO activity on pH. Besides, it allows measuring microamounts of MPO, since the majority of micro-plate spectrophotometers are supplied with filters for 630–650 nm.

These advantages can be used to register the effects of different substances on Tau N-chloramine production by activated neutrophils and differentiated HL-60 cells containing MPO. Thirdly, the rate of CB bleaching is the same for chloride and bromide, which allows measuring MPO activity toward any of the two halogen species. This is the crucial distinction of our proposed method from the one based on oxidation of TMB, since the latter allows revealing Tau N-bromamine without adding KI [18]. The grеat advantage of CB-based assay is the possibility to monitor the kinetics of HOCl production in suspensions of activated neutrophils or HL-60 cells. Fluorescent probe, aminophenyl fluorescein, allows measuring HOCl and HOBr in neutrophils and eosinophils [34], but using this probe requires the flow-cytometry techniques. Among limitations of the new method, one can number the relative instability of CB solutions under direct light, which requires their storage in the dark. Besides, when high concentrations of CP were used in the experiment with sodium-citrate-free medium, CB bleaching was detected even without adding H2O2 (data not shown). However, we observed no significant oxidation of CB when reaction was run in sodium citrate-phosphate buffer, which can be explained by inhibition of CP oxidase activity by citrate ions [35]. A study of the effects of metalcontaining enzymes and oxidants on MPO halogenating activity requires additional control experiments to measure the rate of A650 decrease in a medium containing neither MPO nor KI. In our study, production of Tau N-chloramine by activated cells was estimated by comparing CB bleaching in samples with and without KI. 200 mM CB added to the reaction mixture allows measuring not more than 100 mM of hypochlorite (hypobromite). The same concentration limit for hypochlorite detection was observed when other traditional methods were used to assay MPO halogenating activity. Concentrations of H2O2 in the studies of MPO halogenating activity are usually below100 mM [12–18]. Since the concentration of oxygen in solution is above 100 mM, CB oxidation is not restricted. MS/MS analysis (Table I) of the ion with mass of 362.1346, which supposedly corresponds to CB glycol (Figure 3D), showed that the aromatic system of CB,

Table IV. Kinetic parameters of MPO chlorinating activity in the absence or presence of CP calculated for chloride, bromide, and H2O2. Substrate (concentration of second substrate) NaCl (50 mM H2O2) NaCl (50 mM H2O2) H2O2 (150 mM NaCl) H2O2 (150 mM NaCl) NaBr (50 mM H2O2) NaBr (50 mM H2O2) H2O2 (4 mM NaBr) H2O2 (4 mM NaBr)

CP, mM

Km, mM

kcat, s 1

kcat/Km, s 1  mM 1

Ki, mM

0 1.2 0 1.2 0 1.2 0 1.2

152  3 156  2 0.0087  0.0005 0.0113  0.0007 3.6  0.2 3.8  0.2 0.0056  0.0004 0.0061  0.0005

132  2 68  2 67  2 39  2 73  2 35  2 72  3 39  2

0.87 0.44 7701 3451 20 9 12857 6393

– 1.28  0.09 – 1.67  0.08 – 1.11  0.07 – 1.42  0.09

Reaction mixture contained 200 mM CB, 2 mM Tau, 5 mM KI, and 5 nM MPO in Na-phosphate-citrate buffer at pH 6.2. NaCl (NaBr) and H2O2 concentrations varied.



Figure 6. Formation of Tau N-chloramine upon incubation of neutrophils (gray bars) and differentiated HL-60 cells (white bars) (5  105 and 106 cells per ml) upon PMA stimulation (100 nM), 60 min of incubation. (A) Measurement of oxidation of CB, TMB (expressed as the difference between the samples with and without KI), and TNB. (B) Effect of dapsone (4 mM), 4-aminobenzoic acid hydrazide (ABAH, 4 mM), and CP (2 mM) on Tau N-chloramine production by PMA-activated cells (106 cells per ml) measuring by oxidation of CB, expressed as the difference between the samples with and without KI.

