Protein thiols undergo reversible and irreversible oxidation during chill storage of ground beef as...

Protein Thiols Undergo Reversible and Irreversible Oxidation duringChill Storage of Ground Beef as Detected by 4,4′-DithiodipyridineTine Rysman,† Sisse Jongberg,‡ Geert Van Royen,† Stephanie Van Weyenberg,† Stefaan De Smet,§

and Marianne N. Lund*,‡

†Technology and Food Science Unit, Institute for Agricultural and Fisheries Research (ILVO), Brusselsesteenweg 370, 9090 Melle,Belgium‡Department of Food Science, Faculty of Science, University of Copenhagen, Rolighedsvej 30, 1958 Frederiksberg, Denmark§Laboratory for Animal Nutrition and Animal Product Quality, Faculty of Bioscience Engineering, Ghent University, Proefhoevestraat10, 9090 Melle, Belgium

ABSTRACT: Quantification of protein thiols and disulfides in ground beef during storage under high-oxygen atmosphere at 4°C was performed by thiol detection using 4,4′-dithiodipyridine (4-DPS) before and after disulfide reduction using sodiumborohydride. Two independent storage trials were performed, and in trial 1, only reversible thiol oxidation was observed (thiolloss was 30%). In trial 2, irreversible thiol oxidation occurred during the first days of storage, while further loss of thiols wascaused by reversible disulfide formation (thiol loss was 33%, of which ca. half was lost because of irreversible oxidation). Theresults were compared to sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) analysis of cross-linkedmyosin heavy chain formed by disulfide bonding. Both methods confirmed increasing disulfide formation because of thioloxidation in meat during storage, but the 4-DPS method showed higher disulfide percentages than the SDS−PAGE method(22.2 ± 0.3% and 8.5 ± 1.2%, respectively). The 4-DPS assay provides an accurate method to evaluate the thiol−disulfide redoxstate in meat.

KEYWORDS: meat proteins, protein oxidation, thiols, disulfides, 4,4′-dithiodipyridine

■ INTRODUCTION

Protein oxidation in meat and meat products is known to havean impact on protein functionality, sensory aspects, andnutritional values.1,2 The oxidation of thiol groups (RSH) ofthe cysteine residue in myosin (the most abundant myofibrillarprotein) results in the formation of disulfide cross-links (RSSR)and has shown to alter texture properties, resulting in adecreased tenderness and juiciness of meat.2−6 Furthermore,the structural changes in proteins because of cross-linking mayaffect their recognition sites for proteases, making the oxidizedproteins less susceptible to proteolysis, resulting in decreaseddigestibility.7−9 Thiol oxidation is complex and may lead to theformation of multiple oxidation products, such as disulfides,sulfenic acid, sulfinic acid, sulfonic acid, and thiosulfinates, ofwhich disulfides and sulfenic acid are reversible thiol oxidationproducts.10 Most often oxidation of thiol groups is evaluated byquantification of the loss of thiol groups and not byquantification of thiol oxidation products, but it is generallybelieved that thiol loss results primarily in the formation ofdisulfides (e.g., see ref 11), although no direct evidence for thisstatement can be found in the literature. Inhibition of thioloxidation in meat during storage has been attempted by use ofplant extracts rich in phenolic compounds, which are known toeffectively reduce lipid oxidation but have not been shown to beable to prevent disulfide formation in meat.12 It is thereforeimportant to quantify the amount of reversibly and irreversiblyoxidized thiols to further develop antioxidant strategies to avoidor repair thiol oxidation in meat.

