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Protein Oxidation: Basic Principles and Implications for Meat Quality

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Protein Oxidation: Basic Principles and Implications for

Meat Quality
a c a b a c
Wangang Zhang , Shan Xiao & Dong U. Ahn
a
Department of Animal Science , Iowa State University , Ames , IA , 50011-3150 , USA
b
College of Food Science and Nutritional Engineering , China Agricultural University ,
Beijing , 100083, China
c
Department of Agricultural Biotechnology, Major in Biomodulation , Seoul National
University , Seoul , 151-921 , Korea
Accepted author version posted online: 06 Jul 2012.

To cite this article: Wangang Zhang , Shan Xiao & Dong U. Ahn (2013) Protein Oxidation: Basic Principles and Implications for
Meat Quality, Critical Reviews in Food Science and Nutrition, 53:11, 1191-1201, DOI: 10.1080/10408398.2011.577540

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 Critical Reviews in Food Science and Nutrition, 53:1191–1201 (2013)
Copyright C Taylor and Francis Group, LLC
ISSN: 1040-8398 / 1549-7852 online
DOI: 10.1080/10408398.2011.577540

Protein Oxidation: Basic Principles

and Implications for Meat Quality

WANGANG ZHANG,1,3 SHAN XIAO,1,2 and DONG U. AHN1,3

1
Department of Animal Science, Iowa State University, Ames, IA 50011-3150, USA
2
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, China
3
Department of Agricultural Biotechnology, Major in Biomodulation, Seoul National University, Seoul 151-921, Korea
Downloaded by [Iowa State University] at 05:09 13 September 2013

The involvement of oxidized proteins to the development of biological diseases has been studied for a few decades,
but the effects and the mechanisms of protein oxidation in food systems are largely unknown. Protein oxidation is de-
fined as the covalent modification of a protein induced either by the direct reactions with reactive oxygen species (ROS)
or indirect reactions with secondary by-products of oxidative stress. ROS can cause oxidation in both amino acid side
chains and protein backbones, resulting in protein fragmentation or protein–protein cross-linkages. Although all amino
acids can be modified by ROS, cysteine, and methionine that are the most susceptible to oxidative changes due to high
reaction susceptibility of the sulfur group in those amino acids. Oxidative modifications of proteins can change their physical
and chemical properties, including conformation, structure, solubility, susceptibility to proteolysis, and enzyme activities.
These modifications can be involved in the regulation of fresh meat quality and influence the processing properties of meat
products. Oxidative stress occurs when the formation of oxidants exceeds the ability of antioxidant systems to remove the ROS
in organisms. Increased levels of protein oxidation have been associated with various biological consequences, including
diseases and aging, in humans and other animal species. The basic principles and products of protein oxidation and the
implications of protein oxidation in food systems, especially in meat, are discussed in this review.

Keywords Calpain, carbonyl, meat quality, protein oxidation, reactive oxygen species

INTRODUCTION ketones (Butterfield and Stadtman, 1997). Oxidants can directly

attack the backbone of a protein to cause fragmentation and
Oxidation is one of the major causes for quality deterioration conformational changes in the secondary and tertiary structure
during processing and storage of food products. Lipid oxidation of the protein. Disulfide, dityrosine, and other intermolecular
has been extensively studied in food systems for many years, but bridges induced by oxidation can result in protein aggregation
the influence and the mechanisms of protein oxidation in foods, and polymerization to change their proteolytic properties
especially in fresh meat and meat products are largely unknown. (Martinaud et al., 1997; Morzel et al., 2006). This is mainly due
Oxidation of proteins results in production of various oxidation to an oxidation-induced unfolding process, which increases the
derivatives. The main oxidative modifications of protein takes surface hydrophobicity of the oxidized protein during unfolding
place at the side chains of amino acids, which include thiol (Davies and Delsignore, 1987). These alterations can influence
oxidation, aromatic hydroxylation, and formation of carbonyl physical and chemical properties of proteins including solubil-
groups (Stadtman, 1990). Among all amino acid side chains, ity, hydrophobicity, water-holding capacity, meat tenderness,
cysteine and methionine are the most susceptible to oxidation and gelation functions (Srinivasan and Xiong, 1996; Srinivasan
because they contain reactive sulfur atoms (Shacter, 2000). and Hultin, 1997; Liu and Xiong, 2000; Rowe et al., 2004a,
Sulfur anion is the most powerful nucleophile and is rich in elec- 2004b). In addition, protein oxidation-induced changes may de-
trons, which can be easily removed. Reactive oxygen species crease the bioavailability of amino acid residues and modify the
(ROS) include free radicals (•OH, O2 •−, RS•, and ROO•), non- digestibility of proteins, which negatively affects the nutritional
radical species (H2 O2 and ROOH), and reactive aldehydes and values of meat proteins (Lund et al., 2010). Calpain is believed
to be the major enzyme for the degradation of myofibrillar
Address correspondence to Dong U. Ahn, Department of Animal Science,
proteins and contributes to the development of meat tender-
Iowa State University, 2276 Kildee Hall,, Ames, IA 50011-3150, USA. E-mail: ness and water-holding capacity during postmortem aging
[email protected] (Koohmaraie et al., 1986; Taylor et al., 1995; Kristensen and
1191
 1192 W. ZHANG ET AL.

