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Inactivation of Human Myeloperoxidase by Hydrogen Peroxide

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Inactivation of Human Myeloperoxidase by Hydrogen Peroxide YABBI 6520 No. of Pages 12, Model 5G 14 September 2013 Archives of Biochemistry and Biophysics xxx (2013) xxx–xxx 1 Contents lists available at ScienceDirect Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi 4 5 q 3 Inactivation of human myeloperoxidase by hydrogen peroxide a,b b b a b,⇑ 6 Q1 Martina Paumann-Page , Paul G. Furtmüller , Stefan Hofbauer , Louise N. Paton , Christian Obinger , a 7 Anthony J. Kettle 8 a Centre for Free Radical Research, University of Otago Christchurch, Christchurch, New Zealand 9 b Department of Chemistry, Division of Biochemistry, BOKU – University of Natural Resources and Life Sciences, Muthgasse 18, Vienna, Austria 1110 12 article info abstract 2714 15 Article history: Human myeloperoxidase (MPO) uses hydrogen peroxide generated by the oxidative burst of neutrophils 28 16 Received 18 June 2013 to produce an array of antimicrobial oxidants. During this process MPO is irreversibly inactivated. This 29 17 and in revised form 2 September 2013 study focused on the unknown role of hydrogen peroxide in this process. When treated with low concen- 30 18 Available online xxxx trations of H2O2 in the absence of reducing substrates, there was a rapid loss of up to 35% of its peroxidase 31 activity. Inactivation is proposed to occur via oxidation reactions of Compound I with the prosthetic 32 19 Keywords: group or amino acid residues. At higher concentrations hydrogen peroxide acts as a suicide substrate 33 20 Myeloperoxidase with a rate constant of inactivation of 3.9 Â 10À3 sÀ1. Treatment of MPO with high H O concentrations 34 21 Neutrophils 2 2 resulted in complete inactivation, Compound III formation, destruction of the heme groups, release of 35 22 Oxidative stress 23 Suicide inhibitor their iron, and detachment of the small polypeptide chain of MPO. Ten of the protein’s methionine res- 36 24 Mechanism-based inhibition idues were oxidized and the thermal stability of the protein decreased. Inactivation by high concentra- 37 25 Oxidative modification tions of H2O2 is proposed to occur via the generation of reactive oxidants when H2O2 reacts with 38 26 Compound III. These mechanisms of inactivation may occur inside neutrophil phagosomes when reduc- 39 ing substrates for MPO become limiting and could be exploited when designing pharmacological 40 inhibitors. 41 Ó 2013 The Authors. Published by Elsevier Inc. All rights reserved. 42 43 44 45 Introduction hypothiocyanite. These oxidants kill ingested bacteria but are also 55 implicated in tissue damage associated with numerous inflamma- 56 tory diseases [2]. It has been demonstrated that during phagocyto- 57 46 Neutrophils are the predominant white blood cells in circula- sis the amount of extractable neutrophilic MPO decreases while a 58 47 tion. They are highly specialized for their primary function, the significant fraction of the soluble enzyme is inactivated [3,4]. The 59 48 phagocytosis and destruction of invading pathogens by antimicro- mechanism by which this occurs and its relevance to infection 60 49 bial proteins and reactive oxidants [1]. When stimulated, and inflammation have yet to be investigated in detail. 61 50 neutrophils consume oxygen in a respiratory burst that produces Enzyme inactivation is likely to involve reactions with either 62 51 superoxide and hydrogen peroxide [1]. Simultaneously, these hydrogen peroxide or its products because they have the poten- 63 52 white blood cells discharge the abundant heme enzyme myeloper- 1 tial to oxidatively modify amino acids and the prosthetic heme 64 53 oxidase (MPO) that uses hydrogen peroxide to oxidize chloride, group. It has been demonstrated with several heme peroxidases, 65 54 bromide and thiocyanate to the respective hypohalous acids and including horseradish peroxidase [5,6], ascorbate peroxidase [7] 66 and lactoperoxidase [8], that hydrogen peroxide alone can pro- 67 mote irreversible inactivation. There are also reports that MPO 68 q This is an open-access article distributed under the terms of the Creative is inactivated by hydrogen peroxide in the absence of exogenous 69 Commons Attribution-NonCommercial-No Derivative Works License, which per- 70 mits non-commercial use, distribution, and reproduction in any medium, provided electron donors [9,10]. However, the mechanism of inactivation the original author and source are credited. and the effect on its structural integrity have not been 71 ⇑ Corresponding author. Address: Department of Chemistry at BOKU – University investigated. 72 of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria. Fax: In this study we aimed to show how hydrogen peroxide affects 73 +43 1 47654 6059. the activity of MPO and describe the mechanisms involved in en- 74 E-mail address: [email protected] (C. Obinger). 