Competitive Reduction of Perferrylmyoglobin Radicals by Protein

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Article

Competitive reduction of perferrylmyoglobin radicals by protein thiols and plant phenols Sisse Jongberg, Marianne Nissen Lund, Leif Horsfelt Skibsted, and Michael J. Davies J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf5041433 • Publication Date (Web): 24 Oct 2014 Downloaded from http://pubs.acs.org on November 7, 2014

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Competitive reduction of perferrylmyoglobin radicals by protein

2

thiols and plant phenols

3 4 Sisse Jongberga *, Marianne N. Lunda, Leif H. Skibsteda, Michael J. Daviesb,c.

5 6 7 8 9

a

Department of Food Science, Faculty of Science, University of Copenhagen, Rolighedsvej 30,

10

1958 Frederiksberg, Denmark.

11

b

Heart Research Institute, 7 Eliza Street, Newtown, NSW 2042 Australia.

12

c

Current address: Dept. of Biomedical Sciences, Building 4.5, Panum Institute, University of

13

Copenhagen, Blegdamsvej 3, Copenhagen, 2200 Denmark.

14 15 16

*

17

fax.: +4535333344.

Corresponding author: Sisse Jongberg, email address: [email protected], tel.: +4535332181,

18 19

Table of Contents category: Food and Beverage Chemistry/Biochemistry

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Abstract

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Radical transfer from perferrylmyoglobin to other target species (myofibrillar proteins (MPI) and

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bovine serum albumin (BSA), extracts from green tea (GTE), maté (ME), and rosemary (RE),

23

and three phenolic compounds, catechin, caffeic acid, and carnosic acid) was investigated by

24

electron paramagnetic resonance (EPR) spectroscopy to determine the concentrations of plant

25

extracts required to protect against protein oxidation. Blocking of MPI thiol groups by N-

26

ethylmaleimide was found to reduce the rate of reaction of MPI with perferrylmyoglobin

27

radicals, signifying the importance of protein thiols as radical scavengers. GTE had the highest

28

phenolic content of the three extracts and was most effective as a radical scavenger. IC50 values

29

indicated that the molar ratio between phenols in plant extract and MPI thiols needs to be > 15 in

30

order to obtain efficient protection against protein-to-protein radical transfer in meat. Caffeic

31

acid was found most effective among the plant phenols.

32 33 34 35

Keywords

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Myoglobin, protein radicals, plant phenols, myofibrillar proteins, protein oxidation.

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Introduction

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Oxymyoglobin (MbFe(II)-O2) is the heme protein primarily responsible for the red color of meat.

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This protein undergoes spontaneous autoxidation at a rate dependent on the oxygen pressure, to

41

give superoxide radicals (O2•-) and metmyoglobin (MbFe(III)); the formation of the latter results

42

in a discoloration of meat. Spontaneous or superoxide dismutase-catalyzed disproportionation of

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O2•- gives rise to hydrogen peroxide (H2O2), which can react with metmyoglobin and oxidize this

44

to a (formally) Fe(V)=O species, but which is more accurately described as MbFe(IV)=O

45

heme•+). This heme radical-cation, undergoes rapid electron transfer with the surrounding globin

46

to give a species containing an Fe(IV)=O centre and a globin radical (Mb• Fe(IV)=O; often called

47

perferrylmyoglobin).1,

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pseudoperoxidase activities towards a wide range of substances, and despite their relatively low

49

catalytic activity compared to classical peroxidases, they have been associated with oxidative

50

degradation of proteins and lipids in meat stored under oxidative conditions.3

2

Both the Fe(IV)=O centre and the globin radical(s) possess

51 52

The nature and site of the perferrylmyoglobin radical has been the subject of considerable study

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with evidence presented for multiple sites, as least some of which are in equilibrium with each

54

other via long-range electron transfer. For bovine, equine and sperm whale myoglobin there is

55

evidence for radical formation at both Trp (Trp-14, but not Trp-7) and Tyr (Tyr-103, Tyr-146;

56

and in the case of sperm whale Tyr-151) residues.4-6 In the case of human myoglobin, radical

57

formation at Cys-110 has also been reported;7 this residue is not present in the bovine, equine

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and sperm whale forms. Oxidation is believed to occur at these residues due the ease of oxidation

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of these side-chains compared to other residues, with these reactions resulting in the formation of

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a indolyl radical from Trp-14 (which subsequently undergoes rapid reaction with O2 to give a C-

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3 peroxyl radical on the indole ring),8 phenoxyl radicals from Tyr residues9 and thiyl radical

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from the Cys residue.7 In vitro studies have showed that these globin radicals are capable of

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initiating radical damage to a range of biological systems via one-electron transfer, or hydrogen

64

abstraction reactions, with concomitant loss of the globin radical.9, 10

65 66

Given their high abundance, proteins are considered as one of the major targets for oxidation by

67

both the hypervalent myoglobin species in biological systems including foods and intact

68

mammalian tissues.11 It has been reported that Cys and Tyr residues on myosin are oxidized by

69

perferrylmyoglobin radical(s) to generate thiyl and Tyr phenoxyl radicals, respectively; these

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species are precursors of di-tyrosine and disulfide cross-links.12, 13 The formation of these cross-

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links has been associated with changes in protein structure and function. Myosin contains 16 Cys

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residues, with these being located along the myosin heavy chain (MHC) tail region positioned

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transversely to those on neighboring heavy chains thereby potentially allowing fast formation of

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disulfide bonds when subject to oxidation.14,

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during storage has been related to loss of tenderness,16 clarifying the processes that give rise to

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myosin oxidation and cross-link formation, and processes that modulate or prevent these

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reactions, is of considerable interest with regard to the maintenance of meat quality.

