Biochemistry and Cell Biology
Capacity of ceruloplasmin to scavenge products of the respiratory burst of neutrophils is not altered by the products of reactions catalyzed by myeloperoxidase
Journal: Biochemistry and Cell Biology
Manuscript ID bcb-2017-0277.R1
Manuscript Type: Article
Date Submitted by the Author: 11-Dec-2017
Complete List of Authors: Sokolov, Alexey; FSBSI «Institute of Experimental Medicine»; Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical BiologicalDraft Agency; Saint-Petersburg State University; Centre of Preclinical Translational Research, Almazov National Medical Research Centre Kostevich, Valeria A.; FSBSI «Institute of Experimental Medicine»; Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency Varfolomeeva, Elena; National Research Centre "Kurchatov Institute" B.P. Konstantinov Petersburg Nuclear Physics Institute Grigorieva, Daria V.; Belarusian State University, Department of Biophysics Gorudko, Irina V.; Belarusian State University, Department of Biophysics Kozlov, Stanislav; FSBSI «Institute of Experimental Medicine» Kudryavtsev, Igor V.; FSBSI «Institute of Experimental Medicine»; Far Eastern Federal University Mikhalchik, Elena; Federal Research and Clinical Center of Physical- Chemical Medicine of Federal Medical Biological Agency Filatov, Michael; National Research Centre "Kurchatov Institute" B.P. Konstantinov Petersburg Nuclear Physics Institute Cherenkevich, Sergey N.; Belarusian State University, Department of Biophysics Panasenko, Oleg M.; Federal Research and Clinical Center of Physical- Chemical Medicine of Federal Medical Biological Agency; Pirogov Russian National Research Medical University Arnhold, Juergen ; Institute for Medical Physics and Biophysics, University of Leipzig Vasilyev, Vadim B.; FSBSI «Institute of Experimental Medicine»; Saint- Petersburg State University
Is the invited manuscript for consideration in a Special N/A Issue? :
ceruloplasmin, myeloperoxidase, superoxide dismutase, neutrophils, Keyword: respiratory burst
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Draft
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Capacity of ceruloplasmin to scavenge products of the respiratory burst of neutrophils is not altered by the products of reactions catalyzed by myeloperoxidase. Sokolov A.V.a,b,c,d,*, Kostevich V.A.a,b, Varfolomeeva E.Yu.e, Grigorieva D.V.f, Gorudko I.V.f, Kozlov S.O.a, Kudryavtsev I.V.a,g, Mikhalchik E.V.b, Filatov M.V.e, Cherenkevich S.N.f, Panasenko O.M.b,h, Arnhold J.i, Vasilyev V.B.a,c aFSBSI «Institute of Experimental Medicine», Saint Petersburg 197376, Russia bFederal Research and Clinical Center of Physical Chemical Medicine of Federal Medical Biological Agency, Moscow 119435, Russia cSaint Petersburg State University, Saint Petersburg 199034, Russia dCentre of Preclinical Translational Research, Almazov National Medical Research Centre, Saint Petersburg 197371, Russia eNational Research Centre "Kurchatov Institute" B.P. Konstantinov Petersburg Nuclear Physics Institute, Gatchina 188300, Russia fDepartment of Biophysics, Belarusian State University, Minsk 220030, Belarus gFar Eastern Federal University, Vladivostok 690090, Russia hPirogov Russian National ResearchDraft Medical University, Moscow 117997, Russia iInstitute for Medical Physics and Biophysics, Medical Faculty, University of Leipzig, Leipzig 04107, Germany
Abstract Ceruloplasmin (CP) is a copper containing ferroxidase of blood plasma, acting as an acute phase reactant during inflammation. The effect of oxidative modification of CP induced by oxidants produced by myeloperoxidase, such as HOCl, HOBr and HOSCN, on its spectral, enzymatic and anti inflammatory properties was studied. We monitored chemiluminescence of lucigenin and luminal along with fluorescence of hydroethidine and scopoletin to assay the inhibition by CP of the neutrophilic respiratory burst induced by phorbol 12 myristate 13 acetate (PMA) or formyl methionyl leucyl phenylalanine (fMLP). Superoxide dismutase activity of CP and its capacity to reduce the production of oxidants in respiratory burst of neutrophils remained virtually unchanged upon modifications caused by HOCl, HOBr and HOSCN. Meanwhile, the absorption of type I copper ions at 610 nm became reduced along with a drop of the ferroxidase and amino oxidase activities of CP. Likewise its inhibitory effect on halogenating activity of myeloperoxidase was diminished. Sera of either healthy donors or patients with Wilson disease were co incubated with neutrophils from healthy volunteers. In these experiments, we observed a reverse correlation between the content of CP in sera and the rate of hydrogen peroxide production by activated neutrophils. In conclusion, CP is likely to play a role of an anti
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inflammatory factor tempering the neutrophil respiratory burst in the bloodstream despite the MPO mediated oxidative modifications. Keywords: ceruloplasmin, myeloperoxidase, superoxide dismutase, neutrophils, respiratory burst. Abbreviations: ABTS – 2,2' azino di(3 ethylbenzthiazolinesulphonic acid) disodium salt, CB – celestine blue B, CP – ceruloplasmin, cyt с – cytochrome c, D Glc – D glucose, DCFH DA – 2′,7′ dichlorodihydrofluorescein diacetate, DHR – dihydrorhodamine 123, fMLP − formyl methionyl leucyl phenylalanine, GO – glucose oxidase, MPO – myeloperoxidase, PMA – phorbol 12 myristate 13 acetate, p PD – p phenylenediamine, SOD – superoxide dismutase, TEA – triethanolamine.