Purslow, 2001; Lonergan et al., 2001; Huff-Lonergan and occurs at the α-carbon site of amino acid residues and forms
Lonergan, 2005). Calpain oxidation may arrest its proteolytic stabilized carbon-centered radicals, which react with O2 to pro-
activity and thus negatively influence fresh meat quality during duce alkylperoxyl radicals. These peroxyl radicals either react
postmortem refrigerated storage (Rowe et al., 2004a, 2004b). with HO2 ȧ to form hydroperoxides or are eliminated to gen-
Although great efforts have been exerted in recent years, the erate imines. The hydrolysis of imines can result in backbone
occurrence, mechanisms, and effects of protein oxidation on fragmentation while the decomposition of hydroperoxides can
meat and meat products are still largely unknown. This review cleave peptide bonds through alkoxyl-radical-mediated reac-
mainly discusses the basic principles and products of protein tions (Davies, 2005). The latter reactions can generate amides
oxidation and the implications of protein oxidation in food at the C-terminal side of the N-terminal part of the protein, and
systems especially in meat. α-keto-acyl residues at the N-terminal side of the C-terminal part
of the protein (Stadtman, 1993). In addition, protein fragmenta-
tion can be formed via single backbone cleavage by oxidation
GENERATION OF REACTIVE OXYGEN SPECIES of glutamyl, aspartyl, and prolyl side chains (Essex et al., 2001).
Hydrogen abstraction from γ -carbon atom of a glutamyl residue
ROS can be generated by various processes in biological can lead to the cleavage of peptide bonds and the formation of
system. ROS can be produced during normal metabolism in- N-pyruvyl derivatives, and oxalic acid intermediates (Garrison,
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cluding mitochondrial electron transportation reactions, perox- 1987). Oxidation of prolyl residues can produce two protein
isomal fatty acid metabolism and cytochrome P-450 reactions, fragments and an α-amino butyric acid (Uchida et al., 1990).
and in phagocytic cells especially those involved in terminal For instance, proline oxidation provides a unique mechanism for
respiration (Beckman and Ames, 1998; Shacter, 2000). One- or peptide bond cleavage via the formation of 2-pyrrolidone, which
two-electron reduction from O2 forms superoxide anion (O2 −) later is hydrolyzed to 4-aminobutyric acid (Stadtman, 1993;
and hydrogen peroxide (H2 O2 ), respectively, and eventually Hawkins and Davies, 2001). Additionally, ROS attack on the β-
generate extremely reactive hydroxyl radical (•OH) by reaction position via β-scission reaction can give rise to a carbonyl com-
with other components, which can initiate the oxidative damage pound and an α-carbon radical susceptible to cleavage via the
of proteins, lipids, and nucleic acids. The enzymes catalyzing diamide or α-amidation pathways (Headlam and Davies, 2004).
ROS generation include nitric oxide synthases, NADPH oxi-
dase, prostaglandin synthase, xanthine oxidase, lipoxygenases, Oxidation of Amino Acid Residues
ribonuclueotide reductase, glucose oxidase, myeloperoxidase,
cyclooxygenases, and cytochrome P450 (Shacter, 2000; Davies,
Cysteine and methionine are the most susceptible to oxida-
2005). Modification of proteins also can occur through the addi-
tive modification by all forms of ROS due to their sulfur atoms
tion of oxidatively modified products from lipids, amino acids,
(Garrison, 1987). One- or two-electron oxidation of cysteine can
sugars, and glutathione (Haberland et al., 1988). For example,
form similar end products. One-electron oxidation of cysteine
aldehyde moieties from lipid peroxidation such as malondi-
with radical oxidant can generate thiyl radicals. These species
aldehyde, hydroxynonenal, and acrolein can covalently bind to
have two major pathways: reaction with other thiol/thiolate to
residues of proteins, resulting in protein oxidation (Uchida and
form disulfide, or reaction with O2 to generate thiyl peroxyl
Stadtman, 1994; Requena et al., 1997). In addition, ROS can be
radicals (RSOOȧ) (Wardman and von Sonntag, 1995; Schone-
generated by exposure of organisms to exogenous agents includ-
ich, 2008). The two-electron oxidation between cysteine and
ing ultraviolet (UV) light, irradiation (X-rays, γ -rays, ultravio-
oxidants can result in the formation of sulfenic acid (CysSOH),
let A, and visible light in the presence of a sensitizer), chemical
sulfinic acid (CysSO2 H), and sulfonic acid (CysSO3 H) (Clai-
reagents (metal ions such as Fe2+ and Cu+, HONOO, ozone,
borne et al., 2003). These species are unstable and can yield
N2 O2 , deoxyosones, ketoamines, H2 O2 , HOCl, and HOBr), en-
oxyacids by hydrolysis reactions or disulfide bonds by reacting
vironmental pollutants, drug and their metabolites, xenobiotics,
with another thiol group (Turell et al., 2008). Similarly, methio-
and even cigarette smoke (Shacter, 2000; Davies, 2005; Tetik
nine residues can be easily oxidized by various kinds of oxidant
et al., 2007). In muscle system, pro-oxidative proteins, myo-
species. The major product from methionine oxidation is sulfox-
globin, and hemoglobin can be initiators of protein oxidation as
ide, which can be further oxidized to sulfone (Vogt, 1995; Barelli
reviewed by Baron and Andersen (2002).
et al., 2008). Methionine sulfoxide can be reversed to methio-
nine by enzyme methionine sulfoxide reductase and reducing
CONSEQUENCES OF PROTEIN OXIDATION reagents such as mercaptoethanol and dithiothreitol (Houghten
and Li, 1983; Dean et al., 1997). Methionine oxidation can both
Oxidation of Protein Backbone activate and damage protein functions and regulate homeostasis
in biological systems (Vogt, 1995).
Reactions between nonradical oxidants and protein back- Compared to cysteine and methionine, other amino acids
bone sites are relatively slow. Most of the damages in back- need more stringent conditions to be oxidatively modified.
bone position are induced by radical oxidants via the ·OH- For aromatic amino acids, including histidine, phenylalanine,
dependent hydrogen abstraction (Stadtman, 2001). The reaction tryptophan, and tyrosine, the major reaction is the addition to
 PROTEIN OXIDATION ON MEAT QUALITY 1193