15

As the formation of disulfide bonds in meat

78 79

Phenol-rich extracts from plants and herbs have been shown to protect meat and meat products

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efficiently against lipid oxidation, probably as a result of their capacity to scavenge reactive

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radicals, including those derived from heme-proteins.17 Several studies have shown that such

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extracts can limit oxidation in both fresh meat18-20 and during meat processing.20, 21 However, the

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mechanisms by which these phenols afford protection are not fully elucidated and the relative

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concentrations required for protection (i.e. dose-dependency of the phenolic antioxidants) is a

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critical issue. It has been suggested that protein-to-protein radical transfer may occur at a faster

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rate than protein-to-phenol radical transfer, especially when the antioxidant concentration is

87

low.9 In a model system with 1 % phenol relative to protein (w/w), Jongberg, Lund, Østdal, &

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Skibsted22 demonstrated that a green tea extract could limit radical transfer from

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perferrylmyoglobin to myosin by scavenging the hypervalent myoglobin species.

90 91

In the present study the dose-dependent reduction of perferrylmyoglobin radicals by myofibrillar

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proteins (MPI) and bovine serum albumin (BSA), plant extracts from green tea (GTE), maté

93

(ME), and rosemary (RE), and three phenolic compounds characteristic of those extracts, namely

94

catechin, caffeic acid, and carnosic acid, has been investigated. The reduction of

95

perferrylmyoglobin radical by these proteins, plant extracts, and phenols was monitored directly

96

by electron paramagnetic resonance (EPR) spectroscopy to obtain quantitative information on

97

the concentrations of well-defined phenols and plant extracts required to afford protection

98

against protein oxidation. The role of thiols in perferrylmyoglobin radical reduction was also

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investigated for MPI and BSA using N-ethylmaleimide a modifying agent with a high degree of

100

specificity for thiols.23

101 102

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Materials and Methods

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Chemicals and reagents

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Myoglobin from equine heart (MbFe(III)) of > 90 % purity and bovine serum albumin (BSA) of

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> 96 % purity were obtained from Sigma-Aldrich, Steinheim, Germany. GuardianTM Green Tea

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extract 20M (GTE), and GuardianTM Rosemary extract 202 (RE) were obtained from DuPont

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Nutrition and Biosciences ApS (formerly Danisco A/S), Brabrand, Denmark. Dried maté leaves

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(Ilex paraguariensis) of the Brazilian trade mark Tertúlia® (Ervateria Marca Ltda., Jaborá, Santa

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Catarina) was obtained from a local market in Brasilia, Brazil. Carnosic acid, caffeic acid, and

111

catechin were obtained from Sigma-Aldrich, St. Louis, MO. All other reagents were of analytical

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grade. Double-deionized water (Milipore, Bedford, MA) was used throughout.

113 114

Extraction of phenolics from maté leaves

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An aliquot of 0.5 g maté leaves were mixed with 50 mL double-deionized water in an ultrasonic

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bath for 15 min, centrifuged for 10 min at 936 g, and filtered through WhatmanTM 589/3 filter

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paper (GE Healthcare Life Sciences, Buckinghamshire, UK). These extracts were used on the

118

day of preparation. Phenolic concentration was determined by Folin Ciocalteu’s method as

119

described by Singleton & Rossi24. In brief, maté extract was dissolved in double-deionized water

120

and left to react with Folin-Ciocalteu phenol reagent for 8 min. Subsequently, 20 % sodium

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carbonate was added and the reaction mixture was left to incubate at 20 °C for 2 h. The phenol

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concentration was determined spectrophotometrically at 765 nm against a standard curve

123

prepared from gallic acid. The concentrations are given in gallic acid equivalents (g/100 g dry

124

extract; % w/w), n=5.

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Isolation of myofibrillar proteins

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Myofibrillar protein isolates (MPI) were prepared from pork Longissimus dorsi muscle

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according to Park, Xiong, & Alderton25 with slight modifications as described in Koutina,

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Jongberg, & Skibsted.26

130 131

Protein identification by gel-electrophoresis (SDS-page)

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Freeze-dried MPI was mixed with 0.1 M Tris buffer (pH 7.4, I=1.0) and stirred for 30 min at 20

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°C. The protein suspension was centrifuged at 12.100 g for 1 min and the supernatant was

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collected. Total protein concentration was determined spectrophotometrically by measuring

135

absorbance at 280 nm. The molar extinction coefficient of myosin, A280(1g/l) 0.496 and the molar

136

mass of myosin (520 000) were used to calculate the molar concentration of MPI in µM myosin

137

equivalents. Dissolved MPI was analyzed with or without prior addition of 5 % SDS by SDS-

138

PAGE using NuPAGE® Novex 3-8 % TRIS-Acetate Gels with or without addition of

139

dithiotreitol (DTT) as reducing agent (Invitrogen, Carlsbad, CA) as described by Jongberg et

140

al.22

141 142

Blocking of protein thiol groups with NEM

143

Thiol groups in 10 mg/mL MPI or BSA were blocked by reaction with 100 µM N-

144

ethylmaleimide (NEM) in 0.1 M Tris buffer (pH 7.4, I=1.0) for 90 min at 4°C.12 NEM-blocked

145

samples (MPI-NEM, BSA-NEM) were used on the day of preparation.

146 147

Quantification of thiol groups

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Protein thiols were quantified by reaction with 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB)27

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according to Jongberg, Terkelsen, Miklos, Lund28 with slight modifications. In brief, an aliquot

150

of 835 µl dissolved protein (MPI, MPI-NEM, BSA, or BSA-NEM) was mixed with 165 µl

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10 mM DTNB dissolved in 0.10 M Tris buffer (pH 7.4, I=1.0) and left to react for 30 min

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protected from light at 20 °C. The absorbance at 412 nm was obtained before (ABS412-before) and

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after reaction with DTNB (ABS412-after) for both samples and blanks containing Tris buffer. Thiol

154

concentrations were calculated based on a 5-point standard curve (0.4-83.3 µM) of L-cysteine

155

diluted in Tris buffer. Thiol concentrations are reported as nmol thiol/mg protein.