Introduction One of the effector links of innate immunity is represented by polymorphonuclear leukocytes (neutrophils). The primary response to alien microorganisms is stimulation of neutrophils, which is accompanied by the changes of their form, adhesion, taxis towards the focus of microbial accumulation or of a tissue lesion, Draftphagocytosis of microbes, enhanced oxygen consumption (“respiratory burst”) and degranulation. The latter process is characterized by the flux of enzymes and microbicidal proteins from cytoplasmic granules to the phagosome and also their partial secretion into the extracellular space. In order to restrain a premature activation of neutrophils in the bloodstream, some serum proteins belonging to the acute phase proteins are able to inactivate aggressive components released from activated neutrophils. Under inflammatory conditions, acute phase proteins are additionally released into the circulating
blood. For example, the molar concentration of ceruloplasmin (CP, ferro:O2 oxidoreductase; EC 1.16.3.1), one of the acute phase proteins, raises from 3 µM (basal value) to 10 M (inflammatory value) (Gitlin 1988). However, no unequivocal concept of functions performed by CP in inflammation has been accepted so far, despite its numerous enzymatic and anti inflammatory activities. The distinctive feature of CP is its capacity to oxidize Fe2+ to Fe3+ 2+ (Osaki 1966). Indeed, among all substrates of CP the lowest KM has been determined for Fe . According to the current concept CP and its homologue hephaestin, play an important role in ferroportin mediated export of Fe2+ from enterocytes, hepatocytes and macrophages, and in subsequent incorporation of Fe3+ into transferrin (Musci et al., 2014). Deficiency of the CP gene in humans, manifested as aceruloplasminemia, causes oxidative stress resulting from tissue accumulation of Fe2+ (Vassiliev et al. 2005), yet oxidation of iron is unlikely to be the only role of CP. Enzymatic activities of ferroxidase (Osaki 1966), cuproxidase (Stoj and Kosman 2003), superoxide dismutase (Vasil'ev et al. 1988), glutathione linked peroxidase (Kim and Park 1998)
https://mc06.manuscriptcentral.com/bcb-pubs 2 Biochemistry and Cell Biology Page 4 of 32 and NO oxidase (Shiva et al. 2006) allow CP to prevent production and persistence of oxidants and free radicals. The presence and expression of the CP gene seems essential for survival of neurons in acute phase response to lipopolysaccharide, as injections of the latter to CP knockout mice resulted in iron accumulation and demyelinization with lethal effect (Glezer et al. 2007). In response to infection with Streptococcus pneumoniae synthesis of CP becomes enhanced in endotheliocytes, which suggests its participation in modulation of the blood brain barrier protecting the central nervous system against circulating pathogens and potentially toxic molecules. In case of CP gene knockout mice, this notion is supported by an increased synthesis of P selectin, regulating vascular permeability to neutrophils (Glezer et al. 2007). Such mice do not survive experimental colitis, probably by reason of the absence of anti inflammatory protection normally provided by CP (Bakhautdin et al. 2013). Along with soluble CP secreted by hepatocytes into plasma, its glycophosphoinositol anchored form was found in membranes of a number of cells in neural, immune and other tissues (Salzer et al. 1998; Marques et al. 2012). Synthesis of CP is increased in response to hypoxia, iron deficiency (Mukhopadhyay et al. 2000), copper excess (Martin et al. 2005), and such factors as insulin (Seshadri et al. 2002), thrombinDraft (Yang et al. 2006), estradiol (Voronina and Monakhov 1980), and pro inflammatory cytokines (Mazumder et al. 1997). In the past ten years we described for the first time and partially characterized the high affinity complexes of anionic CP (pI 4.7) with cationic proteins of neutrophilic leukocytes, such as lactoferrin, myeloperoxidase (MPO) (Sokolov et al. 2007a), several serprocidins (elastase, cathepsin G, proteinase 3 and azurocidin) (Sokolov et al. 2007b), and 5 lipoxygenase (Sokolov et al. 2010a). For example, the affinity of CP towards lactoferrin and azurocidin is characterized by Kd ~ 13 nM (Sokolov et al. 2009, 2010b). Both in vitro and in vivo CP can form multimeric complexes including lactoferrin and MPO (Sokolov et al. 2007a; Samygina et al. 2013). Ferroxidase activity of CP becomes enhanced as it forms the complex with lactoferrin (Sokolov et al. 2005a). Its interaction with MPO efficiently inhibits the latter (Segelmark et al. 1997), which has an important physiological significance as the excessive activity of MPO in inflammation provokes halogenative stress via formation of the hypohalous acids HOCl and HOBr (Panasenko et al. 2013). We have shown that inhibition of MPO by CP decreases the pro atherogenic modification of low density lipoproteins (Sokolov et al. 2014a). However, CP does not inhibit the oxidation of thiocyanate by MPO (Sokolov et al. 2014b). Partially proteolyzed CP can inhibit neither chlorinating, nor peroxidase activity of MPO (Panasenko et al. 2008; Sokolov et al. 2008). Stimulation of neutrophils, occurring at phagocytosis in particular, includes activation of NADPH oxidase in their plasma membrane (Segal 2005), and also the arachidonic acid induced
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leukotriene synthesis (Rådmark et al. 2010). The key enzyme of leukotriene synthesis is 5 lipoxygenase that catalyzes the first two reactions converting arachidonic (cis,cis,cis,cis 5,8,11,14 eicosatetraenoic) acid into leukotrienes. In neutrophils, the addition of CP at concentrations higher 10 µg/mL suppressed the activity of 5 lipoxygenase, decreased the phagocytosis of opsonized zymosan and the production of superoxide anion radicals (Sokolov et al. 2010a). As in the case with MPO, no inhibitory effect on 5 lipoxygenase could be achieved if CP was partially proteolyzed (Sokolov et al. 2010a). CP acted as an anti oxidant when added to an in vitro system containing activated neutrophils (Krsek Staples et al. 1993) and monocytes (Betten et al. 2004). Otherwise, low doses of CP can stimulate phagocytosis of neutrophils (Saenko et al. 1994). It is interesting that in patients with localized aggressive periodontitis, whose neutrophils synthesized CP, Fe3+ ions caused an increased production of superoxide anion radicals (Iwata et al. 2009). In our recent paper we demonstrate that increase of CP concentration during pregnancy can decrease the respiratory burst of neutrophils in blood (Varfolomeeva et al. 2016). It is known that CP is damaged by oxidants formed by activated phagocytes (Sharonov et al. 1988; Winyard et al. 1989). However,Draft it remains unknown, how the protective effect of CP is affected by neutrophil derived oxidants. The ability of HOCl, HOBr, and HOSCN to modify amino acid residues in proteins is well known (Hawkins et al. 2003; Pattison et al. 2004; Skaff et al. 2009). Damage of proteins was also documented in reactions with hydrogen peroxide (Sokolov et al. 2012a), superoxide anion radicals and HOCl (Sharonov et al. 1988, 1989). Recently, we demonstrated that albumin modified by products of MPO catalysis (HOCl and HOBr) caused an activation of neutrophils (Mikhal’chik et al. 2013; Gorudko et al. 2014). Therefore, the aim of this study was to investigate the effect of products of reactions catalysed by MPO (HOCl, HOBr, and HOSCN) on enzymatic properties of CP and on the anti inflammatory activity of CP towards activated neutrophils.