containing two auxiliary hydroxyls, remains unchanged upon fragmentation of the ion and the loss of neutral groups (Н2О, C2H4, CHNO, and NH3). The suggestion that glycol is formed upon oxidation of CB is supported by the notion that CP, having a polyphenol oxidase activity, is also able to oxidize CB. Comparison of the measurements of MPO chlorinating activity by our method with those made according to routine protocols revealed that the highest activity of MPO was determined using CB (Table II). When Tau was used as the primary acceptor of MPO-produced oxidants, the values of MPO activity obtained by all methods were close. When a decrease in H2O2 concentration was assayed by means of electrochemical sensor, MPO activity in the absence of Tau dropped 10 times a few seconds after the beginning of the reaction. This is an evidence of inactivation of the enzyme by HOCl produced (data not shown). Not a single method used revealed the high chlorinating

activity of MPO at pH: 7.4. Meanwhile, some evidence of turnover values of about 80 s 1 obtained at neutral pH either with electrochemical sensor [31] or in reaction with NADPH [14] has been published. In our experiments, the CB-based method, the protocol with NADPH, and the assay using electrochemical sensor provided very close values of the enzyme turnover at pH: 7.4. It seems likely that previously published dependences of MPO activity on pH differ from those presented in our paper due to some differences in protocols applied for MPO isolation. MPO used in our experiments was isolated without cationic detergent (cetyltrimethylammonium bromide) routinely applied for extraction of MPO from leukocytes. The drop in MPO activity at neutral pH possibly results from higher lability of the detergent-free enzyme used in this study. Otherwise that phenomenon can be connected with an increase in the yield of Compound II in the reaction of MPO with H2O2 [14].


12 A. V. Sokolov et al. Routinely used synthetic inhibitors of MPO (NaN3, 4-aminobenzoic acid hydrazide, dapsone, and salicylhydroxamic acid) when applied for CB method inhibited the enzyme’s activity at concentrations of 5–100 mM (Table III). These values are in line with the data published in literature [2,3], thus confirming that MPO is the key component of reaction, needed for CB bleaching. The pronounced inhibitory effect of catalase, which scavenges H2O2, results from the high specific activity of the enzyme that deprives MPO of its substrate. Likewise, methionine and cysteine, the scavengers of hypochlorite, strongly diminished CB bleaching when added at concentration close to the expected level of hypochlorite production. This is in line with the notion that the rate constant for reaction of CB with NaOCl in the presence of Tau and KI (1.2105 M 1  s 1) is lower than that in the reaction of hypochlorite with Cys and Met, while the rate at which other amino acid residues react with hypochlorite is slower than that at which the latter reacts with CB. Interestingly, the value of IC50 for thiocyanate corresponds to half of H2O2 concentration used (Table III). The same effect was observed for other concentrations of H2O2 (data not shown), which is probably associated with successful competition of SCNˉ with Clˉ for the active center in MPO, while a compound produced as a result of the reaction of Tau with hypothiocyanate does not oxidize CB. We applied the new CB-based assay to study MPO inhibition by proteins. Plasma proteins, such as albumin, transferrin, and fibrinogen, displayed insignificant capacity to inhibit MPO. Equally, low inhibitory activity was demonstrated by proteolyzed CP that contained no intact 132-kDa protein as judged by SDS-PAGE (Figure 5). This phenomenon can be attributed to the presence in protein molecules of Met and Cys residues competing with Tau for hypochlorite. Non-proteolyzed CP, however, was six times more efficient as MPO inhibitor, though it contains the same number of amino acid residues as its proteolyzed species (Figure 5). The same results were obtained when we used TNB-based method for the measurement of inhibitory effects of CP on MPO [4,5]. This property of CP can be explained by direct contact of proteolysis-sensitive loop (a.a. 883–892) connecting domains 5 and 6 of CP with the heme pocket in MPO, which is a part of its active center [36]. Synthetic peptide RPYLKVNPR identical to the stretch of amino acids 883–892 was an efficient inhibitor of MPO, its activity being only two times lower than that of intact CP (Table III). Studying of the effect of CP on kinetic parameters of MPO halogenating activity revealed no competition of CP with substrates. Indeed, no changes in Km were observed with any of the substrates tested, though kcat for each of them decreased about two times (Table IV). It can be concluded that CP is a non-competitive inhibitor of MPO halogenating activity [37]. For all substrates studied, the ratio kcat/Km characterizing the specificity of an enzyme went down to about half of its initial level in the presence of CP. We have shown previously that CP is a successful competitor of “large” substrates of MPO, such as TMB, and strongly suppresses their oxidation in the peroxidase