156 157

Preparation of freeze-quenched samples

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Myoglobin (30 mg) was dissolved in 1.0 mL 0.1 M Tris buffer (pH 7.4, I=1.0) and purified on a

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Sephadex PD10 column preconditioned with 25 mL 0.1 M Tris buffer. Myoglobin

160

concentrations were determined spectrophotometrically at 525 nm using ε = 7.700 M-1cm-1.29

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Myoglobin (100 µM final concentration) was mixed with proteins, phenols, or plant extracts (for

162

concentrations see text and figure legends) in a total volume of 270 µl in a 1.5 mL Eppendorf

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tube and transferred to an EPR tube (5 mm outside diameter, 227 mm length (Wilmad, NJ) using

164

a syringe with a needle and long plastic tubing. 30 µl of H2O2 (100 µM final concentration) was

165

then added in a similar manner, the samples vortex mixed and frozen in liquid nitrogen 15 sec

166

after H2O2 addition. BSA was dissolved in Tris buffer, and MPI was applied as a suspension in

167

Tris buffer. Stock solution of RE (120 mg/mL) was dissolved in ethanol and subsequently

168

diluted in Tris buffer. GTE and ME were dissolved and diluted directly in Tris buffer. Aqueous

169

stock solutions of caffeic acid (61 mM) and catechin (55 mM) were prepared in Tris buffer /

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ethanol (1:1, v/v). Stock solutions of carnosic acid (107 mM) were made up in acetonitrile and

171

subsequently diluted with Tris buffer.

172 173

Radical detection by Electron Paramagnetic Resonance (EPR) spectroscopy

174

Freeze-quenched samples were mounted in the cavity of an ECS 106 or EMX X-band

175

spectrometer (Bruker, Rheinstetten, Germany) equipped with a quartz liquid nitrogen finger

176

dewar flask filled with liquid nitrogen. Radical spectra were obtained using identical settings

177

with 4 acquisitions averaged. The peak intensity (PI) of the Trp-radical signal was determined by

178

the maximum intensity of the detected peak after baseline subtraction using WINEPR software.  =   . −   

179

The peak intensity was normalized to gain the relative peak intensity (Rel.PI) by using the mean

180

PI of the control samples without any oxidation substrate, indicated as the peak intensity

181

normalization factor (PINorm).  .  =

 ∙ 100 %  !"

182 183

Dose-response analysis and calculation of IC50 values

184

Dose-response analysis was performed by calculating the IC50 values (concentration of reductant

185

to obtain 50 % inhibition of the perferrylmyoglobin radical signal) using Origin Pro 9.1.0©

186

(OriginLab Coperation, Northampton, MA).30 The relative peak intensity was fitted as a function

187

of the logarithm of molecular concentration using Sigmoidal fitting and the DoseResp function.  = '1 +

'2 − '1 1 + 10(!+,-)

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The x values are the logarithm of dose, and the logx0 is the center of the curve, meaning the

189

concentration for half response. Global fit was used to fix the upper (A2) and lower (A1) limits

190

to 100 and 0 %, respectively.

191 192

Statistical data analysis

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All experiments were conducted in triplicates as a minimum unless otherwise stated, and data are

194

expressed as mean ± standard deviation (sd). Statistical analysis were performed using R©

195

version 2.12.1, The R Foundation for Statistical Computing (ISBN 3-900051-07-0). Data were

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analyzed by analysis of variance (ANOVA) using a linear model with mixed effects, where

197

replicates was included as random effects, and concentration was included as systematic effect.

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Student’s t-test using Microsoft Excel was applied for statistical analysis of the IC50 values and

199

variation in relative peak intensity. Significance for all data analysis was assumed at P < 0.05

200 201

Results

202

Characterization of protein radicals

203

MPI was isolated from pork longissimus dorsi muscle and the proteins were identified by the

204

molecular mass after separation by SDS-PAGE (Figure 1). MPI consists of primarily myosin

205

heavy chain (MHC, ~220 KDa), myosin binding protein C (MBP C, ~130 KDa), α-actinin (~100

206

KDa), desmin (~55 KDa), and actin (~42 KDa) according to Lametsch et al.31 These proteins

207

were found to dissolve in the 0.1 M Tris buffer (pH 7.4, I=1.0) both with and without 5% SDS.

208

Although the presence of SDS increased protein solubility, as indicated by the increased protein

209

band intensities, the protein composition of the solutions with and without SDS were the same,

210

and Tris buffer without SDS was therefore used throughout the study.

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Reaction between MbFe(III) and hydrogen peroxide (1:1 molar ratio) for 15 s before freeze-

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quenching in liquid nitrogen at 77 K resulted in the detection of a protein radical by EPR

214

spectroscopy (Figure 2, upper spectra in A, B, C, and D). No signals were observed when any

215

one of the components of the reaction mixture was omitted (data not shown). The observed

216

spectra are interpreted in terms of the presence of a mixture of Trp-derived peroxyl and Tyr-

217

derived phenoxyl radicals with hyperfine coupling constant and g-values as reported previously,9

218

with the major EPR absorptions of each species indicated by TRP and TYR.

219 220

The presence of increasing concentrations of MPI in the reaction mixture of MbFe(III) and

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hydrogen peroxide reduced the intensity of the features assigned to the Trp-peroxyl radical, with

222

this being most noticeable with the low field component (i.e. the left-hand feature) due to the

223

presence of significant overlap of the other components of this signal with features from the Tyr

224

phenoxyl radical. The Trp-peroxyl radical was almost fully extinguished with a concentration of

225

MPI of 14.8 µM (Figure 2, A). The intensity of the EPR absorptions assigned to the Tyr-

226

phenoxyl radical also decreased in the presence of increasing concentrations of MPI (Figure 2,

227

A). These data are interpreted in terms of subsequent reaction of the Mb-derived Trp- and Tyr

228

radicals with the MPI protein, rather than interference with the initial formation of these species

229

on the Mb protein.

230 231

In order to assess whether this process was specific to MPI, similar experiments were carried out

232

in which increasing concentrations of BSA were added to the Mb/hydrogen peroxide system.

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This resulted in a concentration-dependent decrease in the Trp-peroxyl radical signal (Figure 2,

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B). Modification of the Mb-derived Tyr-radical signal was also detected, with increasing

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concentrations of BSA resulting in the detection of a broad signal with increased intensity at an

236

identical g value. This change is interpreted in terms of radical transfer from the

237

perferrylmyglobin species to BSA, with the formation of a mixture of different radical species on

238

the BSA; these are likely to be predominantly Tyr-derived phenoxyl radicals (as the g value

239

remains unchanged) in different environments, with the latter resulting in the loss of spectral

240

resolution (i.e. a broad absorption envelope rather than distinct spectral features).32 As no

241

additional protein radicals were observed in the experiments in which MPI was added to the

242

reaction mixture, it is proposed that the radicals formed on the MPI must be located on residues /

243

sites which do not give rise to EPR-detectable absorptions, or are too short lived, to be detected

244

under the conditions employed. As one potential target residue on the MPI with which the Mb-

245

derived radicals might react are the Cys residues (cf. previous reports on the reaction of Mb-

246

derived radicals with thiols)9 further experiments were carried out with MPI and BSA with

247

blocked Cys residues.