Materials and methods Chemicals The following reagents were used: UNOsphere Q, Bio Gel A1.5m gel “Bio Rad” (USA); ammonium persulfate, arginine, Coomassie R 250, D glucose (D Glc), glucose oxidase (GO),
glycerol, mercaptoethanol, NaN3, Tris “Serva” (Germany); 2,2' azino di(3 ethylbenzthiazolinesulphonic acid) disodium salt (ABTS), catalase, celestine blue B (CB), 4 chloro 1 naphtol, cytochrome c (cyt с) (type VI from horse heart), o dianisidine dihydrochloride, 2′,7′ dichlorodihydrofluorescein diacetate (DCFH DA), dihydrorhodamine 123 (DHR), EDTA,
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ethidium bromide, Fe(NH4)2(SO4)2×6H2O, formyl methionyl leucyl phenylalanine (fMLP), horseradish peroxidase type II, lucigenin, luminol, NaBH4, NaOCl, neomycin, phenylmethylsulfonyl fluoride, p phenylenediamine dihydrochloride (p PD), phenyl Sepharose, phorbol 12 myristate 13 acetate (PMA), resazurin, scopoletin, SDS, Sephacryl S 200 HR, superoxide dismutase (SOD), triethanolamine (TEA), xanthine, xanthine oxidase “Sigma” (USA); acrylamide, N,N' methylenebis(acrylamide), N,N,N',N' tetramethylethylenediamine “MEDIGEN Laboratory” (Russia); heparin “SPOFA” (Poland); Dextran T70 “Roth” (Karlsruhe, Germany). Cyanogen bromide was obtained by bromination of KCN in two phase system «water/dichloroethane». Solution of BrCN in dichloroethane was used to activate Sepharose (or agarose) and further to immobilize heparin and neomycin on BrCN activated gel (Sokolov et al. 2005b). Solid phase synthesis of peptide RPYLKVFNPR (corresponding to amino acid 883 892 stretch in CP) was accomplished in the Research Institute of Extra pure Biopreparations (Saint Petersburg). Peptide was 99.5 % pure as judged by results of HPLC and mass spectrometry. Hydroethidine was synthesized at the Saint Petersburg Nuclear Physics Institute by reducing ethidium bromide with NaBH4 (Filatov et al. 1995). To generate HOBr or HOSCN, equal volumes of equimolar solutions (60Draft mM) of NaOCl and NaBr or NaSCN were mixed in water or 10 mM Na phosphate buffer (pH 7.4). Bleaching of 5 thio 2 nitrobenzoic acid was measured to assay the production of oxidants (van Dalen et al., 1997). All solutions were prepared using apyrogenic deionized water with specific resistance 18.2 M ×cm.
Spectrophotometry Optical spectra and the rate of absorption changes were monitored on a SF 2000 02 («OKB Spectr», Russia). Spectrophotometry was also used for concentration measurements, and the –1 –1 following absorbance coefficients were used: ε430=178000 M cm (Bakkenist et al. 1978), CP – –1 –1 –1 –1 – ε610=9780 M cm (Noyer et al. 1980), H2O2 – ε240=43.6 M cm (Beers and Sizer 1952), ClO –1 –1 – –1 –1 – ε292=350 M cm (Morris 1966), BrO – ε329=345 M cm (Gazda and Margerum 1994).
Protein purification
Stable preparation of monomeric CP with A610/A280 > 0.049, containing more than 95 % of 132 kDa protein was obtained by chromatography of blood plasma on UNOsphere Q and neomycin agarose (Sokolov et al. 2012b). Briefly, 1.4 L of plasma (containing 1 mM phenylmethylsulfonyl fluoride and 0.1 EDTA) was loaded on a PBS equilibrated UNO Sphere Q column (30×2.5 сm, flow rate was 10 mL/min). Chromatography was performed using ice cold solutions. Elution was performed at a rate of 2 mL/min with a linear gradient 0→0.5 M NaCl containing 40 mМ TEA HCl (pH 7.4), the total volume was 200 mL. Blue colored fractions
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were pooled, diluted 1:10 with 20 mM TEA HCl (pH 7.4) and loaded at a flow rate of 5 mL/min on the neomycin agarose column (12×2.5 сm) equilibrated with 40 mМ TEA HCl (pH 7.4).
Proteins were eluted from the column by 120 mL of a linear gradient 0→100 mM CaCl2 containing 40 мМ TEA HCl (pH 7.4) using at a flown rate of 2 mL/min. Blue colored fractions were pooled and concentrated in a VivaSpin 20 unit. It was diluted with 18 mL of 40 mМ TEA HCl (pH 7.4), then again concentrated to 2 mL. Activity of purified CP in reaction with Fe2+ was 1 characterized by KM = 57 M and kcat = 0.98 s (Sokolov et al. 2015c). MPO was purified from human leukocytes by chromatography on heparin Sepharose, phenyl Sepharose and gel filtration
on Sephacryl S 200 HR, which yielded a preparation with A430/A280 (RZ) = 0.85 inherent in homogeneous protein (Sokolov et al. 2010c).
Ceruloplasmin modification by HOCl, HOBr and HOSCN 0.1 3 mM HOCl, HOBr, HOSCN in 50 mM Na phosphate buffer, pH 7.4 was added to CP solution (50 M), to the molar ratios HOX : CP = (2 60) : 1, where X– is Cl–, Br– or SCN–. After 30 min of incubation at 37 °С spectral characteristics of CP, its oxidase activities and the effect on the respiratory burst of neutrophilsDraft were analyzed. Oxidative damage of CP was assayed in vitro by monitoring the changes of its oxidase activity in reaction with o dianisidine in polyacrylamide gel after disc electrophoresis (Davis 1964; Owen and Smith 1961). Reaction mixture contained 1 M CP, 10 nM GO, 100 M D Glc and various combinations of 3 nM MPO, 100 mM NaCl, 1 mM NaBr, 200 M NaSCN in 0.1 M citrate sodium phosphate buffer (рН 6.8). Incubation at 37 ºC lasted for 1 h, after which 200 M taurine and 10 nM catalase were added to the samples.
Enzymatic activity Oxidase activity of CP in reaction with p PD was determined as the difference between the oxidation by CP of p PD (0.05 %) in Na acetate buffer, pH 5.5, in the absence and in the
presence of NaN3 (0.05 %), an efficient inhibitor of CP. Incubation at 37 ºС lasted for 1 h, after
which A530 was measured vs. the control aliquot containing NaN3 (Sokolov et al. 2012b). CP concentration in serum samples was assayed by measuring its oxidase activity with p PD in microplates (Varfolomeeva et al., 2016). 10 l of serum or of purified CP (0 4 M) were loaded into the wells of a 96 well plate, followed by 240 l of freshly prepared 0.2 % p PD (m/v)
solution in 0.4 M Na acetate buffer (pH 5.5). The initial A530 was measured immediately using
plate reader ClarioStar (BMG Labtech, Germany). After 30 min of incubation at 37 °C A530 was measured again. Calibration graph was plotted with purified CP concentration on abscissa and 2 A530 on ordinate (R = 0.997) to calculate CP content in tested samples.