Table 1 Oxidation products of amino acid residue side chains Ischiropoulos and Ai-Mehdi, 1995; Berlett et al., 1996; Viner
Amino acid residues Oxidation products et al., 1999). However, oxidation induced by peroxynitrite
cannot lead to the formation of carbonyl derivatives (Tien
Arginine γ -Glutamyl semialdehyde et al., 1999). The function of sulfhydryl-containing proteins
Cysteine Disulfides, cysteic acid
Glutamic acid Oxalic acid, pyruvate adducts
can be regulated by nitric oxide (NO)-induced S-nitrosylation.
Histidine Aspartate, asparagines, oxo-histidine S-nitrosylation is the covalent attachment of a nitrogen monox-
Leucine 3-, 4-, and 5-hydroxyleucine ide group to the thiol side chain of cysteine (Hess et al., 2005).
Lysine α-Aminoadipic semialdehyde The formation of S-nitrosothiols and mixed disulfides through
Methionine Methionine sulfoxide, methionine sulfone S-nitrosylation can influence catalytic activity of cysteine pro-
Phenylalanine 2-,3-,4-Hydroxyphenylalanine,
3,4-dihydroxyphenylalanine
tease in vivo and in vitro (Xian et al., 2000; Ascenzi et al., 2001;
Proline Glutamic semialdehyde, 4- and Zhang et al., 2008). The proteolytic activity of both m- and μ-
5-hydroxyproline pyroglutamic acid, calpain can be arrested by NO donors at different pH (Michetti
2-pyrrolidone et al., 1995; Koh and Tidball, 2000).
Threonine 2-Amino-3-ketobutyric acid Peroxynitrite can oxidize methionine to both sulfoxide and
Tryptophan 2-,4-,5-,6-, and 7-Hydroxytryptophan,
formylkynurenine, 3-hydroxykynurenine,
ethylene by two via the three pathways (Pryor et al., 1994):
(i) reactions between methionine and peroxynitrous acid lead-
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kynurenine, and nitrotryptophan

Tyrosine 3,4-Dihydroxyphenylalanine, 3-nitrotyrosine, ing to the formation of ethylene by one-electron oxidation path-
tyrosine–tyrosine cross-linkages and way and (ii) a direct bimolecular oxidation between peroxyni-
tyrosine-oxygen-tyrosine trite and methionine resulting in methionine sulfoxide by two-
electron oxidation pathway. Peroxynitrite can nitrate tyrosine
aromatic side chains. The abstraction reactions only occur at het- residues to regulate the interconversion of phosphorylation and
eroatom substitute (Hawkins et al., 2003). The aromatic amino nucleotidylation, which play critical roles in enzyme activities
acid residues are not sensitive to metal-catalyzed oxidation and signal transduction (Hunter, 1995). However, the nitration
systems, but they are the preferred targets of radicals induced of tyrosine and the oxidation of methionine are regulated by the
by radiolysis (Giulivi et al., 2003). Tyrosine can be converted to levels of carbon dioxide (CO2 ). In the absence of CO2 , perox-
dityrosine by UV and γ -irradiation, free radicals, peroxynitrite ynitrite can rapidly oxidize methionine to methionine sulfoxide.
and lipid hydroperoxides (Giulivi and Davies, 1994). The initial In the presence of CO2 , peroxynitrite can react rapidly with CO2
step is the formation of tyrosyl radicals by one-electron oxida- to form the ONOOCO2 − or O2 NOOCO2 − (Lymar and Hurst,
tion of tyrosine. Two monomeric tyrosyl radicals can combine 1995; Berlett et al., 1998).
to form dityrosine, which is relatively stable and resistant to
hydrolysis by lytic enzymes (Giulivi and Davies, 1994). This Formation of Protein Carbonyl Derivatives
dityrosine can lead to the formation of extensive inter- and intra-
protein cross-linkage. Other aromatic amino acids can be oxida- Generation of carbonyls is the most common damage for
tively modified: histidine residues are oxidized to asparagines, oxidized proteins. Four major pathways to generate protein car-
aspartic acid residues, and oxo-histidine; phenylalanine can bonyls include: (i) fragmentation of backbones through the α-
be converted to 2-,3-, and 4-hydroxyphenylalanine and 3,4- amidation pathway and β-scission, (ii) binding of non-protein
dihydroxy-phenylalanine; the oxidation products of tryptophan carbonyl compounds from lipid peroxidation by Michael ad-
are 2-,4-,5-,6-, and 7-hydroxy-tryptophan, formylkynurenine, 3- dition (4-hydroxy-2-nonenal (HNE) and malondialdehyde) to
hydroxykynurenine, kynurenine, and nitrotryptophan (Table 1). protein amino acid side chains including histidine imidazole,
cysteine sulfhydryl, and lysine amino groups (Burcham and
Kuhan, 1996; Berlett and Stadtman, 1997; Refsgaard et al.,
Peroxynitrite-Related Protein Oxidation 2000), (iii) direct oxidation of amino acid side chains includ-
ing arginine, lysine, proline, and threonine (Amici et al., 1989),
Nitric oxideis synthesized during the conversion of L- and (iv) addition of reactive carbonyl derivatives (ketoamines,
arginine to L-citrulline catalyzed by a family of enzymes ketoaldehydes, and deoxyosones) generated by reducing sugars
called nitric oxide synthase (NOS) in human and other and their oxidation products after reacting with lysine (Decker
species. Three isoforms of NOS, which include neuronal NOS et al., 2000).
(NOS1 or nNOS), macrophage (immune)/calcium calmodulin-
independent or inducible NOS (NOS2 or iNOS), and endothe-
lial NOS (eNOS or NOS) have been identified (Stamler and Protein–Protein Cross-linkages
Meissner, 2001). In muscle tissues, the nNOS is the major iso-
form responsible for the production of nitric oxide. Nitric oxide As discussed above, oxidation of protein backbone by hy-
can react with superoxide (·O2 ) to form peroxynitrite, which drogen abstraction can lead to the formation of carbon-centered
can nitrosylate cysteine and nitrate tyrosine, and oxidize me- radicals. In the absence of oxygen, two radicals can combine to-
thionine, tryptophan, and phenylalanine (Beckman et al., 1994; gether to generate cross-linkage within and between proteins
 1194 W. ZHANG ET AL.