248 249

Role of Cys residues on MPI and BSA and concentration dependency in perferrylmyoglobin

250

radical transfer

251

The average number of thiol groups per molecule present in the MPI preparations was found to

252

be 15 (Table 1). As MHC contains 16 Cys residues, whereas other myofibrillar proteins contain

253

fewer (e.g. 6 in actin) this value represents a mean thiol concentration of all proteins combined in

254

the MPI. In contrast, the average number of thiol groups was found to be 0.2 for BSA (Table 1).

255

The sequence of native BSA contains one free Cys, but it is well established that a considerable

256

proportion of these residues are present in modified or derivatized forms in plasma,33 so this

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value of < 1 is not surprising. The protein thiol groups of MPI and BSA were blocked by N-

258

ethylmaleimide (NEM) as described in the Materials and Methods to obtain MPI-NEM and

259

BSA-NEM. The efficiency of thiol-blocking was calculated to be 90 and 75 % for MPI and BSA,

260

respectively (Table 1). The low blocking percentage for BSA may be ascribed to the constrained

261

location of the Cys-34 residue in a cleft in the BSA structure. Higher concentrations of NEM

262

which might result in a greater extent of reaction at the Cys-34 residue were not employed, in

263

order to avoid reaction at other (less reactive, nucleophilic) sites such as Lys residues.23

264 265

These NEM-blocked proteins were subsequently employed in EPR experiments as outlined

266

above for the non-blocked proteins. Whilst blocking of the Cys-34 residues on BSA did not give

267

rise to any noticeable effect on the EPR spectra obtained for the BSA system (Figure 2, D),

268

increasing concentrations of MPI-NEM resulted in the detection of EPR signals from the Mb-

269

derived radicals of diminished intensity (Figure 2, C). These data are interpreted in terms of a

270

key role for the Cys residues of MPI in reaction with the perferrylmyglobin radicals, with

271

subsequent formation of thiyl radicals on the MPI proteins when the Cys residues are not

272

blocked.

273 274

Dose-response curves were generated by plotting the relative peak intensity of the Trp-peroxyl

275

radical signal against the concentration of MPI, BSA, or their NEM-blocked counterparts as a

276

function of their molecular (primary x-axis) or mass-based (secondary x-axis) concentrations

277

(Figure 3). As the half-life of the perferrylmyoglobin radical is short, reactions were stopped

278

after 15 s by freeze-quenching, such that the reactions did not reach equilibrium. The same

279

concentration of myoglobin and hydrogen peroxide were used in all experiments. Hence, the

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dose-response curves represent titration curves of the Mb-derived Trp-peroxyl radical by the

281

added reactive agents, which provide information on the stoichiometry of these reactions.

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The dose-response curve for native MPI was found to be steeper than for MPI-NEM (Figure 3),

284

and the relative peak intensity at 3.5 µM MPI or MPI-NEM was found to be significantly (P =

285

0.014) different. This confirms that blocking the Cys thiol groups dramatically affects the rate of

286

reaction of MPI with the Mb-derived Trp-peroxyl radicals. In contrast, the effect of blocking the

287

Cys-34 residue of BSA observed in these dose-response curves was less dramatic. This may arise

288

from steric interactions due to the partly buried nature of the BSA Cys-34 residue.

289 290

IC50 values were estimated for comparison of the titration data obtained with MPI and BSA, and

291

they indicate that 113.5 µM BSA is required to reduce the Trp-signal by 50% whereas only 1.12

292

µM is required for MPI (Table 2). This suggests that MPI is 100-fold more efficient reducing

293

agent compared to BSA based on the molecular concentration. When considering the size of the

294

protein and comparing by mass-based concentrations only a ~10-fold higher BSA concentration

295

was necessary to reduce the Trp-signal by 50% as compared to MPI. This indicates that the

296

amino acid composition of the target protein is a major factor in the observed reducing capacity.

297

Expressing the IC50 value of MPI or BSA based on the thiol concentration in each protein gives

298

17±3 or 23±3 µM thiols, respectively (calculated e.g. for MPI as 15.1 thiols per molecule ·

299

1.1±0.2 µM MPI). As the two numbers are not significantly different, it indicates that the thiol

300

reactivities of the two proteins are comparable despite the steric hindrance for the BSA thiol.

301

This may arise from the different reaction times used for the two proteins.

302

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Characterization of green tea, maté, and rosemary extracts

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The total phenolic content of the three plant extracts from green tea (GTE), maté (ME), and

305

rosemary (RE) varied with the highest concentration (determined in gallic acid equivalents by

306

the Folin-Ciocalteau assay) for GTE followed by ME and then RE (Table 3). The total phenolic

307

content determined for ME corresponds well with the average total phenolic content of 8.4 g/100

308

g dry matter reported for three commercial maté brands.34

309 310

The phenolic composition of green tea (Camellia sinensis) extract consists primarily of

311

flavonoids, such as catechin, epicatechin, epicatechin gallate, epigallocatechin gallate and

312

quercetin, with lower levels of hydrocinnamic acids, caffeic, coumaric, and ferulic acid.35 Maté

313

(Ilex paraguariensis) extract consists primarily of caffeoyl derivatives, including caffeic acid,

314

chlorogenic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic

315

acid.36 Rosemary (Rosemarinus officialis) extract consists primarily of phenolic diterpenes, such

316

as carnosic acid, carnosol, rosmanol, rosmadial, 12-methoxycarnosic acid, epi- and iso-rosmanol,

317

and the phenolic acids rosmarinic and caffeic acid.35

318 319

Perferrylmyoglobin radical scavenging by green tea, maté, and rosemary extracts

320

Addition of the plant extracts to the reaction mixture of MbFe(III) and hydrogen peroxide

321

showed that all plant extracts were able to reduce the perferrylmyoglobin radicals (Figure 4). The

322

EPR spectra demonstrated that both the Tyr-phenoxyl and Trp-peroxyl radicals were reduced in

323

the presence of increasing concentrations of these extracts. This is presumed to result in the

324

generation of phenoxyl- / catechol-derived radicals on the phenols present in the extracts, though

325

these transients were not detected. The absence of EPR signals from such extract-derived

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radicals is not surprising as both a number of species are likely to be formed from such complex

327

mixtures, and these species are likely to undergo rapid subsequent reaction (dimerization or

328

disproportionation) due to their low molecular mass and unhindered nature.