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Ferroxidase activity was assayed in reaction of 15 nM CP with 150 M
Fe(NH4)2(SO4)2×6H2O in 0.4 Na acetate buffer, pH 5.5. After 10 min incubation, ferrozin was added to the reaction mixture to the final concentration of 150 M, and A564 was monitored vs. the control aliquot with NaN3 (Pulina et al. 2010). Superoxide dismutase activity of CP was assayed as its capacity to decelerate resazurin reduction by superoxide anion radicals generated in potassium phosphate buffer, pH 7.4, containing 100 nM xanthine oxidase, 150 M xanthine and 50 M resazurin. The rate of resazurin reduction (A604/min) by superoxide anion radicals was measured upon addition of 100, 200 and 400 nM CP. In control experiments, applying the same components except resazurin, the effect of CP on xanthine oxidase activity was evaluated by monitoring urate production
(A295/min). To study an effect of CP and synthetic peptide RPYLKVFNPR (corresponding to the fragment 883 892 of the CP sequence) on oxidation of ABTS by MPO (Sokolov et al., 2015a), we used a mixture of 3 nM MPO, 0.1 mM H2O2 and 1 mM ABTS in 0.1 M sodium acetate buffer, pH 6.0 and various amounts of CP or peptide (50 1600 nM). Draft Isolation of neutrophils Polymorphonuclear leukocytes (neutrophils) were isolated from fresh donor blood containing 1.2 g/l EDTA as follows. Erythrocytes were precipitated by 3 % dextran T 500 in 0.85 % NaCl (40 mL of solution per 100 mL of blood) at room temperature. Leukocyte and platelet enriched plasma was applied on the layer of Ficoll Paque (density 1.077 g/L) in a 50 mL centrifuge tube. Upon centrifugation for 30 min at 500 g in density gradient the precipitate contained granulocyte fraction with admixture of erythrocytes. Red blood cells were lyzed in a hypotonic buffer consisting of 114 mM NH4Cl, 7.5 mM KHCO3 and 100 M EDTA. Granulocytes were twice washed in PBS (1.06 mМ KH2PO4, 155 mМ NaCl, 2.7 mМ KCl, 2.96 mМ Na2HPO4, pH 7,4) and resuspended in D PBS (0.49 mМ MgCl2, 2.7 mМ KCl, 1.15 mМ K2HPO4, 138 mМ NaCl,
9.58 mМ NaH2PO4, pH 7.4) with D Glc (1 g/l). All experiments with neutrophils were accomplished on the day of blood sampling.
Measurements of H2O2 generation by neutrophils using scopoletin
H2O2 production by neutrophils was measured using the scopoletin/peroxidase fluorescent technique (Timoshenko et al. 1998; Gorudko et al. 2011). Briefly, suspension of neutrophils (2 × 6 10 cells/mL in PBS, containing 1 mM CaCl2, 0.5 mM MgCl2) was supplemented with 1 M scopoletin (a fluorescent substrate of peroxidase), 20 g/mL horseradish peroxidase, and 1 mM
NaN3 (catalase and MPO inhibitor) and was challenged with native or modified CP at various
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concentrations to activate plasma membrane NADPH oxidase. For inhibitor studies neutrophils were pre incubated for 5 min at 37 °C with CP (0 4.5 M) or tested serum before the addition of
fMLP. H2O2 mediated oxidation of scopoletin was recorded as a decrease of its fluorescence at 460 nm (excitation was at 350 nm) during 20 min at 37 °C using a SOLAR CM2203 fluorescence spectrometer (SOLAR). The maximal slope of the recorded traces was calculated
and referred to as the rate of H2O2 generation by cells.
• Assay of O2 production by neutrophils using cytochrome c • Production of O2 was continuously recorded by monitoring the SOD inhibitable reduction of cyt с as previously described (Timoshenko et al. 1998). Neutrophils, resuspended in PBS, • containing 1 mM CaCl2, 0.5 mM MgCl2, pH 7.4, were stimulated to produce O2 by adding native or modified CP at various concentrations. The mixtures containing neutrophils (2 × 106 cells/mL) and 75 µM cyt c were incubated at 37 °C with or without SOD (25 µg/mL). The increase of absorption at 550 nm was monitored using a PB2201 spectrophotometer. Neutrophil • • 6 O2 production was finally expressed as nanomoles of O2 per 2 × 10 neutrophils in 15 min. Draft Luminol and lucigenin dependent chemiluminescence MPO and NADPH oxidase dependent oxygenation activity of neutrophils was assayed on the basis of chemiluminescence arising from luminol and lucigenin as the chemoluminigenic used as substrates. The intensity of emission reflects the MPO dependent formation of HOCl and • NADPH oxidase dependent formation of O2 (Panasenko et al. 2016). Briefly, suspension of neutrophils (final concentration 0.4 × 106 cells/mL) was distributed into polystyrene tubes containing Krebs Ringer buffer (pH 7.4), luminol or lucigenin (0.2 mM), which was followed by adding PMA and then native or modified CP (1 6 M). Luminescence response was measured using a single photon luminometer (LKB Wallac Luminometer 1251, Finland).
Monitoring respiratory burst in blood using flow cytometry with hydroethidine Respiratory burst reaction was monitored not later than 8 h after blood sampling. Such prolonged sample storage did not affect the results (Filatov et al. 1995). The samples (1 mL) were put into test tubes containing 40 units of heparin. Prior to incubation an equal volume of PBS was added into a test tube. Finally, hydroethidine was added to achieve the final concentration of 140 g/mL. The reaction was induced by adding 10 ng/mL of PMA and ran for 80 min at 37 °C. A control portion was the same, but without PMA. Fluorescence intensity spread was measured immediately as the reaction is stopped. A standard flow cytometer
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Registering respiratory burst of neutrophils by flow cytometry with DHR and DCFH DA
The effect of native or modified CP (0.2 2 M) on H2O2 production by neutrophils was determined by flow cytometry (Vlasova et al. 2016). Fluorogenic probe DCFH DA or DHR was added to neutrophils (5 × 106 cells/mL) at a concentration of 10 µM. After 45 min incubation with 50 nM PMA samples were diluted 1:20 in PBS for flow cytometer analysis. Fluorescence intensity was measured using a flow cytometer NaviousTM (Beckman Coulter). Ten thousand leukocytes were registered in each sample.
Monitoring the HOCl production by neutrophils Neutrophils (5×105 cells/mL) wereDraft resuspended in 10 mM phosphate buffer, pH 7.4, containing 140 mM NaCl, 10 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2, 1 mg/mL D Glc and 20 mM taurine (Sokolov et al. 2015b). An effect of native or modified CP (0.2 2 M) on HOCl production by cells 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 placing the 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 l) was mixed with 50 l of 0.5 mM CB (with/without 25 M KI) in 96 well plates. A650 was registered on a plate reader (CLARIOstar, BMG LABTECH, Germany). HOCl production by cells was calculated using calibration plots reflecting dependence between A650 and NaOCl (2 40 M) added in 96 well plates with 10 mM phosphate buffer, pH 7.4, containing 140 mM NaCl, 10 mM
KCl, 1 mM CaCl2, 0.5 mM MgCl2, 1 mg/mL D Glc, 20 mM taurine, and 100 M CB (with/without 5 M KI).