cycle, but it is a weak inhibitor of “small” substrates’ oxidation (guaiacol) [5]. Taking into account these results, the absence of competition for MPO active site between CP and “small” chloride, bromide, and H2O2 is not surprising. In the case of hydrogen peroxide, CP also inhibits MPO peroxidase activity in a non-competitive manner [5]. A decrease of kcat in the presence of CP seems to be related to its ability to favor transformation of MPO active forms (Compounds I and II) into the native form of the enzyme. Similar conclusion was drawn in a recent paper dedicated to the interpretation of a mechanism by which CP inhibits MPO [38]. The proposed method allows studying kinetic parameters of halogenating activity of MPO and mechanisms by which it is inhibited by various compounds, and screening a broad spectrum of potential MPO inhibitors both in MPO solutions and in suspensions of MPO-containing cells. The latter possibility seems promising as it can help in finding an efficient control over MPO activity in case of inflammation, usually associated with oxidative/halogenative stress. Acknowledgments The authors are grateful to Professor V. N. Kokryakov (Institute of Experimental Medicine, St. Petersburg) for granted materials, valuable advice, and constructive discussions; to Professor A.A. Karyakin and Ph.D. A.V. Borisova (“RUSENS”) for granted H2O2-sensors. Estimation of rate constants was done with kind assistance of Dr. A.V. Bulatov (Department of Analytical Chemistry, Faculty of Chemistry, Saint-Petersburg State University). This study was supported by RFBR grants No. 13-0401186, 14-04-00807, 14-04-90007, and 15-04-03620, and by the RAMS Program “Human Proteome”. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. References [1] Panasenko OM, Gorudko IV, Sokolov AV. Hypochlorous acid as a precursor of free radicals in living systems. Biochemistry (Moscow) 2013;78:1466–1489. [2] Malle E, Furtmüller PG, Sattler W, Obinger C. Myeloperoxidase: a target for new drug development? Br J Pharmacol 2007;152:838–854. [3] Kettle AJ, Winterbourn CC. Mechanism of inhibition of myeloperoxidase by anti-inflammatory drugs. Biochem Pharmacol 1991;41:1485–1492. [4] Panasenko OM, Chekanov AV, Vlasova II, Sokolov AV, Ageeva KV, Pulina MO, Cherkalina OS, Vasil’ev VB. Influence of ceruloplasmin and lactoferrin on the chlorination activity of leukocyte myeloperoxidase assayed by chemiluminescence. Biophysics 2008;53:268–272.


[5] Sokolov AV, Ageeva KV, Pulina MO, Tcherkalina OS, Samygina VR, Vlasova II, et al. Ceruloplasmin and myeloperoxidase in complex affect the enzymatic properties of each other. Free Radic Res 2008;42:989–998. [6] Gorudko IV, Cherkalina OS, Sokolov AV, Pulina MO, Zakharova ET, Vasil’ev VB, Cherenkevich SN, Panasenko OM. New approaches to the measurement of the concentration and peroxidase activity of myeloperoxidase in human blood plasma. Bioorg Khim 2009;35:629–639. [7] Vlasova II, Arnhold J, Osipov AN, Panasenko OM. pHdependent regulation of myeloperoxidase activity. Biochemistry (Mosc) 2006;71:667–677. [8] Vlasova II, Sokolov AV, Arnhold J. The free amino acid tyrosine enhances the chlorinating activity of human myeloperoxidase. J Inorg Biochem 2012;106:76–83. [9] Green TR, Fellman JH, Eicher AL, Pratt KL. Antioxidant role and subcellular location of hypotaurine and taurine in human neutrophils. Biochim Biophys Acta 1991;1073:91–97. [10] Weiss SJ, Klein R, Slivka A, Wei M. Chlorination of taurine by human neutrophils. Evidence for hypochlorous acid generation. J Clin Invest 1982;70:598–607. [11] Stapleton PP, O’Flaherty L, Redmond HP, Bouchier-Hayes DJ. Host defense–a role for the amino acid taurine? J Parenter Enteral Nutr 1998;22:42–48. [12] Kettle AJ, Winterbourn CC. The mechanism of myeloperoxidase-dependent chlorination of monochlorodimedon. Biochim Biophys Acta 1988;957:185–191. [13] Chesney JA, Mahoney JR Jr, Eaton JW. A spectrophotometric assay for chlorine-containing compounds. Anal Biochem 1991;196:262–266. [14] Auchère F, Capeillère-Blandin C. NADPH as a co-substrate for studies of the chlorinating activity of myeloperoxidase. Biochem J 1999;343:603–613. [15] Kettle AJ, Winterbourn CC. Assays for the chlorination activity of myeloperoxidase. Methods Enzymol 1994;233: 502–512. [16] Flemmig J, Arnhold J. Interaction of hypochlorous acid and myeloperoxidase with phosphatidylserine in the presence of ammonium ions. J Inorg Biochem 2010;104:759–764. [17] Ching TL, de Jong J, Bast A. A method for screening hypochlorous acid scavengers by inhibition of the oxidation of 5-thio2-nitrobenzoic acid: application to anti-asthmatic drugs. Anal Biochem 1994;218:377–381. [18] Dypbukt JM, Bishop C, Brooks WM, Thong B, Eriksson H, Kettle AJ. A sensitive and selective assay for chloramine production by myeloperoxidase. Free Radic Biol Med 2005;39:1468–1477. [19] Sokolov AV, Pulina MO, Ageeva KV, Ayrapetov MI, Berlov MN, Volgin GN, et al. Interaction of ceruloplasmin, lactoferrin, and myeloperoxidase. Biochemistry (Mosc) 2007; 72:409–415. [20] Sokolov AV, Kostevich VA, Romanico DN, Zakharova ET, Vasilyev VB. Two-stage method for purification of ceruloplasmin based on its interaction with neomycin. Biochemistry (Mosc) 2012;77:631–638. [21] Bakkenist AR, Wever R, Vulsma T, Plat H, van Gelder BF. Isolation procedure and some properties of myeloperoxidase from human leucocytes. Biochim Biophys Acta 1978;524:45–54.