329 330

The relative capability of the different extracts to reduce the Mb Trp-peroxyl radical was

331

examined by determining dose-response curves based on the concentration of gallic acid

332

equivalents in the extracts (Figure 4, left lower panel) or the mass of extract used (Figure 4, right

333

lower panel). Full reduction of the Trp-peroxyl radical signal was detected with each extract and

334

there was no significant difference in the gallic acid equivalents between the three extracts

335

required to achieve this (Figure 4, left lower panel). In contrast, when the reducing capacity of

336

the extracts was assessed based on the amount of extract added, much lower levels of GTE (~10

337

mg/L) was necessary to reduce the radical signal by 50% compared to ME and RE, where ~40

338

mg/L and ~60 mg/L, respectively, were necessary (Figure 4, right lower panel). These data

339

indicate that the total phenolic content has a more marked impact on the reducing capacity of

340

these extracts against the perferrylmyglobin radicals than the specific composition of the

341

phenolic compounds present in each extract.

342 343

Perferrylmyoglobin radical scavenging by catechin, caffeic acid and carnosic acid

344

In the light of the above data, further experiments were carried out with a number of specific

345

phenolic compounds (catechin, caffeic acid, and carnosic acid) to determine their relative

346

efficacies with regard to reaction with Mb radicals. The EPR spectra obtained from reaction

347

mixtures containing these three phenolic compounds showed that all of them were able to

348

scavenge perferrylmyoglobin radicals (Figure 5). The spectra obtained indicate that no additional

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phenoxyl radicals from the added compounds were formed as a result of radical transfer, as no

350

additional EPR signals were observed. However, significant reductions in the intensity of the

351

Mb-derived radicals were observed, indicating that reaction had occurred. Even at the highest

352

phenol concentrations, a weak residual signal were detected (Figure 5, upper panels). However,

353

as identical signals were observed at elevated concentrations (up to 100 µM catechin, data not

354

shown) the modifications are more likely to be an artefact rather than a secondary phenoxyl

355

radical.9, 37

356 357

Dose-response curves obtained for increasing concentrations of these plant phenols, presented in

358

molar concentration of the phenolic compounds, showed that the phenols had markedly different

359

reducing capacities (Figure 5, lower panel). Caffeic acid was a far more efficient scavenger of

360

the perferrylmyoglobin radicals as compared to catechin, and carnosic acid was least efficient.

361

These phenols were selected as representatives of the extracts, GTE, MA, and RE, as they are the

362

dominant phenol in each extract. However, whilst the reducing capacity of the extracts was

363

shown to be primarily dependent on the total phenolic content, and showed little variation with

364

regard to phenol composition, these phenols when present alone showed marked variations.

365 366

Discussion

367

Protection against meat protein oxidation by phenol-rich extracts

368

The reducing capacity of the proteins, plant extracts, and phenols was quantified by their IC50

369

values (Table 2), and may be used to predict whether a specific phenol-rich extract may protect

370

against protein oxidation. Comparing the IC50 values between the two proteins showed that the

371

proteins in the MPI are ~100-fold more efficient as compared to BSA, and after blocking the

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372

thiol groups by NEM this ratio dropped to ~20, indicating that the thiol groups are highly

373

significant for the reducing capacity. No significant differences were observed between the plant

374

extracts, indicating that the exact phenolic composition of these extracts is not the most

375

important factor with regard to the overall reducing capacity, and that the total amount of phenol

376

is important for these extracts where several phenols are present in mixtures. In fact, other

377

constituents of the extracts, such as fillers or fibers, may be of higher importance, facilitating

378

radical transfer processes in the extract.

379 380

A study including the same GTE and RE extracts was recently presented by Jongberg et al.22

381

Phenoxyl radicals are stabilized when coordinated to divalent metal ions, such as Zn2+, hereby

382

preventing the dimerization reaction and enhancing the radical signals.38,

383

Jongberg et al.22 showed increased phenoxyl radical intensities of RE as compared to GTE after

384

reaction with the perferrylmyglobin radical in the presence of Zn(II), and this increase was

385

explained by more efficient radical-derived polymerization reaction of the phenols contained in

386

the GTE as compared to the RE. This is of major significance for the application of such extracts

387

to foods, as the addition of “antioxidative” ingredients may add flavors or aromas to the given

388

food product. In order to minimize any off-flavors derived from the extract, a high total phenolic

389

content and high ability to terminate radical processes by polymerization is beneficial, and

390

radical transfer processes in the extract is of importance in this respect.

39

The results of

391 392

In the present study, it was shown that with the isolated, specific phenols there is considerable

393

variation in IC50 values, with caffeic acid being significantly more efficient than catechin, which

394

in turn was significantly more efficient than carnosic acid. All three phenols examined contain

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catechol moieties (Figure 6), which are important for their radical scavenging activity.35 Caffeic

396

acid, which contains a single catechol moiety, showed the most efficient reducing capacity

397

towards perferrylmyoglobin radicals when compared with catechin, which contains two catechol

398

moieties, one on the B- and one on the A-ring in the flavonoid structure. Hence, the number of

399

catechol groups may not be the most significant contributor to the reducing capacity observed,

400

and other properties may be important for effective Mb radical scavenging. The carboxylate

401

groups of caffeic and carnosic acids are powerful electron delocalizing groups that affect the pKa

402

values of the neighboring phenolic hydroxyl groups. The carboxylic acids are deprotonated in the

403

reaction mixture (pH 7.4) as the pKa1c of caffeic acid and carnosic acid are 4.36 and 4.9,

404

respectively.40, 41 In contrast, catechin is protonated in the reaction mixture as the pKa of the most

405

acidic groups, the catechol and phloroglucinol moieties, are 8.7 and 9.7 for the B-ring and A-

406

ring, respectively.42 The presence of the ionized form of the phenol enhanced the reducing

407

capacity as an increased electron density enhances the ability of the molecule to donate electrons.