(Stadtman, 2001). Oxidation and nitrosylation of sulfhydryl digestibility partly due to protein aggregation, whereas modestly
groups and tyrosine residues can lead to the generation of disul- oxidized proteins are easily digested by corresponding proteases
fide and dityrosine bonds, respectively, resulting in cross-linked (Levine et al., 1981; Wolff and Dean, 1986; Davies et al., 1987;
proteins (Giulivi and Davies, 1993). Carbonyl derivatives aris- Smuder et al., 2010). It is possible that conformational changes
ing from direct or indirect oxidation reactions can react with can expose some buried peptide bonds for enzyme hydroly-
lysine amino groups within a protein or between different pro- sis and increase degradation susceptibility. Otherwise, strong
teins leading to the formation of protein cross-linkages (Shen oxidation environment can result in the formation of protein
et al., 1996). When myofibrillar proteins of pork were suspended cross-linkage, which further leads to protein aggregation. These
in an iron-catalyzed oxidizing system or an H2 O2 -activated cross-linkages and aggregates are more resistant to enzymatic
metmyoglobin oxidizing system, the myosin was shifted from degradation compared to native protein forms (Davies et al.,
head–head associations to tail–tail cross-links due to the forma- 1987). Oxidation of methionine especially buried methionine in
tion of disulfide bonds (Xiong et al., 2010). The cross-linking protein hydrophobic region is highly possible to change protein
sites of myosin heavy chain was in light meromyosin segment structures. In actin, chloramine-T-induced methionine oxida-
through disulfide bonds, while no cross-linking was found in tion changed its conformation, and increased its surface hy-
heavy meromyosin after chicken myofibrils were exposed to hy- drophobicity. Oxidation of buried methionine can disrupt the
droxyl radical-generating system (Ooizumi and Xiong, 2006). non-covalent interactions within actin and decrease the stability
Downloaded by [Iowa State University] at 05:09 13 September 2013

In three different oxidizing systems (iron-catalyzed oxidizing of actin filament (Dalle-Donne et al., 2002).
system, H2 O2 -activated metmyoglobin oxidizing system, and
linoleic acid-oxidizing system) for porcine myofibrillar pro-
METHODS TO DETERMINE PROTEIN OXIDATION
teins, disulfide bonds were reported to be mainly responsible
IN FOOD SYSTEMS
for protein cross-linkage while malonaldehyde was hardly in-
volved in the formation of cross-linkage (Xiong et al., 2009).
These cross-linkages can change the properties of proteins such 2,4-Dinitrophenylhydrazine (DNPH) Derivatization Method
as structure, function, and susceptibility to degradation as dis-
cussed in the following section. Although carbonyl compounds are not the oxidation
products of some amino acid residues such as histidine,
phenylalanine, and tryptophan, DNPH derivatization method
that detect carbonyl compounds has been developed as a
Modification of Conformation, Structure, and Function convenient and regular method to quantify the levels of protein
oxidation in food system (Oliver et al., 1987; Levine et al.,
Oxidants can attack proteins to disrupt the physical and 1994). In this method, DNPH reacts with the carbonyl groups
chemical forces, which contribute to the stability of unmodi- of proteins to generate hydrazones and the absorbance is read
fied proteins. These attacks can change the primary structure at 370 nm (Levine et al., 1990). The carbonyl content was
of proteins, which can further distort secondary and tertiary calculated as nmol/mg protein using an absorption coefficient
structure of proteins. Eventually, changes in primary, secondary, of 22,000 M−1cm−1 (Levine et al., 1994). In food systems,
and tertiary structures can induce proteins to unfold to ran- carbonyl groups can also be generated by other pathways
dom conformations (Davies and Delsignore, 1987). Amino acid including metal-catalyzed oxidation of specific amino acid side
residues exposed on surface are more readily oxidized com- chains and the addition of sugars or lipid oxidation products
pared to buried ones, but oxidation of these surface residues such as 4-hydroxynonenal and malondialdehyde (Stadtman
seems to have no significant effects on protein conformation and Berlett, 1998), which can lead to overestimation of protein
(Levine et al., 1999). Oxidation of buried side chains can lead oxidation (Estévez et al., 2008). However, DNPH derivatization
to unfolding, reduced thermal stability, increased or decreased method has been utilized as a useful and meaningful marker for
surface hydrophobicity, and conformational changes (Liu and the accumulation of oxidized proteins in living tissues of human
Xiong, 1996; Gao et al., 1998). The formation of disulfide bonds and other species partly due to the simplicity and convenience
can contribute to the stability of the native conformation of pro- of this method. This method has been successfully applied in
teins by changing their thermodynamics. Oxidation of aromatic meat systems including raw fresh meat, meat emulsions, and
residues can lead to addition of some charged groups, which in- meat products to evaluate the degree of protein oxidation in the
crease the hydrophilic status for these residues. These changes products (Mercier et al., 1998; Haak et al., 2006; Estévez et al.,
may force oxidized aromatic residues to be present on protein 2007; Lund et al., 2007, 2008; Zhang et al., 2010).
surface resulting in protein conformation change (Chao et al.,
1997). For example, oxidation on catalytic cysteine 252 of ty-
rosine phosphatase-like phytase could result in conformation Fluorescence Spectroscopy Method
changes in other amino acid residues and P-loop of this pro-
tein (Gruninger et al., 2008). There are still some arguments Fluorescence spectroscopy has been reported to be effective
about the susceptibility of enzyme-induced degradation of ox- and specific to measure the destruction of tryptophan residues
idized protein. Generally, heavily oxidized proteins can lower of apolipoprotein B in copper-induced low-density lipoprotein
 PROTEIN OXIDATION ON MEAT QUALITY 1195