Statistical analysis Experiments were repeated at least three times (n ≥ 3) and mean values were calculated as
Хm = (1/n)ΣXi, where Хi is a value of each successive sample. The standard error was expressed
2 ∑ ()− XX mi * * ()n −1 as S /n, where S = , and the confidence interval was calculated as Xm ±
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* 1/2 (S /n )tn 1,1 α/2, for which t was found in the table of values on condition that in our experiments
α = 0.05. General linear regression and Pearson’s correlation were used to evaluate IC50 and the
relationship between CP added to serum and the H2O2 production by activated neutrophils.
Results Oxidative modification of CP by products of MPO catalysis i.e. HOCl, HOBr and HOSCN. In the first series of experiments, myeloperoxidase derived hypo(pseudo)halous acids, important products of activated neutrophils, were incubated with CP to investigate their effects on selected CP functions. Increasing concentrations of HOCl and HOBr, but not of HOSCN, decreased the catalytic activity of CP in oxidation of Fe2+ (Fig. 1a) and p PD (Fig. 1b). In contrast, SOD activity of modified CP slightly grew with increasing concentrations of all tested hypo(pseudo)halous acids as compared to non modified protein (Fig. 1c). Interaction of CP with HOCl, HOBr and HOSCN was also followed by gradual decrease of the absorption at 610 nm, which is peculiar for type I copper ions (Fig. 2). There was a stronger decrease of absorbance at 610 nm in case of HOCl and HOBr, as compared to HOSCN. The increase of absorption in the range Draft300 400 nm upon modification of CP with HOCl and HOBr, but not in case of HOSCN (Fig. 2), results likely from formation of chloramines and bromamines that absorb in this spectral region (Zgliczyński et al. 1971). Treatment of CP with HOSCN did not affect its capacity to inhibit the peroxidase activity of MPO. In contrast, HOCl and HOBr reduced this antioxidant activity of CP (Fig. 3, Table 1). Similar results were obtained using the inhibitory CP peptide RPYLKVFNPR(883 892). Thus, the MPO products HOCl and HOBr are able to disturb the catalytic function of CP by modifying critical amino acids. Effects of HOCl, HOBr and HOSCN, produced in a system containing MPO, D Glc, GO, halogenides and thiocyanate, on oxidase activity of CP were additionally studied (Fig. 4). Thiocyanate protected CP against the damage caused by hypohalous acids (HOCl and HOBr) generated in the catalytic cycle of MPO.
Effect of modified CP on respiratory burst of neutrophils. Next, we studied the effect of CP, modified by hypo(pseudo)halous acids, on the activation of neutrophils. Doses of native CP corresponding to concentrations of this protein in plasma are • known to inhibit the production of O2 during the respiratory burst of neutrophils (Sokolov et al. 2010a). In our experiments the effect of modified CP on the activity of neutrophils was evaluated by monitoring the production of superoxide anion radicals, hydrogen peroxide and hypochlorous acid (Table 2). Regardless of the type of oxidative modification, CP preserves the capacity to
https://mc06.manuscriptcentral.com/bcb-pubs 10 Biochemistry and Cell Biology Page 12 of 32 scavenge oxidants produced during the respiratory burst of neutrophils. For better comparison, the IC50 values were calculated for the inhibition of the respiratory burst by non modified and modified CP. Any method we used to monitor the respiratory burst gave virtually the same value of IC50 either for intact CP or for the protein modified by various oxidants (Table 2). The effect of CP on NADPH oxidase activity of neutrophils can be the result of its capacity to scavenge oxidants. It can be caused also by the changes occurring in transmembrane signaling following the interaction of CP with the cells (Kataoka and Tavassoli 1985). To clarify the issue, we studied the activator induced release of hydrogen peroxide by neutrophils pre incubated with CP. As shown in Fig. 5, we observed no significant difference between the rate of fMLP induced oxidation of scopoletin by neutrophils in the control group and the cells washed after incubation with CP. This result allows suggesting that CP reduces the respiratory burst due to its interaction with oxidants discharged into the medium upon activation of neutrophils. When Cu,Zn SOD was added together with fMLP to the suspension of neutrophils, the rate of scopoletin oxidation increased noticeably (Fig. 5, d). This result favors the notion that fMLP • induced the production of O2 , which is followed by dismutation of the latter to H2O2. The process was almost completely inhibitedDraft by 2 M CP. When CP was added to the suspension of neutrophils, no significant amounts of hydrogen peroxide were formed, in contrast to the effect of Cu,Zn SOD. When fMLP activated neutrophils were washed with PBS to eliminate CP, the release of hydrogen peroxide resumed (Fig 5, c). Abrogation of the inhibitory effect of CP by washing is contrary to the data about the presence of CP receptors on neutrophils (Kataoka and Tavassoli 1985). Hence, apparently the SOD like activity of CP is responsible for the inhibition of the • respiratory burst. The difference between O2 dismutation by Cu,Zn SOD and CP is the end product, which is likely to be water in case of CP, but not hydrogen peroxide as with Cu,Zn SOD.
We observed a reverse correlation between the rate of H2O2 production by activated neutrophils and the concentration of CP in sera of either healthy donors or patients with Wilson disease (r = –0.92), if samples of sera were added to neutrophils prior to activation (Fig. 6). Similar reverse correlation between CP concentration and activation of neutrophils was shown in our recent study of pregnant women’s blood (Varfolomeeva et al. 2016). Flow cytometry with hydroethidine did not show any difference between suppression of the respiratory burst in activated neutrophils by modified and non modified CP (Fig. 7). Thus, CP inhibits the respiratory burst of neutrophils under conditions that are very similar to those in the bloodstream.
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Discussion In late 1980 ies Lyzlova’s group showed that among plasma proteins CP has the strongest • potential as the scavenger of O2 and HOCl that are, respectively, the substrate and the product of reactions catalyzed by MPO (Govorova et al. 1986; Sharonov et al. 1988, 1989). Shortly after that a protein inhibitor of MPO with М ~ 150 kDa was isolated from serum (Yea et al. 1991), but only some years later it was identified as CP (Segelmark et al. 1997). All hypo(pseudo)halous acids used for CP modification react with Cys residues. An increase of the SOD activity of modified CP might result from alteration of its type I copper properties caused by oxidation of Cys residues. It is known that for CP to act as SOD, the enzyme needs only part of its catalytic center, i.e. type II and III of copper ions coordinated by His residues (Vasil'ev et al. 1988). HOCl and HOBr interact also with Met, Tyr and other amino acid residues. HOSCN interacts only with Cys at sufficient rate, but not with other residues (Hawkins et al. 2003; Pattison et al. 2004; Skaff et al. 2009). The changes in oxidase properties of CP are probably due to the preferential targets of HOCl and HOBr, which are Met residues. Properly these residues participate in coordination of type I copper that plays the key role in oxidation of Fe2+, p PD and o dianisidine. ElectrophoreticDraft mobility of modified CP changed (Fig. 4), most likely due to partial oxidation of its disulfide bonds. A small stimulatory effect of HOSCN on p PD oxidase activity of CP might result from the residual HOSCN reacting with p PD (Fig. 2, b).
