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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
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Competitive reduction of perferrylmyoglobin radicals by protein
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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
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give superoxide radicals (O2•-) and metmyoglobin (MbFe(III)); the formation of the latter results
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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
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to a (formally) Fe(V)=O species, but which is more accurately described as MbFe(IV)=O
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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
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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
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other via long-range electron transfer. For bovine, equine and sperm whale myoglobin there is
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evidence for radical formation at both Trp (Trp-14, but not Trp-7) and Tyr (Tyr-103, Tyr-146;
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and in the case of sperm whale Tyr-151) residues.4-6 In the case of human myoglobin, radical
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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
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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
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both the hypervalent myoglobin species in biological systems including foods and intact
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mammalian tissues.11 It has been reported that Cys and Tyr residues on myosin are oxidized by
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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
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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.
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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
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(ME), and rosemary (RE), and three phenolic compounds characteristic of those extracts, namely
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catechin, caffeic acid, and carnosic acid, has been investigated. The reduction of
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perferrylmyoglobin radical by these proteins, plant extracts, and phenols was monitored directly
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by electron paramagnetic resonance (EPR) spectroscopy to obtain quantitative information on
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the concentrations of well-defined phenols and plant extracts required to afford protection
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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
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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
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day of preparation. Phenolic concentration was determined by Folin Ciocalteu’s method as
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described by Singleton & Rossi24. In brief, maté extract was dissolved in double-deionized water
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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
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prepared from gallic acid. The concentrations are given in gallic acid equivalents (g/100 g dry
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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
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mass of myosin (520 000) were used to calculate the molar concentration of MPI in µM myosin
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equivalents. Dissolved MPI was analyzed with or without prior addition of 5 % SDS by SDS-
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PAGE using NuPAGE® Novex 3-8 % TRIS-Acetate Gels with or without addition of
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dithiotreitol (DTT) as reducing agent (Invitrogen, Carlsbad, CA) as described by Jongberg et
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al.22
141 142
Blocking of protein thiol groups with NEM
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Thiol groups in 10 mg/mL MPI or BSA were blocked by reaction with 100 µM N-
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ethylmaleimide (NEM) in 0.1 M Tris buffer (pH 7.4, I=1.0) for 90 min at 4°C.12 NEM-blocked
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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
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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
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concentrations were calculated based on a 5-point standard curve (0.4-83.3 µM) of L-cysteine
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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
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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
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a syringe with a needle and long plastic tubing. 30 µl of H2O2 (100 µM final concentration) was
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then added in a similar manner, the samples vortex mixed and frozen in liquid nitrogen 15 sec
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after H2O2 addition. BSA was dissolved in Tris buffer, and MPI was applied as a suspension in
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Tris buffer. Stock solution of RE (120 mg/mL) was dissolved in ethanol and subsequently
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diluted in Tris buffer. GTE and ME were dissolved and diluted directly in Tris buffer. Aqueous
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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
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subsequently diluted with Tris buffer.
172 173
Radical detection by Electron Paramagnetic Resonance (EPR) spectroscopy
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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
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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
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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©
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(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
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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©
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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
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were found to dissolve in the 0.1 M Tris buffer (pH 7.4, I=1.0) both with and without 5% SDS.
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Although the presence of SDS increased protein solubility, as indicated by the increased protein
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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
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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
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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-
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ethylmaleimide (NEM) as described in the Materials and Methods to obtain MPI-NEM and
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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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349
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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395
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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441
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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446
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
454 455
1. George, P.; Irvine, D. H. The reaction between metmyoglobin and hydrogen peroxide.
456 457
Biochem. J. 1952, 52, 511-517. 2. Gibson, J. F.; Ingram, D. J. E.; Nicholls, P. Free Radical Produced in the Reaction of
458 459
Metmyoglobin with Hydrogen Peroxide. Nature 1958, 181, 1398-1399. 3. Kroger-Ohlsen, M.; Carlsen, C. U.; Andersen, M. L.; Skibsted, L. H. Pseudoperoxidase
460
activity of myoglobin: Pigment catalyzed formation of radicals in meat systems. In
461
ACS symposium series 807, Free Radicals in Food, Morelle, M.; Shahidi, F.; Ho,
462
C., Eds.; American Chemical Society: Washington DC, 2002; pp 138-150.
463
4. Davies, M. J. Identification of a globin-free radical in equine myoglobin treated with
464
peroxides. Biochim. Biophys. Acta, Bioenerg. 1991, 1077, 86-90.
465
5. Gunther, M. R.; Kelman, D. J.; Corbett, J. T.; Mason, R. P. Self-peroxidation of
466
Metmyoglobin Results in Formation of an Oxygen-reactive Tryptophan-centered
467
Radical. J. Biol. Chem. 1995, 270, 16075-16081.