oxidation (Giessauf et al., 1995). The decrease of tryptophan flu- strong correlation supports that both AAS and GGS can be rep-
orescence was determined at an emission wavelength of 331 nm resentative of the total amount of carbonyl contents and thus
and an excitation wavelength of 282 nm. In a lecithin-liposome can be used as the markers for the protein oxidation in food
oxidation system, fluorescence spectroscopy was sensitive to systems (Estévez et al., 2009). Compared to DNPH and fluores-
detect the decrease of tryptophan and lysine in bovine serum cence spectroscopy, the newly developed LC-ESI-MS method
albumin (BSA) (Heinonen et al., 1998). This fluorescence spec- can further detect particular compounds that have been proved
troscopy method could also be used to detect compounds formed to be effective and reliable protein oxidation indicators. The de-
by the lipid oxidation products and amino acid groups of proteins tection of these compounds is very significant because it enables
with special spectral properties (Viljanen et al., 2004). Recently, us to better comprehend the particular mechanisms involved in
fluorescence spectroscopy has been used to study the oxidation the oxidative degradation of myofibrillar proteins and the fate
of myofibrillar proteins by measuring the loss of tryptophan of specific amino acids in food systems.
and the increase of protein carbonyl compounds in an oil-in-
water emulsion system (Estévez et al., 2008). Emulsions with
rapeseed oil and myofibrillar proteins from porcine longissimus PROTEIN OXIDATION AND MEAT QUALITY
dorsi were dissolved in phosphate buffer. The decrease of tryp-
tophan could be determined by the emission spectra recorded
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Protein Oxidation in Fresh Meat During Postmortem Aging

from 300 to 400 nm at the excitation wavelength of 283 nm
(Estévez et al., 2008). They reported that tryptophan fluores- In postmortem muscle, protein oxidation has been gradu-
cence was decreased in the early period of 10-day storage while ally recognized as an important factor for meat quality. During
the fluorescence of protein carbonyls was generated in late stage postmortem storage, muscle has a decreased ability to maintain
as secondary protein oxidation products (Estévez et al., 2008). its antioxidant defense system, which can lead to the increased
accumulation of reactive oxygen and nitrogen species (Renerre
et al., 1996). These species can cause increased levels of protein
Liquid Chromatography-Electrospray Ionization-Mass oxidation, which can modify protein structure and function in-
Spectrometry (LC-ESI-MS) cluding enzyme activity. These changes are possibly involved in
mediating the conversion of muscle to meat, and thus, regulating
Recently, Estévez and his research group have developed a meat quality.
new method to detect special protein oxidation products, α- Protein oxidation can naturally occur during the postmortem
aminoadipic semialdehydes (AAS) and γ -glutamic semialde- aging and refrigerated storage. Using fluorescence microscopy
hydes (GGS), and applied this method to meat products as well to detect carbonyl content in muscle cells, Astruc et al. (2007)
as other food proteins (Armenteros et al., 2009; Estévez et al., determined the localization of protein oxidation in bovine rectus
2009). AAS is an oxidative product from the deamination of abdominis with three different treatments (chemical oxidation
lysine and GGS is generated from the oxidation of arginine reagents of Fe2+/H2 O2 , refrigerated storage at 4◦ C, and cook-
and proline residues through Maillard reaction (Requena et al., ing at 100oC). All of these treatments induced protein oxidation
2003). Akagawa et al. (2005) developed a high-performance liq- at both the periphery and the inside of muscle cells. Particu-
uid chromatography (HPLC) method to detect AAS and GGS larly, the amount of protein carbonyl increased fourfold from
in BSA in vitro after derivatizing the protein with NaCNBH3 during 10 days of refrigerated storage. In addition, the levels
and the p-amino-benzoic acid (ABA). For the first time, Ar- of protein oxidation at day 10 after refrigerated storage were
menteros et al. (2009) employed ABA derivatization procedure similar to the levels of oxidation induced by 1 mM of oxidant
and a LC-ESI-MS technique to detect the oxidation products mixture (FeSO4 and H2 O2 ). Protein oxidation was not equally
of specific amino acids in meat products. AAS and GGS were distributed in muscle cells. The peripheral area corresponding
successfully detected in ground meat, dry-cured ham, dry-cured to the cell membrane and to a region in close contact with the
loin, dry-cured sausage, and cooked sausage. Among the meat cell membrane showed higher extent of protein oxidation com-
products, dry-cured ham and dry-cured sausages showed the pared to an inner area containing myofibrils and sarcoplasmic
highest GGS, while the amount of AAS was higher in ground proteins (Astruc et al., 2007). The authors concluded that pro-
meat and cooked sausage than other meat products (Armenteros tein oxidation was initiated from the muscle membrane to the
et al., 2009). This indicates that AAS and GGS are the form inside of muscle cells during postmortem storage. Lipophilic
of oxidation products in lysine, arginine and proline residues of antioxidants such as vitamin E may be powerful reagents to
myofibrillar proteins in fresh meat and processed meat products. limit the deterioration by protein oxidation (Rowe et al., 2004a;
In other food proteins including α-lactalbumin, soy proteins, and Astruc et al., 2007). Martinaud et al. (1997) also found that car-
BSA, LC-ESI-MS plus ABA derivatisation procedure was ef- bonyl content increased in longissimus lumborum (70% from
fective in determining the contents of AAS and GGS derived 3.1 to 5.1 nmol/mg protein) and diaphragma pedialis (44% from
from the oxidation by Fe3+ and H2 O2 (Estévez et al., 2009). 4.8 to 6.9 nmol/mg protein) of beef, respectively, during a 10-
The DNPH measurements were significantly correlated with day aging period. Lambs raised on concentrated diet produced
the amount of AAS and GGS determined by LC-ESI-MS. The greater amount of protein carbonyls than those on pasture during
 1196 W. ZHANG ET AL.