In our experiments CP efficiently inhibited the production of H2O2 by activated neutrophils, depriving MPO of its substrate. This effect seems to be a prerequisite condition for CP to retain its MPO inhibiting activity performed essentially by non fragmented protein, while hydrogen peroxide is able to destroy the molecule (Sokolov et al. 2012a). The apparent role of CP as a physiological inhibitor of MPO allows suggesting its participation in a number of pathological processes, inflammation being the most vivid. Recently we studied sophisticated interrelations among CP, MPO and thrombin in synovial fluid of rheumatoid arthritis patients and raised again the important issue of CP molecule’s integrity for its functioning (Sokolov et al. 2015c). Yet, many details of that functioning remain unclear, and one of those is the effect of deleterious products formed in inflammation foci on CP. Results of the present study clarify some of such details. CP readily forms complexes with quite a few proteins released into plasma and other fluids (Sokolov et al. 2007a, 2007b, 2010a, 2015c; Samygina et al., 2013; Kostevich et al. 2015; Skarżyńska et al. 2017). Such interactions mostly help a partner protein to accomplish its functions (Sokolov et al. 2005a, 2014b) and can increase the enzymatic activity of CP. However, some interactions with other proteins or non protein molecules can entail partial or complete loss of its functions (Hawkins et al. 2003; Dutra et al. 2005; Sokolov et al., 2015c). This study
https://mc06.manuscriptcentral.com/bcb-pubs 12 Biochemistry and Cell Biology Page 14 of 32 enlarges our knowledge of the ‘safety margins’ for interactions of CP, at least with respect to MPO. It was shown that interaction of C reactive protein with MPO does not prevent CP from efficient inhibition of the latter (Xu et al. 2014). Our study showed that blood plasma, from which CP had been eliminated by immunoprecipitation, has a decreased capacity to inhibit peroxidase activity of MPO, but the latter retains its ability to utilize hydrogen peroxide for oxidation of the more physiological substrate i.e. thiocyanate (Sokolov et al. 2014b). Indeed, thiocyanate oxidation by MPO with the lowest KM and the highest specificity is not suppressed in the presence of CP (van Dalen 1997). In this study we used a model system with GO generating H2O2 to show that brominating and chlorinating reactions catalyzed by MPO decrease the oxidase activity of CP (Fig. 3), while thiocyanate prevents its modification. We also observed that HOCl and HOBr produced by MPO, reduced the activity of CP in oxidation of Fe2+ and p PD caused by, but its SOD like activity slightly increased (Fig. 1), which coincided with the capacity of modified CP to inhibit the respiratory burst in neutrophils (Table 2). Decrease of ferroxidase activity of CP after modification by HOCl and HOBr may affect the iron metabolism. However, the impact ofDraft this observation in hypoferremia during acute inflammation along with the well known effect of hepcidin needs to be further investigated. Neither CP, nor its peptide (883 892) could inhibit MPO, once they were modified by HOCl or HOBr (Fig. 3, Table 1). We showed previously that CP cannot act as an efficient inhibitor of MPO activity in reactions with bromide and thiocyanate (Sokolov et al. 2014b, 2015a). In view that upon treatment with HOBr and HOCl, but not with HOSCN, the capacity of the CP peptide RPYLKVFNPR(883 892) to inhibit MPO peroxidase activity decreased, it can be suggested that bromination and/or chlorination damages an amino acid residue that is crucial for interaction with the active center of MPO. It seems likely that such residue is Tyr885. Apparently, the MPO mediated bromination and chlorination are able to damage an amino acid residue in CP, most likely Tyr885, which is crucial for interaction with the active center of MPO. Indeed, among amino acids of the peptide 883 892 (RPYLKVFNPR) tyrosine has a sufficient high rate constant with HOCl and HOBr (Hawkins et al. 2003; Pattison et al. 2004). The CP peptide RPYLKVFNPR(883 892) displays a strong regulatory effect in PMA activated neutrophils as an inducer of superoxide formation and an inhibitor of leukotriene synthesis (Golenkina et al. 2017). In contrast, mild oxidant HOSCN, the production of which by MPO is not inhibited by CP, caused neither noticeable decrease of any oxidase activity of CP, nor its capacity to inhibit MPO and the respiratory burst of neutrophils. An important conclusion from these results is that CP seems capable of favoring the oxidation by MPO of thiocyanate, but not chloride. As a result,
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hypothiocyanite is formed, which is relatively harmless for human cells, but is efficient as an antibacterial agent (Chandler and Day 2012).
Acknowledgements The authors are grateful to Prof. V.N. Kokryakov (Institute of Experimental Medicine) for the generously provided buffy coat of healthy donors. This study was supported by Russian Scientific Foundation Grant 17 75 30064 (isolation and purification of CP and MPO), Russian Foundation for Basic Research Grants 15 04 03620 (characterization of CP oxidative modification), 17 04 00530 (effect of CP on respiratory burst of neutrophils) and by the RAMS Program «Human Proteome» (plate reader ClarioStar).