408

Nevertheless, Silva et al.40 found that structural modification of the ethylenic side chain on the

409

aromatic ring of caffeic acid to yield alkyl esters actually increased the radical scavenging

410

activity towards DPPH• (2,2-diphenyl-1-picrylhydrazyl radical). The same authors concluded

411

that no relationship between the radical scavenging activity and pKa exists, and suggested that

412

other physicochemical properties, such as the redox potential, could be of major importance for

413

the radical scavenging capacities.40

414 415

A third factor to consider with regards to the reducing capacity is the size and three-dimensional

416

structure of the phenols (Figure 6). Caffeic acid has a planar structure, which may allow it to

417

react at protein sites that are inaccessible to larger more bulky molecules. An example of this

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418

could be catechin, in which the C-ring allows the B-ring to be in pseudoaxial position, which

419

may decrease the reactivity of the phenol with the protein radical due to steric hindrance.

420

Similarly, the carboxylic group of carnosic acid is also in an axial position, and this may reduce

421

the reactivity towards the protein radicals. A final factor to consider is the radical-radical

422

interactions resulting in the polymerization of phenolic compounds that terminate radicals and

423

regenerate the hydroxyl groups to regenerate their radical scavenging activity. Carnosic acid

424

contains only one unsubstituted carbon atom in the aromatic ring, and may not polymerize to the

425

same extent as catechin and caffeic acid, which both contains multiple unsubstituted carbons.

426

Hence, the low reducing capacity of carnosic acid may be due to the limited ability to regenerate

427

the phenolic moiety. However, in extracts multiple components are present and other radical

428

transfer mechanisms may be active.

429 430

Overall, the results based on the IC50 values indicate that MPI is 15-fold more efficient as

431

scavenger of the perferrylmyoglobin radical as compared to GTE or RE based on the molecular

432

concentration of the components. This may reflect the different chemical reactivity of a thiol

433

compared to a phenol, and suggests that the molar ratio between GTE and MPI needs to be > 15

434

in order to obtain efficient protection against protein-to-protein radical transfer.

435 436

Polyphenols have been shown, based on kinetic experiments and modelling, to protect lipids

437

against oxidation by meat pigments in the gastric tract.43 In other complex systems, such as meat

438

emulsions, 100 ppm GTE, corresponding to 1.6% GTE per MPI, has been found to protect

439

against protein thiol loss.28 In the present study, the mass-based concentration of MPI and GTE

440

needed to reduce the Trp-radical signal by 50% were 0.6 g/L (Figure 3) and 8 mg/L (Figure 4),

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respectively. Calculation of the mass to mass percentage of GTE per MPI gives 1.3%, which is

442

within the concentration range of GTE per MPI in the meat emulsion study mentioned above.

443

This indicates that the model system applied may allow an effective preliminary evaluation of

444

the amounts of plant extracts needed to inhibit protein oxidation in meat and meat products.

445

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Acknowledgements

447

The authors are grateful for the technical assistance of Bente Danielsen from Faculty of Science,

448

University of Copenhagen. The authors also thank The Danish Council for Independent Research

449

Technology and Production within The Danish Agency for Science Technology and Innovation

450

for granting the project entitled: “Antioxidant mechanisms of natural phenolic compounds

451

against protein cross-link formation in meat and meat systems” (11-117033).

452

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Journal of Agricultural and Food Chemistry

References

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1. George, P.; Irvine, D. H. The reaction between metmyoglobin and hydrogen peroxide.

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Biochem. J. 1952, 52, 511-517. 2. Gibson, J. F.; Ingram, D. J. E.; Nicholls, P. Free Radical Produced in the Reaction of

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Metmyoglobin with Hydrogen Peroxide. Nature 1958, 181, 1398-1399. 3. Kroger-Ohlsen, M.; Carlsen, C. U.; Andersen, M. L.; Skibsted, L. H. Pseudoperoxidase

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activity of myoglobin: Pigment catalyzed formation of radicals in meat systems. In

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ACS symposium series 807, Free Radicals in Food, Morelle, M.; Shahidi, F.; Ho,

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C., Eds.; American Chemical Society: Washington DC, 2002; pp 138-150.

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4. Davies, M. J. Identification of a globin-free radical in equine myoglobin treated with

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peroxides. Biochim. Biophys. Acta, Bioenerg. 1991, 1077, 86-90.

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5. Gunther, M. R.; Kelman, D. J.; Corbett, J. T.; Mason, R. P. Self-peroxidation of

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Metmyoglobin Results in Formation of an Oxygen-reactive Tryptophan-centered

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Radical. J. Biol. Chem. 1995, 270, 16075-16081.

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6. Libardi, S. H.; Skibsted, L. H.; Cardoso, D. R. Oxidation of carbon monoxide by

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perferrylmyoglobin. J. Agric. Food Chem. 2014, 62, 1950-1955. 7. Witting, P. K.; Douglas, D. J.; Mauk, A. G. Reaction of human myoglobin and H2O2.

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Involvement of a thiyl radical produced at cysteine 110. J. Biol. Chem. 2000, 275,

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8. DeGray, J. A.; Gunther, M. R.; Tschirret-Guth, R. A.; Ortiz de Montellano, P. R.; Mason,

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9. Irwin, J. A.; Ostdal, H.; Davies, M. J. Myoglobin-Induced Oxidative Damage: Evidence for

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Radical Transfer from Oxidized Myoglobin to Other Proteins and Antioxidants.

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Peroxide Forms a Peroxyl Radical Which Oxidizes Substrates. J. Biol. Chem. 1994,

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11. Davies, M. J.; Dean, R. T. Radical-mediated protein oxidation; Oxford Science

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13. Frederiksen, A. M.; Lund, M. N.; Andersen, M. L.; Skibsted, L. H. Oxidation of porcine

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myosin by hypervalent myoglobin: The role of thiol groups. J. Agric. Food Chem.