storage (Santé-Lhoutellier et al., 2007). The carbonyl content in related with meat tenderness at 1, 3, and 7 days of postmortem
turkey M. sartorius and M. pectoralis increased to 2.53 and 1.83 storage. Increased shear force associated with protein oxidation
nmol/mg protein, respectively, after 9 days of postmortem re- was suggested to be related to the aggregation of myofibrillar
frigerated storage under oxygen permeable conditions (Mercier proteins or due to the inhibition of proteolytic enzyme activity
et al., 1998). (Rowe et al., 2004a). Vitamin E was found to be effective in de-
creasing the amount and the extent of oxidation of sarcoplasmic
proteins. In a parallel study, Rowe et al. (2004b) determined the
High-Oxygen Modified Atmosphere Packaging effects of irradiation-induced oxidation on protein degradation
and Protein Oxidation and calpain activity in beef. Compared to nonirradiated beef-
steaks, irradiated samples had more intact and less degraded
Modified atmosphere packaging (MAP) with high oxygen products of desmin after 3, 7, and 14 days of refrigerated stor-
content (70–80%) has been widely used in fresh meat retail age. The decreased proteolysis was likely due to the inhibi-
market due to the bright red color in high-oxygen MAP system tion of μ-calpain activity by irradiation (Rowe et al., 2004b).
(Eilert, 2005; Seyfert et al., 2005). Studies have been focus- In nonirradiated samples, steaks from vitamin E-supplemented
ing on the changes of fresh meat quality and microorganisms animals showed faster degradation of troponin-T than those
induced by MAP, but very limited research on the effects of from control animals not supplemented with vitamin E. The
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MAP on protein oxidation. In high-oxygen packaging systems, differences in protein degradations were associated with higher
oxygen could possibly lead to protein oxidation and thus influ- Warner–Bratzler shear force in irradiated steaks than nonirradi-
ence fresh meat quality during postmortem chill storage. Kim ated steaks at 1, 3, 7, and 14 days postirradiation. Yoon (2003)
et al. (2010) reported that beef steaks of longissimus lumborum reported that low-dose γ -irradiation was associated with higher
in high-oxygen MAP system showed higher star probe val- cook loss and greater shear force in chicken breast meat during
ues and lower subjective tenderness scores than samples from 14 days of refrigerated storage.
vacuum packaging. The decreased tenderness was associated
with the formation of cross-linkages between myosin and titin
and was independent to the postmortem protein degradation
and the activity of μ-calpain (Kim et al., 2010). Similar re- Protein Oxidation and the Calpain System
sults were reported in porcine longissimus dorsi when muscle
samples were packaged in 70% oxygen and 30% CO2 (Lund The calpain system is generally believed to be responsi-
et al., 2007). High oxygen in the packaging system resulted in ble for regulating the degradation of proteins in postmortem
lower tenderness, which could be explained by protein oxida- muscle, and is known to be associated with meat tender-
tion. Cross-linkage between myosin heavy chains by disulfide ness and water-holding capacity during postmortem storage
bonds and decreased amounts of free thiol groups were shown (Koohmaraie, 1992; Huff-Lonergan et al., 1996; Huff-Lonergan
in high-oxygen package systems compared to skintight pack- and Lonergan, 2005; Zhang et al., 2006). Three members of
aging (Lund et al., 2007). Clausen et al. (2009) reported that the calpain system are the proteases m- and μ-calpain and
high-oxygen MAP resulted in decreased tenderness, lower val- their inhibitor calpastatin (Goll et al., 2003). Calpains autolyze
ues of myofibril fragment index, and higher levels of protein in the presence of calcium and this autolysis is indicative of
carbonyl compounds in beef longissimus dorsi, indicating the their proteolytic activation in postmortem muscles (Geesink and
possible association between tenderness and protein oxidation Koohmaraie, 1999). Since both m- and μ-calpain are cysteine
and proteolysis. Based on these limited studies, MAP with high protease, oxidation may regulate their proteolytic activities, and
oxygen has negative influence on fresh meat quality, especially thus, influence fresh meat quality.
tenderness and color. However, whether the decreased tender- Guttmann et al. (1997) reported that 100 μM H2 O2 strongly
ness is due to the oxidation of proteins or the reduced levels of inhibited μ-calpain-induced proteolysis including fluorescent
protein degradation is still inconclusive. peptide and the microtubule-associated protein tau at all calcium
concentrations. The autolysis rate and the extent of either 80 or
30 kDa subunits were not influenced by oxidation conditions
Irradiation-Induced Protein Oxidation and Meat Quality regardless of the calcium concentrations. In another study, cal-
pain activity was inhibited in SH-SY5Y human neuroblastoma
Irradiation is highly effective in controlling pathogenic mi- cells by oxidants doxorubicin. The autolytic rate for large sub-
croorganism in meat, and thus extending the shelf life of meat. unit from 80 to 76 kDa also decreased during oxidative stress,
However, irradiation can produce free radicals including •OH but oxidants did not influence the extent of calpain autolysis
and O2 −, which potentially increase the oxidation of proteins (Guttmann and Johnson, 1998).
and lipids in meat products (Jo and Ahn, 2000; Nam and Ahn, Carlin et al. (2006) further determined the effects of ox-
2003). Rowe et al. (2004a, 2004b) reported that the oxidation idation on the activity of purified m- and μ-calpain and the
of myoglobin played important roles in the discoloration of ir- interaction between μ-calpain and calpastatin. Proteolytic ac-
radiated beefsteaks. Carbonyl contents of myofibrillar and sar- tivity of μ-calpain and m-calpain was shown to be inhibited by
coplasmic proteins in irradiated beefsteaks were negatively cor- H2 O2 -induced oxidation at different pH including 6.0, 6.5, and
 PROTEIN OXIDATION ON MEAT QUALITY 1197