468
6. Libardi, S. H.; Skibsted, L. H.; Cardoso, D. R. Oxidation of carbon monoxide by
469 470
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.
471
Involvement of a thiyl radical produced at cysteine 110. J. Biol. Chem. 2000, 275,
472
20391-20398.
473
8. DeGray, J. A.; Gunther, M. R.; Tschirret-Guth, R. A.; Ortiz de Montellano, P. R.; Mason,
474
R. P. Peroxidation of a specific tryptophan of metmyoglobin by hydrogen peroxide.
475
J. Biol. Chem. 1997, 272, 2359-2362.
23 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
476
9. Irwin, J. A.; Ostdal, H.; Davies, M. J. Myoglobin-Induced Oxidative Damage: Evidence for
477
Radical Transfer from Oxidized Myoglobin to Other Proteins and Antioxidants.
478
Arch. Biochem. Biophys. 1999, 362, 94-104.
479
10. Kelman, D. J.; DeGray, J. A.; Mason, R. P. Reaction of Myoglobin with Hydrogen
480
Peroxide Forms a Peroxyl Radical Which Oxidizes Substrates. J. Biol. Chem. 1994,
481
269, 7458-7463.
482
11. Davies, M. J.; Dean, R. T. Radical-mediated protein oxidation; Oxford Science
483 484
Publications: Oxford, 2003. 12. Lund, M. N.; Luxford, C.; Skibsted, L. H.; Davies, M. J. Oxidation of myosin by heme
485
proteins generates myosin radicals and protein cross-links. Biochem. J. 2008, 410,
486
565-574.
487
13. Frederiksen, A. M.; Lund, M. N.; Andersen, M. L.; Skibsted, L. H. Oxidation of porcine
488
myosin by hypervalent myoglobin: The role of thiol groups. J. Agric. Food Chem.
489
2008, 56, 3297-3304.
490
14. Uniprot Consortium, Myosin heavy chain 2b, MYH4_PIG. URL (http://www. uniprot.
491 492
org/uniprot/Q9TV62) (2014, July 7). 15. Xiong, Y. L.; Park, D.; Ooizumi, T. Variation in the cross-linking pattern of porcine
493
myofibrillar protein exposed to three different oxidative environments. J. Agric.
494
Food Chem. 2009, 57, 153-159.
495
16. Lund, M. N.; Lametsch, R.; Hviid, M. S.; Jensen, O. N.; Skibsted, L. H. High-Oxygen
496
Packaging Atmosphere Influences Protein Oxidation and Tenderness of Porcine
497
longissimus dorsi during Chill Storage. Meat Sci. 2007, 77, 295-303.
24 ACS Paragon Plus Environment
Page 24 of 40
Page 25 of 40
498
Journal of Agricultural and Food Chemistry
17. Shah, M. A.; Bosco, S. J. D.; Mir, S. A. Plant extracts as natural antioxidants in meat and
499 500
meat products. Meat Sci. 2014, 98, 21-33. 18. Rodríguez-Carpena, J. G.; Morcuende, D.; Estévez, M. Avocado by-products as inhibitors
501
of color deterioration and lipid and protein oxidation in raw porcine patties
502
subjected to chilled storage. Meat Sci. 2011, 89, 166-173.
503
19. Rababah, T.; Hettiarachchy, N.; Horax, R.; Eswaranandam, S.; Mauromoustakos, A.;
504
Dickson, J.; Niebuhr, S. Effect of electron beam irradiation and storage at 5C on
505
thiobarbituric acid reactive substances and carbonyl content in chicken breast meat
506
infused with antioxidants and selected plant extracts. J. Agric. Food Chem. 2004,
507
52, 8236-8241.
508
20. Lara, M. S.; Gutierrez, J. I.; Timón, M.; Andrés, A. I. Evaluation of two natural extracts
509
(Rosmarinus officinalis L. and Melissa officinalis L.) as antioxidants in cooked pork
510
patties packed in MAP. Meat Sci. 2011, 88, 481-488.
511
21. Jongberg, S.; Tørngren, M. A.; Gunvig, A.; Skibsted, L. H.; Lund, M. N. Effect of green
512
tea or rosemary extract on protein oxidation in Bologna type sausages prepared
513
from oxidatively stressed pork. Meat Sci. 2013, 93, 538-546.
514
22. Jongberg, S.; Lund, M. N.; Ostdal, H.; Skibsted, L. H. Phenolic antioxidant scavenging of
515
myosin radicals generated by hypervalent myoglobin. J. Agric. Food Chem. 2012,
516
60, 12020-12028.
517
23. Lundblad, R. L. Techniques in protein modification; CRC Press: Boca Raton, FL, 1995.
518
24. Singleton, V. L.; Rossi, J. A. Colorimetry of total phenolics with phosphomolybdic-
519
phosphotungstic acid reagents. Am. J. Enol. Vitic. 1965, 16, 144-158.