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14. Uniprot Consortium, Myosin heavy chain 2b, MYH4_PIG. URL (http://www. uniprot.

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org/uniprot/Q9TV62) (2014, July 7). 15. Xiong, Y. L.; Park, D.; Ooizumi, T. Variation in the cross-linking pattern of porcine

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myofibrillar protein exposed to three different oxidative environments. J. Agric.

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Food Chem. 2009, 57, 153-159.

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16. Lund, M. N.; Lametsch, R.; Hviid, M. S.; Jensen, O. N.; Skibsted, L. H. High-Oxygen

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Packaging Atmosphere Influences Protein Oxidation and Tenderness of Porcine

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longissimus dorsi during Chill Storage. Meat Sci. 2007, 77, 295-303.

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17. Shah, M. A.; Bosco, S. J. D.; Mir, S. A. Plant extracts as natural antioxidants in meat and

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meat products. Meat Sci. 2014, 98, 21-33. 18. Rodríguez-Carpena, J. G.; Morcuende, D.; Estévez, M. Avocado by-products as inhibitors

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of color deterioration and lipid and protein oxidation in raw porcine patties

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subjected to chilled storage. Meat Sci. 2011, 89, 166-173.

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19. Rababah, T.; Hettiarachchy, N.; Horax, R.; Eswaranandam, S.; Mauromoustakos, A.;

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Dickson, J.; Niebuhr, S. Effect of electron beam irradiation and storage at 5C on

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thiobarbituric acid reactive substances and carbonyl content in chicken breast meat

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infused with antioxidants and selected plant extracts. J. Agric. Food Chem. 2004,

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20. Lara, M. S.; Gutierrez, J. I.; Timón, M.; Andrés, A. I. Evaluation of two natural extracts

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(Rosmarinus officinalis L. and Melissa officinalis L.) as antioxidants in cooked pork

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patties packed in MAP. Meat Sci. 2011, 88, 481-488.

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21. Jongberg, S.; Tørngren, M. A.; Gunvig, A.; Skibsted, L. H.; Lund, M. N. Effect of green

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tea or rosemary extract on protein oxidation in Bologna type sausages prepared

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from oxidatively stressed pork. Meat Sci. 2013, 93, 538-546.

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22. Jongberg, S.; Lund, M. N.; Ostdal, H.; Skibsted, L. H. Phenolic antioxidant scavenging of

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myosin radicals generated by hypervalent myoglobin. J. Agric. Food Chem. 2012,

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60, 12020-12028.

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23. Lundblad, R. L. Techniques in protein modification; CRC Press: Boca Raton, FL, 1995.

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24. Singleton, V. L.; Rossi, J. A. Colorimetry of total phenolics with phosphomolybdic-

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phosphotungstic acid reagents. Am. J. Enol. Vitic. 1965, 16, 144-158.

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25. Park, D.; Xiong, Y. L. Oxidative modification of amino acids in porcine myofibrillar

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protein isolates exposed to three oxidizing systems. Food Chem. 2007, 103, 607-

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26. Koutina, G.; Jongberg, S.; Skibsted, L. H. Protein and lipid oxidation in Parma ham during

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production. J. Agric. Food Chem. 2012, 60, 9737-9745.

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27. Ellman, G. L. Tissue Sulfhydryl Groups. Arch. Biochem. Biophys. 1959, 82, 70-77.

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28. Jongberg, S.; Terkelsen, L. S.; Miklos, R.; Lund, M. N. Green tea extract impair meat

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emulsion properties by disturbing protein disulfide cross-linking. Meat Sci. 2015,

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100, 2-9.

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29. Andersen, H. J.; Skibsted, L. H. Kinetics and Mechanism of Thermal Oxidation and

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Photooxidation of Nitrosylmyoglobin in Aqueous Solution. J. Agric. Food Chem.

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1992, 40, 1741-1750.

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30. Kajer, T. B.; Fairfull-Smith, K. E.; Yamasaki, T.; Yamada, K.; Fu, S.; Bottle, S. E.;

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Hawkins, C. L.; Davies, M. J. Inhibition of myeloperoxidase- and neutrophil-

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mediated oxidant production by tetraethyl and tetramethyl nitroxides. Free Radical

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31. Lametsch, R.; Larsen, M. R.; Essén-Gustavsson, B.; Jensen-Waern, M.; Lundstrom, K.;

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Lindahl, G. Postmortem changes in pork muscle protein phosphorylation in relation

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to RN genotype. J. Agric. Food Chem. 2011, 59, 11608-11615.

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32. Østdal, H.; Skibsted, L. H.; Andersen, H. J. Formation of long-lived protein radicals in the

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reaction between H2O2-activated metmyoglobin and other proteins. Free Radical

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Biol. Med. 1997, 23, 754-761.

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33. Turell, L.; Radi, R.; Alvarez, B. The thiol pool in human plasma: The central contribution

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of albumin to redox processes. Free Radical Biol. Med. 2013, 65, 244-253. 34. Bravo, L.; Goya, L.; Lecumberri, E. LC/MS characterization of phenolic constituents of

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mate (Ilex paraguariensis, St. Hil.) and its antioxidant activity compared to

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commonly consumed beverages. Food Res. Int. 2007, 40, 393-405.

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35. Brewer, M. S. Natural antioxidants: Sources, compounds, mechanisms of action, and

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potential applications. Comp. Rev. Food Sci. Food Saf. 2011, 10, 221-247. 36. Heck, C. I.; De Mejia, E. G. Yerba mate tea (Ilex paraguariensis): A comprehensive

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review on chemistry, health implications, and technological considerations. J. Food

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Sci. 2007, 72, R138-R151.

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37. Østdal, H.; Andersen, H. J.; Davies, M. J. Formation of long-lived radicals on proteins by

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radical transfer from heme enzymes - A common process? Arch. Biochem. Biophys.

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1999, 362, 105-112.

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38. Chen, Y. R.; Chen, C. L.; Chen, W.; Zweier, J. L.; Augusto, O.; Radi, R.; Mason, R. P.