7.5. Interestingly, H2 O2 decreased the inhibition of μ-calpain ferent oxidation systems including chelated ferric iron, H2 O2 ,
activity by calpastatin at pH 6.5 and 7.5, and allowed μ-calpain and ascorbate. The shear force and true strength of cooked gels
to degrade desmin in the presence of calpastatin (Carlin et al., increased by 70 and 20%, respectively (Srinivasan and Hultin,
2006). They reported oxidation decreased the conversion of 1997). In beef heart surimi, antioxidant washing (propyl gallate
80 kDa subunit to 76 kDa without changing the extent of autol- and α-tocopherol) of minced muscle caused a decreased abil-
ysis of large subunit. Both intact 80 kDa and autolyzed 76 kDa ity of gel formation and increased levels of protein carbonyl
products of large subunit of μ-calpain were sensitive to ox- compared to water washing treatment (Srinivasan and Xiong,
idative environment. Inactivation of intact μ-calpain could be 1996). The increased gel-forming ability due to protein oxida-
reversed by a reducing agent such as dithiothreitol (Guttmann tion may be associated with the formation of cross-linkage be-
et al., 1997). The inhibition of μ-calpain activity was possi- tween polypeptides and between proteins. These cross-linkages
bly associated with the formation of a disulfide bond between can decrease the mobility of gel network and stabilize other
Cys115 and Cys108 within active site by LC-MS/MS analysis non-covalent bonds within the gel matrix (Decker et al., 2000).
(Lametsch et al., 2008). This disulfide bond was also detected in Protein oxidation can induce protein polymerization and
calpain purified from porcine meat and in calpain incubated with aggregation, and thus, change their digestibility, which nega-
reducing agent β-mercaptoethanol, indicating that the cysteine tively influences the nutritional values of muscle foods. Protein
within the active site is highly susceptible to the formation of oxidation can change the intermolecular and intramolecular in-
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intramolecular disulfide bond. All these studies provide strong teractions within a protein, and thus, influence their confor-
evidences that protein oxidation is involved in regulating fresh mation. These changes generally can increase the surface hy-
meat quality through mediating the autolysis and proteolytic drophobicity of a protein due to changes in the tertiary struc-
activity of calpains. ture. In addition, protein oxidation is associated with the for-
mation of dimmer, trimer, and polymer, and other inter- and
intra-cross-linkage, which can further lead to protein aggrega-
tion (Hanan and Shaklai, 1995). High levels of oxidation can
Protein Oxidation and Protein Functionality in Meat result in protein denaturation and precipitation, which is asso-
and Meat Products ciated with decreased protein solubility. Earlier studies showed
that linoleic acid and linoleic acid hydroperoxides could de-
Protein oxidation-induced structural modification can change stroy A-band structure of thick filament in fish and cause the
their functionality and thus influence products quality. During myosin denaturation. After incubation with hydroperoxides for
processing of meat products, mechanical strength can destroy 2.5 hours, solubility of myofibrillar protein can be decreased by
the integral structure of cells and break up antioxidant defense 90% due to the decreased extractability (Jarenback and Lilje-
systems resulting in high susceptibility to protein oxidation. Af- mark, 1975). The solubility of myofibrillar proteins from turkey
terturkey white muscles wereincubated withoxidants (iron chlo- white muscle decreased by 32 and 36%, respectively, when
ride or copper chloride) for 6 hours, the strength of gel formed the proteins were added in an oxidant system containing iron,
from myofibrillar proteins decreased from 0.76 to 0.05 and 0.08, copper, and ascorbate (Decker et al., 1993). Sarcoplasmic pro-
respectively. The water-holding capacity of gels oxidized by iron teins are also susceptible to oxidative modifications. Rowe et al.
and copper also decreased by 23 and 10%, respectively (Decker (2004a) found that irradiation of beef at 6.4 kGy induced pro-
et al., 1993). Liu et al., (2010) reported that pork longissimus tein oxidation in sarcoplasmic proteins and resulted in decreased
muscle showed lower water-holding capacity after incubation protein extractability after 4 and 14 days of refrigerated storage.
with FeCl3 /ascorbate/H2 O2 at 4◦ C. Changes in water-holding Lipid free radicals caused the aggregation and denaturation of
capacity and product yield were consistent with the increases of myoglobin and decreased protein solubility in bovine myosin-
protein carbonyl content and cross-linkage among both myofib- methyl linoleate emulsion system (Nakhost and Karel, 1983).
rillar and sarcoplasmic proteins. The decreased water-holding Beef longissimus thoracis showed high levels of protein oxida-
capacity could be explained by the enlarged extracellular space tion when they were cooked to the surface temperature of 65, 96,
between adjacent fibers in the oxidized muscle samples (Liu and 207◦ C (Gatellier et al., 2010). High temperature resulted in
et al., 2010). Effects of protein oxidation on their functional higher levels of protein carbonyl content and lower contents of
properties were also studied by incubating muscles with antiox- tryptophan and tyrosine. The amounts of cysteine, methionine,
idant. In minced beef heart muscle, myofibrillar proteins showed and tyrosine decreased significantly in ion-catalyzed oxidizing
increased shear stress and viscosity after they were washed with system, while the amount of cysteine, glycine, histidine, ala-
combined antioxidants containing 0.02% propyl gallate, 0.2% nine, leucine, lysine, and the total amounts of amino acids were
sodium ascorbate, and 0.2% sodium tripolyphosphate. The an- significantly lower in metmyoglobin-oxidizing system after the
tioxidant washing improved other functionality of myofibrillar myofibrillar protein isolate was incubated with those oxidants
proteins including greater storage, less moduli within 50–60◦ C, for 24 hours at 4◦ C (Park and Xiong, 2007). Oxidation of these
and stronger gels (Wan et al., 1993). However, protein oxida- amino acids along with other essential amino acids including
tion is not always associated with deleterious effects on protein lysine, histidine, arginine, and threonine could decrease the nu-
functionality. Minced cod muscle showed improved gelation tritional values of meat and meat products (Liu and Xiong,
and emulsification characteristics when exposed to three dif- 2000).
 1198 W. ZHANG ET AL.