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Sokolov, A.V., Ageeva, K.V., Pulina, M.O., Zakharova, E.T., and Vasilyev, V.B. 2009. Effect of lactoferrin on oxidative features of ceruloplasmin. Biometals. 22(3): 521–529. doi:10.1007/s10534 009 9209 4. Sokolov, A.V., Golenkina, E.A., Kostevich, V.A., Vasilyev, V.B., and Sud'ina, G.F. 2010a. Interaction of ceruloplasmin and 5 lipoxygenase. Biochemistry (Moscow). 75(12): 1464–1469. doi:10.1134/S0006297910120072. Sokolov, A.V., Kostevich, V.A., Kozlov, S.O., Donskyi, I.S., Vlasova, I.I., Rudenko, A.O., Zakharova, E.T., Vasilyev, V.B., and Panasenko, O.M. 2015b. Kinetic method for assaying the halogenating activity of myeloperoxidase based on reaction of celestine blue B with taurine halogenamines. Free Radic. Res. 49(6): 777–789. doi:10.3109/10715762.2015.1017478. Sokolov, A.V., Kostevich, V.A., Romanico, D.N., Zakharova, E.T., and Vasilyev, V.B. 2012b. Two stage method for purification of ceruloplasmin based on its interaction with neomycin. Biochemistry (Moscow). 77(6): 631–638. doi:10.1134/S0006297912060107. Sokolov, A.V., Kostevich, V.A., Runova, O.L., Gorudko, I.V., Vasilyev, V.B., Cherenkevich, S.N., and Panasenko, O.M. 2014a. Proatherogenic modification of LDL by surface bound myeloperoxidase. Chem Phys Lipids.Draft 180: 72–80. doi:10.1016/j.chemphyslip.2014.02.006. Sokolov, A.V., Kostevich, V.A., Zakharova, E.T., Samygina, V.R., Panasenko, O.M., and Vasilyev, V.B. 2015a. Interaction of ceruloplasmin with eosinophil peroxidase as compared to its interplay with myeloperoxidase: Reciprocal effect on enzymatic properties. Free Radic. Res. 49(6): 800–811. doi:10.3109/10715762.2015.1005615. Sokolov, A.V., Pulina, M.O., Ageeva, K.V., Ayrapetov, M.I., Berlov, M.N., Volgin, G.N., Markov, A.G., Yablonsky, P.K., Kolodkin, N.I., Zakharova, E.T., and Vasilyev, V.B. 2007a. Interaction of ceruloplasmin, lactoferrin, and myeloperoxidase. Biochemistry (Moscow). 72(4): 409–415. doi:10.1134/S0006297907040074. Sokolov, A.V., Pulina, M.O., Ageeva, K.V., Runova, O.L., Zakharova, E.T., and Vasilyev, V.B. 2007b. Identification of leukocyte cationic proteins that interact with ceruloplasmin. Biochemistry (Moscow). 72(8): 872–877. doi:10.1134/S0006297907080093. Sokolov, A.V., Pulina, M.O., Zakharova, E.T., Shavlovski, M.M., and Vasilyev, V.B. 2005a. Effect of lactoferrin on the ferroxidase activity of ceruloplasmin. Biochemistry (Moscow). 70(9): 1015–1019. doi:10.1007/s10541 005 0218 9. Sokolov, A.V., Solovyov, K.V., Kostevich, V.A., Chekanov, A.V., Pulina, M.O., Zakharova, E.T., Shavlovski, M.M., Panasenko, O.M., and Vasilyev, V.B. 2012a. Protection of ceruloplasmin by lactoferrin against hydroxyl radicals is pH dependent. Biochem. Cell Biol. 90(3): 397–404. doi:10.1139/o2012 004.
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Sokolov, A.V., Zakahrova, E.T., Kostevich, V.A., Samygina, V.R., and Vasilyev, V.B. 2014b. Lactoferrin, myeloperoxidase, and ceruloplasmin: complementary gearwheels cranking physiological and pathological processes. Biometals. 27(5): 815–828. doi:10.1007/s10534 014 9755 2. Sokolov, A.V., Zakharova, E.T., Shavlovskiĭ, M.M., and Vasil'ev, V.B. 2005b. Isolation of stable human ceruloplasmin and its interaction with salmon protamine. Bioorg. Khim. 31(3): 269–279. doi:10.1007/s11171 005 0033 5 Stoj, C., and Kosman, D.J. 2003. Cuprous oxidase activity of yeast Fet3p and human ceruloplasmin: implication for function. FEBS Lett. 554(3): 422–426. doi:10.1016/S0014 5793(03)01218 3. Timoshenko, A.V., Kayser, K., and Gabius, H.J. 1998. Lectin triggered superoxide/H2O2 and granule enzyme release from cells. Meth. Mol. Med. 9: 441–451. doi:10.1385/0 89603 396 1:441. van Dalen, C.J., Whitehouse, M.W., Winterbourn, C.C., and Kettle, A.J. 1997. Thiocyanate and chloride as competing substrates for myeloperoxidase. Biochem. J. 327(2): 487–492. doi:10.1042/bj3270487. Draft Varfolomeeva, E.Y., Semenova, E.V., Sokolov, A.V., Aplin, K.D., Timofeeva, K.E., Vasilyev, V.B., and Filatov, M.V. 2016. Ceruloplasmin decreases respiratory burst reaction during pregnancy. Free Radic. Res. 50(8): 909–919. doi:10.1080/10715762.2016.1197395. Vasil'ev, V.B., Kachurin, A.M., and Soroka, N.V. 1988. Dismutation of superoxide radicals by ceruloplasmin details of the mechanism. Biokhimiia. 53: 2051–2058. Vassiliev, V., Harris, Z.L., and Zatta, P. 2005. Ceruloplasmin in neurodegenerative diseases. Brain Res. Brain Res. Rev. 49(3): 633–640. doi:10.1016/j.brainresrev.2005.03.003. Vlasova, I.I., Mikhalchik, E.V., Barinov, N.A., Kostevich, V.A., Smolina, N.V., Klinov, D.V., and Sokolov, A.V. 2016. Adsorbed plasma proteins modulate the effects of single walled carbon nanotubes on neutrophils in blood. Nanomedicine. 12(6): 1615–1625. doi:10.1016/j.nano.2016.02.012. Voronina, O.V., and Monakhov, N.K. 1980. Estradiol induced formation of the polyribosomal complex synthesizing ceruloplasmin in rats. Biokhimiia. 45(6): 1010–1016. Winyard, P.G., Hider, R.C., Brailsford, S., Drake, A.F., Lunec, J., and Blake, D.R. 1989. Effects of oxidative stress on some physiochemical properties of caeruloplasmin. Biochem. J. 258(2): 435–445. doi:10.1042/bj2580435. Xu, P.C., Li, Z.Y., Yang, X.W., Zhao, M.H., and Chen, M. 2014. Myeloperoxidase influences the complement regulatory function of modified C reactive protein. Innate. Immun. 20(4): 440– 448. doi:10.1177/1753425913508164.
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Legends to figures. Figure 1. Effect of modification of CP (50 M) by HOCl, HOBr and HOSCN on its ferroxidase (a), p PD oxidase (b) and SOD (c) activity. Data are presented as mean value and confidence interval (α=0.05), n=6. The activity of CP before modification was set to 1, * indicates significant differences (α=0.05) between the effect HOCl (HOBr) and HOSCN. Figure 2. Absorption spectra of CP (50 M) after adding HOCl, HOBr and HOSCN (0 600 M). Optical pathway 1 cm, solutions of HOCl, HOBr and HOSCN (0 600 M) were used as the control. Data are presented a mean of 3 curves.