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Formation of protein tyrosine ortho-semiquinone radical and nitrotyrosine from

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cytochrome c-derived tyrosyl radical. J. Biol. Chem. 2004, 279, 18054-18062.

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39. Dalsgaard, T. K.; Nielsen, J. H.; Brown, B. E.; Stadler, N.; Davies, M. J. Dityrosine, 3,4-

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dihydroxyphenylalanine (DOPA), and radical formation from tyrosine residues on

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milk proteins with globular and flexible structures as a result of riboflavin-mediated

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photo-oxidation. J. Agric. Food Chem. 2011, 59, 7939-7947.

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40. Silva, F. A. M.; Borges, F.; Guimarães, C.; Lima, J. L. F. C.; Matos, C.; Reis, S. Phenolic

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acids and derivatives: Studies on the relationship among structure, radical

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scavenging activity, and physicochemical parameters. J. Agric. Food Chem. 2000,

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48, 2122-2126.

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41. Bentayeb, K.; Rubio, C.; Batlle, R.; Nerín, C. Direct determination of carnosic acid in a

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new active packaging based on natural extract of rosemary. Anal. Bioanal. Chem.

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2007, 389, 1989-1996.

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42. Herrero-Martínez, J. M.; Sanmartin, M.; Rosés, M.; Bosch, E.; Ráfols, C. Determination of

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43. Lorrain, B.; Dangles, O.; Genot, C.; Dufour, C. Chemical modeling of heme-induced lipid

573

oxidation in gastric conditions and inhibition by dietary polyphenols. J. Agric. Food

574

Chem. 2010, 58, 676-683.

575

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Captions

577 578

Figure 1. SDS-PAGE analysis of porcine myofibrillar protein isolate (MPI) suspended in 0.1 M

579

Tris buffer (pH 7.4, I=1.0) with or without addition of 5 % sodium dodecyl sulfate (SDS).

580

Reduced samples were treated with dithiothreitol (DTT) prior to running the gel. A, B, C

581

represent triplicate suspensions, and Mw indicates a mixed protein standard with known

582

molecular masses indicated on the figure. Myosin heavy chain (MHC), myosin binding protein C

583

(MBP C), α-actinin, desmin and actin were identified based on their molecular masses.

584 585

Figure 2. EPR spectra of perferrylmyoglobin radicals generated by reaction with hydrogen

586

peroxide in 1:1 molar ratio (100 µM) alone or in the presence of (A) MPI, (B) BSA, (C) MPI-

587

NEM, or (D) BSA-NEM in increasing concentrations. The signal assigned to the Trp-peroxyl

588

radical and Tyr-phenoxyl radical are indicated by arrows in the Control- MPI EPR spectra (upper

589

left panel). The spiking observed on some spectra are experimental artefacts caused by gas

590

bubbles.

591 592

Figure 3. Dose-response curves of the relative peak intensity (Rel.PI, mean ± standard deviation,

593

n ≥ 3) of the Trp-peroxyl radical as a function of the molar concentrations of MPI and MPI-NEM

594

(left panel), and BSA and BSA-NEM (right panel). The secondary (top) x-axis presents the

595

mass-based concentration of the proteins.

596 597

Figure 4. Upper panels: EPR spectra of perferrylmyoglobin radicals generated by reaction with

598

hydrogen peroxide in 1:1 molar ratio (100 µM) alone or in the presence of green tea (GTE), maté

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Journal of Agricultural and Food Chemistry

599

(ME), or rosemary (RE) extracts in increasing concentrations. Lower panels: Dose-response

600

curves of the relative peak intensity (Rel.PI, mean ± standard deviation, n ≥ 3) of the Trp-peroxyl

601

radical as a function of the molar- (left panel) or mass-based (right) concentrations of green tea,

602

maté, or rosemary extract.

603 604

Figure 5. Upper panels: EPR spectra of perferrylmyoglobin radicals generated by reaction with

605

hydrogen peroxide in 1:1 molar ratio (100 µM) alone or in the presence of catechin (Cat), caffeic

606

acid (Caf.A), carnosic caid (Car.A) in increasing concentrations. Lower panel: Dose-response

607

curves of relative peak intensity (Re.PI, mean ± standard deviation, n ≥ 3) of the Trp-peroxyl

608

radical as a function of the molar concentrations of catechin, caffeic acid, carnosic caid.

609 610

Figure 6: Chemical structures of catechin, caffeic acid, and carnosic acid.

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Tables Table 1. Concentration of thiol groups in proteins presented as mean±sd (n=3) and efficiency of thiol-blocking by N-ethylmaleimide (NEM). Protein MPI MPI-NEM BSA BSA-NEM

[thiols] nmol/mg protein 29.0 ± 2.8 3.1 ± 0.9 3.6 ± 1.0 0.9 ± 1.0

# thiols per protein moleculea 15.1 ± 1.2 1.6 ± 0.4 0.2 ± 0.1 0.1 ± 0.1

Efficiency of thiol blocking

a

90 % 75 %

# thiols per molecule are calculated from the concentration of thiols divided by the molecular weight of BSA or MPI using myosin as reference.

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Table 2. IC50 values for the reduction of perferrylmyoglobin radical by proteins, extracts and phenols presented as mean±sd (n≥3). Letters (a-d) denotes significant difference (P < 0.05) between samples. Sample MPI MPI-NEM BSA BSA-NEM Green Tea extract Maté extract Rosemary extract Catechin Caffeic acid Carnosic acid

IC50 (µM) 1.1 ± 0.2c 4.5 ± 2.8bc 114 ± 12a 94 ± 40a 11.0 ± 4.3b 15.0 ± 8.3b 14.0 ± 5.9b 2.2 ± 1.2c 0.3 ± 0.1d 58 ± 43ab

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Table 3. Total phenolic content in extracts presented as mean±sd (n=3). Extract Green tea extract Maté extract Rosemary extract

Total phenolic content (% w/w)* 23.8 ± 1.3a 7.7 ± 0.9 4.8 ± 0.1a

* g gallic acid equivalents/100 g extract a Jongberg, Lund, Østdal, & Skibsted (23).

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