75 1 Abbreviations used: MPO, myeloperoxidase; Por, porphyrin; TMB, 3,30,5,50-tetra- zyme inactivation. We demonstrate that MPO is highly sensitive methylbenzidine; DTPA, diethylenetriaminepentaacetic acid; cetrimide, alkyltrimeth- to inactivation by hydrogen peroxide. Irreversible inactivation of 76 ylammonium bromide; FOX, ferrous oxidation in xylenol orange; PVDF, MPO was accompanied by changes in its secondary and tertiary 77 polyvinylidene difluoride; CID, collision induced dissociation; ECD, electronic circular architecture including breakage of covalent heme linkages and 78 dichroism; DSC, differential scanning calorimetry; GdnHCl, guanidinium disruption of its subunit structure. 79 hydrochloride. 0003-9861/$ - see front matter Ó 2013 The Authors. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.abb.2013.09.004 Please cite this article in press as: M. Paumann-Page et al., Arch. Biochem. Biophys. (2013), http://dx.doi.org/10.1016/j.abb.2013.09.004 YABBI 6520 No. of Pages 12, Model 5G 14 September 2013 2 M. Paumann-Page et al. / Archives of Biochemistry and Biophysics xxx (2013) xxx–xxx 80 Materials and methods and glucose (1 mg/ml) produced hydrogen peroxide in 50 mM 139 phosphate buffer, pH 7.4. The flux of hydrogen peroxide was linear 140 81 Reagents over 60 min and increasing concentrations of glucose oxidase (0.1– 141 2.5 lg/mL glucose oxidase) gave a linear increase in production of 142 82 Human MPO (lyophilized and highly purified, Reinheitszahl hydrogen peroxide (9.4 lM–285.5 lM per h). 143 83 0.84) was obtained from Planta Natural Products (http:// 84 www.planta.at) and the concentration was determined spectro- À1 85 photometrically with a molar extinction coefficient of 91,000 M Spectral analysis of myeloperoxidase 144 86 cmÀ1 per heme [11]. Hydrogen peroxide (30% analytical grade) 87 was purchased from Biolab (Aust) Ltd. and concentrations were Spectra of 1.5 lM MPO in 50 mM phosphate buffer, pH 7.4, con- 145 88 determined spectrophotometrically using a molar extinction coef- taining 100 lM DTPA were recorded after reactions were started 146 À1 À1 0 0 89 ficient of 43.6 M cm at 240 nm [12]. TMB (3,3 ,5,5 -tetrameth- by adding hydrogen peroxide. To determine how peroxidase or 147 90 ylbenzidine), xylenol orange, D-sorbitol, halogenation substrates influenced the degradation of the heme 148 91 diethylenetriaminepentaacetic acid (DTPA), alkyltrimethylammo- groups of MPO, 200 lM ascorbate or 5 mM bromide plus, 5 mM 149 92 nium bromide (cetrimide), glucose, catalase from bovine liver, bo- methionine, respectively were also added as indicated below. The 150 93 vine superoxide dismutase, glucose oxidase from Aspergillus niger, involvement of superoxide was checked by adding 20 lg/mL SOD 151 0 94 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p -disulfonic acid to a separate set of reactions. 152 95 monosodium salt hydrate (ferrozine) and L-ascorbate were pur- 96 chased from Sigma. Dimethylformamide and ferrous ammonium 97 sulfate were from J.T. Baker. SDS–PAGE analyses 153 98 All spectrophotometric assays were performed on an Agilent 99 8453 diode array spectrophotometer. For enhanced chemilumines- To follow the impact of hydrogen peroxide on the structural 154 100 cence Amersham™ ECL Plus Western Blotting Detection System integrity of myeloperoxidase, 1.5 lM MPO in 50 mM sodium phos- 155 101 from GE Healthcare was used. phate buffer, pH 7.4, and 100 lM DTPA was incubated with 1.5 mM 156 hydrogen peroxide. Aliquots were taken at 5, 10, 25, 40 and 60 min 157 102 Myeloperoxidase activity assays and residual hydrogen peroxide was removed by adding 20 lg/ml 158 catalase. Samples of 15 lL were added to non-reducing and reduc- 159 103 The residual peroxidase activity of MPO was determined by ing sample loading buffers, respectively (final concentrations: 2% 160 104 measuring its ability to oxidize TMB. MPO (32 nM) in 50 mM so- SDS, 10% glycerol, 125 mM Tris–HCl buffer, pH 6.5, and for reduc- 161 105 dium phosphate buffer, pH 7.4, and 100 M DTPA was incubated ing SDS–PAGE 1% b-mercaptoethanol). Samples were loaded with- 162 106 at various protein to hydrogen peroxide ratios and aliquots were out prior heating and resolved by 8–20% gradient SDS– 163 107 taken over time to assess the decline in peroxidase activity. Super- polyacrylamide gel electrophoresis. Gels were stained with Coo- 164 108 oxide dismutase was added to a separate number of experiments massie Brilliant Blue R-250 for 60 min and subsequently destained. 165 109 at a concentration of 20 g/mL to investigate the requirement for Gels were scanned using ChemiDocÒ XRS (Bio-Rad). 166 110 superoxide in enzyme inactivation caused by hydrogen peroxide. Furthermore, 1.5 lM MPO in 50 mM sodium phosphate buffer, 167 111 Residual peroxidase activity was measured by adding a 25 lL ali- pH 7.4, and 100 lM DTPA was incubated with the following hydro- 168 112 quot to 850 lL of 200 mM sodium acetate buffer, pH 5.4, contain- gen peroxide: protein ratios: 1:1, 5:1, 10:1, 40:1, 100:1, 167:1, 169 113 ing 0.01% cetrimide, 100 lL of 20 mM TMB in DMF (made fresh 500:1, 1000:1 and 2000:1 for 2 h at room temperature.
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