Figure 3. Plots used to calculate values of IC50 (nM) for inhibition of MPO catalyzed peroxidase reaction with ABTS by (a) CP and (b) its peptide (883 892). Data were obtained for non modified inhibitors and those modified by 4 molar (CP peptide) and 20 molar (CP) excess of HOCl, HOBr or HOSCN. Logarithmic scale is used to plot concentrations of CP and its peptide. The activity of MPO before adding CP or CP peptide was set to 1. Figure 4. Disc electrophoresis in polyacrylamide gel of CP (2 g per lane). 1 M of CP was incubated for 1 h at 37 ºC with combinations of 10 nM glucose oxidase (GO) and 0.1 mM of D glucose (D Glc), 3 nM MPO, 100 mMDraft NaCl, 1 mM NaBr, 0.2 mM NaSCN in 0.1 M citrate sodium phosphate buffer (pH 6.8). After incubation 0.2 mM taurine and 10 nM catalase were added. Gel was soaked in o dianisidine to reveal CP oxidase activity. After three repeats the most typical and demonstrable gel was chosen. Figure 5. Effect of CP on hydrogen peroxide production by neutrophils upon stimulation with 100 nM fMLP. a. Typical fluorescence spectrum of scopoletin obtained after activation of
neutrophils in the presence of CP (2 M) and in its absence (control). b. The rate of H2O2 production at various CP concentrations (n=4). c. Scopoletin fluorescence after washing with PBS the suspension of neutrophils from added CP. d. Effects of CP (2 M) and Cu,Zn SOD (25 g/mL) compared.
Figure 6. Relationship between H2O2 production by stimulated neutrophils and the CP concentration in serum added to neutrophil prior stimulation. All measurements were performed in triplicate. Figure 7. Inhibitory effect of native and modified CP (2 M) on the oxidative burst of PMA stimulated neutrophils. Activation of neutrophils was assessed with hydroethidine and expressed as mean fluorescence intensity (a) (n = 5, confidence interval α=0.05) or analysed by flow cytometry (b – f). Typical histograms demonstrate the distribution of hydroethidine fluorescence of neutrophils in the absence of CP (b), or presence of native CP (c), HOCl (d), HOBr (e), or HOSCN (f) modified CP.
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Figure 1. Effect of modification of CP (50 M) by HOCl, HOBr and HOSCN on its ferroxidase (a), p-PD- oxidase (b) and SOD (c) activity. Data are presented as mean value and confidence interval (α=0.05), n=6. The activity of CP before modification was set to 1, * - indicates significant differences (α=0.05) between the effect HOCl (HOBr) and HOSCN.
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Figure 2. Absorption spectra of CP (50 µM) after adding HOCl, HOBr and HOSCN (0 - 600 µM). Optical pathway 1 cm, solutions of HOCl, HOBr and HOSCN (0-600 µM) were used as the control. Data are presented a mean of 3 curves.
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Figure 3. Plots used to calculate values of IC50 (nM) for inhibition of MPO-catalyzed peroxidase reaction with ABTS by (a) CP and (b) its peptide (883-892). Data were obtained for non-modified inhibitors and those modified by 4-molar (CP peptide) and 20-molar (CP) excess of HOCl, HOBr or HOSCN. Logarithmic scale is used to plot concentrations of CP and its peptide. The activity of MPO before adding CP or CP peptide was set to 1.
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Figure 4. Disc-electrophoresis in polyacrylamide gel of CP (2 µg per lane). 1 µM of CP was incubated for 1 h at 37 ºC with combinations of 10 nM glucose oxidase (GO) and 0.1 mM of D-glucose (D-Glc), 3 nM MPO, 100 mM NaCl, 1 mM NaBr, 0.2 mM NaSCN in 0.1 M citrate-sodium-phosphate buffer (pH 6.8). After incubation 0.2 mM taurine and 10 nM catalase were added. Gel was soaked in o-dianisidine to reveal CP oxidase activity. After three repeats theDraft most typical and demonstrable gel was chosen.
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Figure 5. Effect of CP on hydrogen peroxide production by neutrophils upon stimulation with 100 nM fMLP. a. Typical fluorescence spectrum of scopoletin obtained after activation of neutrophils in the presence of CP (2 µM) and in its absence (control). b. The rate of H2O2 production at various CP concentrations (n=4). c. Scopoletin fluorescence after washing with PBS the suspension of neutrophils from added CP. d. Effects of CP (2 µM) and Cu,Zn-SOD (25 µg/mL) compared.
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Figure 6. Relationship between H2O2 production by stimulated neutrophils and the CP concentration in serum added to neutrophil prior stimulation. All measurements were performed in triplicate.
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Figure 7. Inhibitory effect of native and modified CP (2 M) on the oxidative burst of PMA-stimulated neutrophils. Activation of neutrophils was assessed with hydroethidine and expressed as mean fluorescence intensity (a) (n = 5, confidence interval α=0.05) or analysed by flow cytometry (b – f). Typical histograms demonstrate the distribution of hydroethidine fluorescence of neutrophils in the absence of CP (b), or presence of native CP (c), HOCl- (d), HOBr- (e), or HOSCN- (f) modified CP.
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Table 1. Values of IC50 (nM) for inhibition of MPO-catalyzed peroxidase reaction with ABTS by CP and its peptide (883-892). Data were obtained for non-modified inhibitors and those modified by 4-molar (CP peptide) and 20-molar (CP) excess of HOCl, HOBr or HOSCN. MPO inhibitor Non-modified Modified by HOCl HOBr HOSCN CP 38±6 650±40 840±60 49±13 CP peptide (883-892) 158±14 920±60 1260±80 186±18
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Table 2. Values of IC50 ( Μ) for inhibition of by CP of neutrophilic respiratory burst, also upon modification of the enzyme by 20-molar excess of HOCl, HOBr or HOSCN. Oxidant produced by activated CP CP+HOCl CP+HOBr CP+HOSCN neutrophils (method of detection)
• O2 (cytochrome c) 0.17±0.02 0.14±0.02 0.17±0.02 0.19±0.03
• O2 (chemiluminescence of lucigenin) 3.1±0.2 3.2±0.2 3.5±0.3 2.9±0.3
• O2 (flow cytometry with hydroethidine) 1.1±0.06 1.2±0.07 1.1±0.08 1.3±0.09
H2O2 (flow cytometry with DHR) 0.47±0.04 0.52±0.03 0.48±0.04 0.49±0.03
H2O2 (flow cytometry with DCFH) 1.7±0.2 1.5±0.1 1.8±0.2 1.9±0.3
H2O2 (scopoletin) 0.94±0.08 0.86±0.09 0.85±0.10 0.99±0.09
HOCl (chemiluminescence of luminol) 4.3±0.3 4.5±0.2 4.8±0.4 4.1±0.3 HOCl (celestine blue B) Draft0.66±0.03 0.69±0.04 0.68±0.03 0.69±0.04
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