Interactions between Protein and Lipid Oxidation respectively, as the oxymyoglobin concentrations increased
from 0.25 to 0.625 and 2.5 mg/ml after 1 hour incubation at 37◦ C
Oxidation of oxymyoglobin to metmyoglobin results in (Chan et al., 1997). Chan et al. (1997) also reported that H2 O2
meat discoloration, while lipid oxidation leads to the produc- instead of O2 − was directly involved in regulating the interaction
tion of off-flavor and decrease nutritional values of meat and of oxidation of oxymyoglobin and lipids. In conclusion, protein
meat products. The mechanisms behind and the control of in- oxidation-induced changes can influence fresh meat quality dur-
teractions between myoglobin and lipid oxidation have been ing postmortem aging and subsequent protein functionality of
reviewed by Baron and Andersen (2002), and recently by Faust- meat products. Addition of antioxidant such as vitamin E in diet
man et al. (2010). Many studies have focused on the interac- and other technological strategies could be effective to limit de-
tions between protein and lipid oxidation, and increased evi- teriorative effects of both lipid and protein oxidation in muscle
dence supports the idea that oxidation products from protein foods as recently reviewed by Lund et al. (2010).
and lipid can further increase the oxidation in a reciprocal
manner (Faustman et al., 2010). Amino acids are more sus-
ceptible to damage by secondary products of lipid oxidation CONCLUSIONS
such as aldehydes compared to primary products of lipid ox-
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idation such as hydroperoxides. Secondary products derived Protein oxidation induced by ROS can cause the modifica-
from lipid oxidation can interact with the amino acid residues tion of backbones and side chains of proteins. These modifi-
of proteins regulating their structure and function. For exam- cations lead to the structural changes at the levels of primary,
ple, hydroperoxides and aldehydes produced from lipid oxida- secondary, and tertiary structure of proteins. These structural
tion can react with lysine residues to lead to the formation of changes can induce conformational and functional modifica-
pyrrole derivatives (Hidalgo et al., 1998). The derivatives of tions of proteins including enzyme activity. Protein oxidation
interactions between lipid oxidation products and amino acid especially μ-calpain oxidation can influence the conversion of
residues can cause the formation of cross-linkage between pro- muscle to meat and thus contribute to the variation in meat qual-
teins. The reaction between 4,5-epoxy-2-alkenals and the amino ity including tenderness and water-holding capacity. Oxidative
groups of amino acids results in the formation of N-substituted modification of proteins can change their properties including
pyrroles (II), which can polymerize rapidly and lead to the de- solubility, nutritional values, gel-forming ability, surface hy-
velopment of brown color (Zamora et al., 2000). Lysine moi- drophobicity, and water-binding activity. Interactions between
eties of proteins could react with γ -hydroxy-α,β-unsaturated protein and lipid oxidation can lead to further oxidation in a
epoxides generated from lipid oxidation and form some concomitant and a reciprocal manner. Although great efforts
compounds including 4-(propylamino)-trans-2-hexene-1,5-diol have been exerted to explore the influence of protein oxidation
and N 2-acetyl-N 6-(1,5-dihydroxy-trans-2-hexen-4-yl)-l-lysine in muscle foods recently, the occurrence and the mechanisms
4-methylcoumar-7-ylamide in a model system with trans- of protein oxidation in meat systems are still largely unclear.
4,5-epoxy-trans-2-hexen-1-ol (EH) and propylamine (Lederer, Development of new, accurate methods to detect the modifica-
1996). After beef and porcine oxymyoglobin were incubated tions of amino acid residues in proteins will help understanding
with HNE, two histidine residues of porcine myoglobin while the mechanisms and the products of protein oxidation in meat
four histidine residues of beef myoglobin were attached with systems. The roles of protein oxidation on fresh meat quality
HNE via Michael addition (Suman et al., 2006). Adduction in early postmortem stage should be further studied especially
of HNE to oxymyoglobin changed the tertiary structure and with the consideration that oxidation of some key enzymes may
promoted its susceptibility to oxidation (Maheswarappa et al., regulate the rate of glycogen metabolism and the concentration
2009). Lipid oxidation as a promoter of protein oxidation can of calcium in cytoplasm of muscle cells in early postmortem
be evidenced by feeding animals with different status of unsatu- stage. In addition, the interaction between protein oxidation and
rated fatty acids (Nute et al., 2007). Zhang et al. (2010) reported lipid oxidation and how combinations of these modifications
that consumption of oxidized oil was related to increased levels modulate muscle food quality should be better explained. Un-
of protein carbonyl and decreased meat quality including lipid derstanding the mechanisms of protein oxidation will enable us
oxidation and drip loss in breast meat of broiler chickens. to produce muscle foods with better appearance, sensory quality,
Some proteins in muscle foods especially myoglobin can act and nutritional values in future.
as pro-oxidants that initiate and accelerate lipid oxidation (Baron
and Anderson, 2002). Increased levels of oxygen in packag-
ing systems of beefsteaks were associated with increased lipid
oxidation and decreased color stability (Zakrys et al., 2008). ACKNOWLEDGMENTS
Strong negative correlations were found between the content of
2-thiobarbituric acid reactive substances (TBARS) and the con- This study was supported jointly by Iowa State University and
centration of oxymyoglobin especially in high-oxygen packag- WCU (World Class University) program (R31-10056) through
ing groups (Zakrys et al., 2008). In a myoglobin-liposome sys- the National Research Foundation of Korea funded by the Min-
tem, the TBARS values increased by 12.5-, 17.1-, and 19.0-fold, istry of Education, Science and Technology.
 PROTEIN OXIDATION ON MEAT QUALITY 1199

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