The degree of protein disulfide cross-linking in meat andmeat products can be evaluated by means of protein separationby sodium dodecyl sulfate−polyacrylamide gel electrophoresis(SDS−PAGE).13 However, measurement of the protein bandpixel intensity on the SDS−PAGE gel is merely semi-quantitative, and irreversible thiol oxidation cannot beevaluated by this method. A sensitive method to quantifydisulfide formation is thus needed to obtain clear insight intothe thiol−disulfide chemistry in meat and meat products. Thespectrophotometric measurement of the loss of free thiolgroups in muscle proteins has been widely used as an indicatorof protein oxidation.14 The quantification of protein disulfidesis somewhat more complex, because disulfide bonds must firstbe cleaved before detecting the newly formed thiols. To cleaveall disulfide bonds, a strong and efficient reducing agent mustbe selected. Furthermore, this reducing agent must not cross-react with the thiol detection agent.15

An approach for measuring thiols and disulfides is shown inFigure 1. Thiols can be detected by reaction with a thiol-specificdetection agent with useful spectrophotometric properties(Figure 1A). The most common thiol detection reagent is5,5′-dithiobis(2-nitrobenzoic acid) (DTNB or Ellman’s re-agent), which forms a disulfide bond with free thiol groups,releasing a yellow NTB dianion (2-nitro-5-thiobenzoate) with

Received: July 18, 2014Revised: October 31, 2014Accepted: November 9, 2014Published: November 9, 2014

Article

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© 2014 American Chemical Society 12008 dx.doi.org/10.1021/jf503408f | J. Agric. Food Chem. 2014, 62, 12008−12014

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maximum absorbance at 412 nm in the pH range from 6 to9.5.14,15 An alternative to DTNB is 4,4′-dithiodipyridine (4-DPS), which reacts in a similar way with thiol groups, forming4-thiopyridone (4-TP) that absorbs at 324 nm. The small sizeof this detection reagent makes it easier to reach poorlyaccessible thiols within the protein core. 4-TP also has a higherextinction coefficient than NTB, suggesting that 4-DPS is amore sensitive thiol detection agent than DTNB.16 Further-more, 4-TP is stable in lower pH ranges (pH 3−7), makingthiol detection possible at low pH as in common reversed-phase high-performance liquid chromatography (HPLC)conditions.17 Ruan et al.18 found 4-DPS to be less affected bypH and denaturants compared to DTNB and, thus, found 4-DPS more suitable and reliable for thiol detection in soyprotein. The measurement of disulfide bonds is based on thereduction of disulfides, followed by detection of the newlyreduced thiols (Figure 1B). The amount of disulfides can thenbe calculated by subtracting the amount of free thiols from totalthiols. To reduce disulfide bonds during SDS−PAGE,dithiothreitol (DTT) is often used. This reagent is less suitablefor disulfide quantification because of its slow reaction rate andthe cross-reactivity with thiol detection agents.15,17 Liu et al.19

have quantified disulfide bonds in myofibrillar proteins basedon a method described by Damodaran.20 In this procedure,disulfides are cleaved by adding an excess of sodium sulfite.However, oxidative sulfitolysis of disulfide bonds is slow andrarely quantitative in the absence of catalysts.15,21 An alternativereducing agent is sodium borohydride (BH), a highly reactiveand strong reductant, whose main advantage is that its excesscan be removed by acidification.15,22

The aim of the present study was to investigate the extent ofreversible and irreversible thiol oxidation in ground beef duringstorage. A sensitive and reliable method for the quantificationof protein disulfides in meat was established on the basis ofdisulfide reduction with sodium borohydride and thioldetection with 4-DPS. The method was subsequently usedfor quantification of reversible and irreversible thiol oxidation inground beef during storage in high-oxygen modified atmos-phere packaging (HiOx MAP) and compared to SDS−PAGE-based methodology.