Assay for halogenating activity of MPO 13 [22] Noyer M, Dwulet FE, Hao YL, Putnam FW. Purification and characterization of undegraded human ceruloplasmin. Anal Biochem 1980;102:450–458. [23] Beers RF Jr, Sizer IW. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem 1952;195:133–140. [24] Morris JC. The acid ionization constant of HOCl from 5° to 35°. J Phys Chem 1966;70:3798–3805. [25] Gazda M, Margerum DW. Reactions of monochloramine with Br2, Br3-, HOBr, and OBr-: formation of bromochloramines. Inorg Chem 1994;33:118–123. [26] Eyer P, Worek F, Kiderlen D, Sinko G, Stuglin A, SimeonRudolf V, Reiner E. Molar absorption coefficients for the reduced Ellman reagent: reassessment. Anal Biochem 2003;312:224–227. [27] Bulatov AV, Petrova AV, Vishnikin AB, Moskvin AL, Moskvin LN. Stepwise injection spectrophotometric determination of epinephrine. Talanta 2012;96:62–67. [28] Borisova AV, Karyakina EE, Cosnier S, Karyakin AA. Current-free deposition of prussian blue with organic polymers: Towards improved stability and mass production of the advanced hydrogen peroxide transducer. Electroanalysis 2009;21:409–414. [29] Sokolov AV, Golenkina EA, Kostevich VA, Vasilyev VB, Sud’ina GF. Interaction of ceruloplasmin and 5-lipoxygenase. Biochemistry (Mosc) 2010;75:1464–1469. [30] Senthilmohan R, Kettle AJ. Bromination and chlorination reactions of myeloperoxidase at physiological concentrations of bromide and chloride. Arch Biochem Biophys 2006;445: 235–244. [31] van Dalen CJ, Whitehouse MW, Winterbourn CC, Kettle AJ. Thiocyanate and chloride as competing substrates for myeloperoxidase. Biochem J 1997;327:487–492. [32] Furtmüller PG, Burner U, Jantschko W, Regelsberger G, Obinger C. Two-electron reduction and one-electron oxidation of organic hydroperoxides by human myeloperoxidase. FEBS Lett 2000;484:139–143. [33] Furtmüller PG, Burner U, Obinger C. Reaction of myeloperoxidase compound I with chloride, bromide, iodide, and thiocyanate. Biochemistry 1998;37:17923–17930. [34] Flemmig J, Zschaler J, Remmler J, Arnhold J. The fluoresceinderived dye aminophenyl fluorescein is a suitable tool to detect hypobromous acid (HOBr)-producing activity in eosinophils. J Biol Chem 2012;287:27913–27923. [35] Pribyl T. Serum polyphenol oxidase activity (ceruloplasmin) in conventional laboratory animals and man. Folia Biol (Praha) 1978;24:136–141. [36] Samygina VR, Sokolov AV, Bourenkov G, Petoukhov MV, Pulina MO, Zakharova ET, et al. Ceruloplasmin: macromolecular assemblies with iron-containing acute phase proteins. PLoS One 2013;8:e67145. [37] Sokolov AV, Zakahrova ET, Kostevich VA, Samygina VR, Vasilyev VB. Lactoferrin, myeloperoxidase, and ceruloplasmin: complementary gearwheels cranking physiological and pathological processes. Biometals 2014;27:815–828. [38] Chapman AL, Mocatta TJ, Shiva S, Seidel A, Chen B, Khalilova I, et al. Ceruloplasmin is an endogenous inhibitor of myeloperoxidase. J Biol Chem 2013;288:6465–6477.