25 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
520
25. Park, D.; Xiong, Y. L. Oxidative modification of amino acids in porcine myofibrillar
521
protein isolates exposed to three oxidizing systems. Food Chem. 2007, 103, 607-
522
616.
523
26. Koutina, G.; Jongberg, S.; Skibsted, L. H. Protein and lipid oxidation in Parma ham during
524
production. J. Agric. Food Chem. 2012, 60, 9737-9745.
525
27. Ellman, G. L. Tissue Sulfhydryl Groups. Arch. Biochem. Biophys. 1959, 82, 70-77.
526
28. Jongberg, S.; Terkelsen, L. S.; Miklos, R.; Lund, M. N. Green tea extract impair meat
527
emulsion properties by disturbing protein disulfide cross-linking. Meat Sci. 2015,
528
100, 2-9.
529
29. Andersen, H. J.; Skibsted, L. H. Kinetics and Mechanism of Thermal Oxidation and
530
Photooxidation of Nitrosylmyoglobin in Aqueous Solution. J. Agric. Food Chem.
531
1992, 40, 1741-1750.
532
30. Kajer, T. B.; Fairfull-Smith, K. E.; Yamasaki, T.; Yamada, K.; Fu, S.; Bottle, S. E.;
533
Hawkins, C. L.; Davies, M. J. Inhibition of myeloperoxidase- and neutrophil-
534
mediated oxidant production by tetraethyl and tetramethyl nitroxides. Free Radical
535
Biol. Med. 2014, 70, 96-105.
536
31. Lametsch, R.; Larsen, M. R.; Essén-Gustavsson, B.; Jensen-Waern, M.; Lundstrom, K.;
537
Lindahl, G. Postmortem changes in pork muscle protein phosphorylation in relation
538
to RN genotype. J. Agric. Food Chem. 2011, 59, 11608-11615.
539
32. Østdal, H.; Skibsted, L. H.; Andersen, H. J. Formation of long-lived protein radicals in the
540
reaction between H2O2-activated metmyoglobin and other proteins. Free Radical
541
Biol. Med. 1997, 23, 754-761.
26 ACS Paragon Plus Environment
Page 26 of 40
Page 27 of 40
542
Journal of Agricultural and Food Chemistry
33. Turell, L.; Radi, R.; Alvarez, B. The thiol pool in human plasma: The central contribution
543 544
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
545
mate (Ilex paraguariensis, St. Hil.) and its antioxidant activity compared to
546
commonly consumed beverages. Food Res. Int. 2007, 40, 393-405.
547
35. Brewer, M. S. Natural antioxidants: Sources, compounds, mechanisms of action, and
548 549
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
550
review on chemistry, health implications, and technological considerations. J. Food
551
Sci. 2007, 72, R138-R151.
552
37. Østdal, H.; Andersen, H. J.; Davies, M. J. Formation of long-lived radicals on proteins by
553
radical transfer from heme enzymes - A common process? Arch. Biochem. Biophys.
554
1999, 362, 105-112.
555
38. Chen, Y. R.; Chen, C. L.; Chen, W.; Zweier, J. L.; Augusto, O.; Radi, R.; Mason, R. P.
556
Formation of protein tyrosine ortho-semiquinone radical and nitrotyrosine from
557
cytochrome c-derived tyrosyl radical. J. Biol. Chem. 2004, 279, 18054-18062.
558
39. Dalsgaard, T. K.; Nielsen, J. H.; Brown, B. E.; Stadler, N.; Davies, M. J. Dityrosine, 3,4-
559
dihydroxyphenylalanine (DOPA), and radical formation from tyrosine residues on
560
milk proteins with globular and flexible structures as a result of riboflavin-mediated
561
photo-oxidation. J. Agric. Food Chem. 2011, 59, 7939-7947.
562
40. Silva, F. A. M.; Borges, F.; Guimarães, C.; Lima, J. L. F. C.; Matos, C.; Reis, S. Phenolic
563
acids and derivatives: Studies on the relationship among structure, radical
27 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
564
scavenging activity, and physicochemical parameters. J. Agric. Food Chem. 2000,
565
48, 2122-2126.
566
41. Bentayeb, K.; Rubio, C.; Batlle, R.; Nerín, C. Direct determination of carnosic acid in a
567
new active packaging based on natural extract of rosemary. Anal. Bioanal. Chem.
568
2007, 389, 1989-1996.
569
42. Herrero-Martínez, J. M.; Sanmartin, M.; Rosés, M.; Bosch, E.; Ráfols, C. Determination of
570
dissociation constants of flavonoids by capillary electrophoresis. Electrophor. 2005,
571
26, 1886-1895.
572
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
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Chem. 2010, 58, 676-683.
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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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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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