■ MATERIALS AND METHODSChemicals. All chemicals were of analytical grade or the highest

available purity. Citric acid was purchased from Carl Roth GmbH(Karlsruhe, Germany). Disodium hydrogen phosphate was purchasedfrom Panreac Quimica, S.A.U. (Castellar del Valles, Barcelona, Spain).Trisodium citrate dihydrate, sodium dodecyl sulfate (SDS), hydro-chloric acid, tris(hydroxymethyl)-aminomethane (Tris), 1-octanol,sodium hydroxide, and bovine serum albumin (BSA) were obtainedfrom Merck (Darmstadt, Germany). Guanidine hydrochloride(GuHCl), L-cysteine, and sodium borohydride were purchased fromSigma-Aldrich Co. (St. Louis, MO). 4-DPS and oxidized glutathione(GSSG) were obtained from Acros Organics (Geel, Belgium).

NuPAGE Novex 3−8% Tris−acetate gels, lithium dodecyl sulfate(LDS) sample buffer, SDS Tris−acetate running buffer, and MolecularProbes SYPRO Ruby Protein Gel Stain were purchased fromInvitrogen (Carlsbad, CA). Precision Plus Protein Standard All Bluewas obtained from Bio-Rad Laboratories, Inc. (Hercules, CA). DTTwas purchased from Applichem GmbH (Darmstad, Germany). Waterwas prepared using a Millipore Mill-Q purification system (MilliporeCorp., Bedford, MA).

Sampling of HiOx MAP Ground Meat. Two independentstorage trials (trials 1 and 2) were performed for sampling of HiOxMAP ground beef. For trial 1, ground beef stored in HiOx MAP wasobtained from a local Danish supermarket. The meat had been packedunder modified atmosphere (high oxygen) the day before andconsisted of nine packages of 400 g each. Three packages (replicates A,B, and C) were opened, and the meat was vacuum-packed in portionsof 50 g and stored at −80 °C until analysis (day 0). The remaining sixpackages were stored in darkness at 4 °C until day 4 or 9, at whichpoint another three replicates (A, B, and C) were collected, vacuum-packed, and frozen to −80 °C.

For trial 2, similar sampling was performed for freshly ground beefobtained from a local Belgian butcher, ground 3 days after slaughter ofthe animal. The meat was packed under modified atmosphere (70%O2 and 30% CO2) into 15 packages of 450 g each and stored at 4 °Cuntil sampling day. On days 0, 3, 6, 9, and 12, three packages(replicates A, B, and C) were opened, vacuum-packed in portions of75 g, and stored at −80 °C until analysis.

Influence of pH and Denaturant on the Molar ExtinctionCoefficients of 4-TP. A total of 24 buffers were prepared, by varyingthe buffering capacity, the pH value, and the presence of a denaturant.Six buffers with a low buffering capacity were prepared by mixing 0.1M citric acid and 0.2 M Na2HPO4 to pH values of 2.5, 3.5, 4.5, 5.5, 6.5,and 7.5. Likewise, six high-capacity buffers were prepared by mixing 1M citric acid and 1 M trisodium citrate dihydrate to theaforementioned pH values. SDS buffers were prepared by adding 5%(w/v) SDS to the low-capacity buffers. The same was performed forguanidine hydrochloride by adding 6 M GuHCl to the high-capacitybuffers. Concentrations of L-cysteine ranging from 2.5 to 500 μM wereprepared in all buffers. Thiol detection with 4-DPS was performed onthe basis of the study by Riener et al.,16 by diluting 500 μL of L-cysteine solution with 2 mL of the corresponding buffer and adding500 μL of 4-DPS solution (4 mM 4-DPS in 12 mM HCl). Theabsorbances were measured at 324 nm against water before theaddition of 4-DPS (Apre) and after exactly 30 min of reaction with 4-DPS in the dark at room temperature (Apost). The absorbancecorresponding to the thiol concentration was calculated by subtractingApre and Ablank (2.5 mL of buffer + 500 μL of 4-DPS solution) fromApost. Molar extinction coefficients (ε) were calculated from the slopeof the standard curves according to the Lambert−Beer law (A is theabsorbance, and is the path length).

ε=A c (1)

Optimization of Disulfide Reduction. The efficiency of thedisulfide reduction was tested by the reduction of knownconcentrations (25−200 μM) of commercially available GSSG. Theexpectation was to find thiol concentrations in the form of reducedglutathione (GSH) double the aforementioned molarities (50−400μM). GSSG was dissolved in 6 M GuHCl in 100 μM Tris buffer (pH8.0), because this was the buffer system that was chosen to solubilizethe meat proteins. The method was developed on the basis of disulfidereduction according to Hansen et al.17 The reduction was performedby adding 100 μL of 30% (w/v) alkaline borohydride to 3 mL ofsample. After incubation at 50 °C, the excess of borohydride wasremoved with 6 M hydrochloride (final concentration of 1.8 M) atroom temperature. Different conditions and parameters were tested byvarying the solvent for sodium borohydride (1, 2, and 4 M NaOH),the incubation time for the reduction with borohydride (15, 30, 60,and 150 min), and the hydrolysis time of the residual borohydride (2,10, 20, 30, 40, 45, 60, 90, 150, 165, and 180 min).

Quantification of Free and Total Thiols in MAP Ground BeefUsing 4-DPS. The vacuum-packed meat was thawed in water at room

Figure 1. Approach for detection of (A) free thiols and (B) totalthiols. Thiols destined for detection are denoted S* (modified withpermission from ref 15. Copyright 2009 Elsevier).

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temperature for 30 min before homogenizing 1.0 g of meat in 25 mLof 6 M GuHCl in 100 mM Tris buffer (pH 8.0) using an Ultra Turrax.The homogenates were centrifuged (for 20 min at 5311g and 4 °C),and supernatants were filtered (qualitative filter paper, particleretention of 11 μm). Samples were kept on ice at all times. Theprotein concentration of the filtrates was determined spectrophoto-metrically at 280 nm using a five-point standard curve prepared fromBSA. Disulfide reduction was performed according to the optimizedprocedure, as described in the previous section. Hence, an aliquot of 3mL of filtrate was subjected to disulfide reduction by adding 50 μL of1-octanol (as an anti-foaming agent) and 100 μL of freshly prepared30% (w/v) sodium borohydride in 1 M NaOH. After incubation at 50°C for 30 min, an aliquot of 1.35 mL of 6 M HCl was added, followedby stirring for 10 min.Free and total thiols were determined with 4-DPS in the non-

reduced and reduced filtrates, respectively. An aliquot of 500 μL offiltrate was mixed with 2 mL of 6 M GuHCl in 1 M citric acid buffer(pH 4.5) and 500 μL of 4-DPS solution (4 mM 4-DPS in 12 mMHCl). The absorbance was measured at 324 nm against 6 M GuHCl in1 M citric acid buffer (pH 4.5) before the addition of 4-DPS (Apre) andafter exactly 30 min of reaction with 4-DPS in the dark at roomtemperature (Apost). A mixture of 2.5 mL of 6 M GuHCl in 1 M citricacid buffer (pH 4.5) and 500 μL of 4-DPS solution was prepared as ablank sample (Ablank). The absorbance corresponding to the thiolconcentration was calculated by subtracting Apre and Ablank from Apost.The thiol concentration was calculated on the basis of a five-pointstandard curve ranging from 2.5 to 500 μM L-cysteine in 6 M GuHClin 1 M citric acid buffer (pH 4.5). The thiol content was expressed asnanomoles of thiol per milligram of protein. Because treatment ofdisulfide bonds with sodium borohydride yields two thiol groups,23 thedisulfide content was calculated as half of the difference between totaland free thiols.SDS−PAGE Analysis. The meat from trial 1 was subjected to

SDS−PAGE analysis. The meat was thawed at room temperature for30 min before an aliquot of 1.0 g of meat was homogenized in 25 mLof 5% (w/v) SDS dissolved in 100 mM Tris buffer at pH 8.0 using anUltra Turrax. The homogenates were placed in a water bath at 80 °Cfor 30 min, followed by centrifugation for 20 min at 950g. Thesupernatant was filtered (particle retention of 5−13 μm), and theprotein concentration was determined spectrophotometrically at 280nm using a standard curve prepared from BSA. The homogenates wereanalyzed by gel electrophoresis using NuPAGE Novex 3−8% Tris−acetate gels according to the instructions of the manufacturer(Invitrogen, Carlsbad, CA). Reduced samples were prepared bymixing 1.6 μL of diluted sample (2.0 mg of protein/mL) and 14.4 μLof loading solution, which was prepared from 60 μL of LDS samplebuffer, 24 μL of 1.0 M DTT, and 132 μL of Milli-Q water. The non-reduced samples were prepared by mixing 1.6 μL of diluted sample(2.0 mg of protein/mL) and 14.4 μL of loading solution, which wasprepared from 60 μL of LDS sample buffer and 156 μL of Milli-Qwater. Aliquots of 10 μL of reduced or non-reduced samples as well as3 μL of Precision Plus Protein Standard All Blue marker were loadedto the wells of the gels. Electrophoresis was run for 90 min at 150 V incassettes containing ice-cold SDS Tris−acetate running buffer. Afterelectrophoresis, the gels were fixed overnight in fixation solution (50%ethanol and 7% acetic acid) at room temperature on a rocking table.Following staining overnight by the fluorescence SYPRO Ruby ProteinGel Stain, the gels were photographed by a charge-coupled device(CCD) camera (Raytest, Camilla II, Straubenhardt, Germany). Theprotein concentration of the bands were estimated by the pixelintensity as determined by the freeware GelAnalyzer by Dr. IstvanLazar, www.gelanalyzer.com. Band intensities were quantified from thevolume of the pixel intensity when analyzing the gels withGelAnalyzer. To correct for total protein content of each sample,the pixel intensities of cross-linked myosin heavy chain (CL-MHC)and myosin heavy chain (MHC) were divided by the total pixelvolume of all bands in each lane of the gel to obtain the relativeintensity of CL-MHC and MHC for each sample. The degree of cross-linking was calculated on the basis of gels containing both reduced andnon-reduced samples and was considered equal to the ratio of the

relative intensity of CL-MHC in the non-reduced samples to therelative intensity of MHC in the reduced samples at day 0 (CL-MHCnon‑red/MHCred, day 0, %).

Statistical Analysis. Statistical analysis was performed using SAS9.3 package (SAS Institute, Inc., Cary, NC). Recovery percentageswere analyzed using a linear model with the concentration as fixedeffect. Free and total thiols were analyzed using a linear model with thestorage time (days), treatment (with or without reduction), and theirinteraction as fixed effects. Disulfides and degrees of cross-linking wereanalyzed using a linear model with the storage time as fixed effect. Thecorrelation between the 4-DPS assay and SDS−PAGE was assessedusing Pearson correlation. Effects were considered significant at p <0.05.

■ RESULTS AND DISCUSSIONInfluence of pH and Denaturant on the Molar

Extinction Coefficients of 4-TP. To determine the optimalpH for thiol detection with 4-DPS, two types of buffer solutionswith a low and high buffering capacity were prepared thatranged from pH 2.5 to 7.5. It is strictly necessary to includeprotein denaturants in the buffer during analysis of thiols inmeat to prevent precipitation of some of the cross-linkedprotein. In the absence of protein denaturants, such precipitantswould not be included in the assay, resulting in erroneousquantifications. The effect of denaturants was therefore testedby adding 5% SDS and 6 M GuHCl to the low- and high-capacity buffers, respectively. Molar extinction coefficients werecalculated from the slope of a L-cysteine standard curve, whichwas subjected to the 4-DPS assay at all pH and denaturantconditions.According to Riener et al.,16 the molar extinction coefficient

of 4-TP is 21 400 M−1 cm−1 at pH 7.0. As shown in Figure 2,

the molar extinction coefficients in all low- and high-capacitybuffers without denaturants stay close to this value. However,adding denaturants to the buffers clearly narrows the pH rangein which the molar extinction coefficients are stable. This is inagreement with the findings by Hansen and Winther,15 whofound that the presence of denaturants decreased the alkylationrate of NTB by N-ethylmaleimide. The lowest 4-TPabsorptions were measured at pH 2.5 with buffers added 5%SDS and 6 M GuHCl (ε = 8500 and 13 800 M−1 cm−1,respectively). The amount of GuHCl was not soluble at pH 6.5and 7.5; thus, Figure 2 shows no results for these conditions.Overall, optimal molar extinction coefficients were found in

buffers containing denaturants ranging from pH 3.5 to 5.5. Two

Figure 2. Molar extinction coefficients of 4-DPS as a function of pH.



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factors were considered to select the optimal working buffer.First, the pH value in the samples was very low afteracidification in the reduction step. Therefore, a high-capacitybuffer was needed to ensure correct pH adjustment. Second, astrong denaturant is crucial to ensure good solubility of meatproteins. We therefore chose to use 6 M GuHCl in 1 M citricacid buffer at pH 4.5 as a working buffer for determining thefree and total thiols in meat using 4-DPS.Optimization of Disulfide Reduction. It was necessary to

optimize the disulfide reduction in the buffer systems chosenfor the meat samples, and this was established by determinationof recovery percentages for the reduction of GSSG based onincubation at 50 °C with alkaline borohydride, followed byremoval of residual borohydride by acidification. The reductionprocedure was tested by combining different sodium hydroxideconcentrations with varying times of incubation and hydrolysis,as described above. For each combination, a knownconcentration of GSSG in 6 M GuHCl in 100 mM Tris buffer(pH 8.0) and a blank (6 M GuHCl in 100 mM Tris buffer atpH 8.0) were subjected to the reduction assay. Twice as muchGSH as GSSG in molar concentrations was expected; thus, therecovery percentages were calculated according to eq 2.

= ×recovery percentage (%)thiols quantifiedthiols expected

100(2)

It is well-known that sodium borohydride rapidly decomposesat neutral or acidic pH.24 Therefore, solutions of 30% (w/v)borohydride in 1, 2, and 4 M NaOH were freshly prepared forthe reduction of GSSG. After mixing 3 mL of sample with 100μL of BH dissolved in 1, 2, and 4 M NaOH, the pH wasapproximately 8.6, 11.0, and 12.0, respectively. AlthoughPatsoukis and Georgiou25 highlighted that a high alkalineenvironment (pH 12.0 rather than pH 8.0) is needed foroptimal disulfide reduction by borohydride, we found similarrecovery percentages with the 1 M NaOH treatment as with 2and 4 M NaOH (data not shown). The lack of correlationbetween our results and those reported by Patsoukis andGeorgiou25 may be due to the use of different buffers andderivatization agents.After the addition of BH, the mixtures were incubated at 50

°C as recommended by Patsoukis and Georgiou.25 The optimalincubation time for effective reduction depends upon theconcentration and reactivity of BH and the accessibility of theprotein disulfides. Incubation must take long enough to let BHreach and reduce all disulfides. Prolonged incubation, however,might promote reoxidation of the newly formed thiols.22

Furthermore, NaBH4 can decompose over time, even atalkaline pH.22,26 For the current method development, oxidizedglutathione was incubated with BH during 15, 30, 60, and 150min and optimal recovery percentages were found with 30 minof incubation (data not shown).Subsequent to incubation, the residual BH was removed by

acidification with 6 M HCl, bringing the final concentration ofHCl in the mixture to 1.8 M. Acidification of NaBH4completely discharges the reactive hydride, yielding hydrogengas (H2)

15,27 and causing the protein solution to foam. Thisfoaming was avoided by adding a small amount of 1-octanol.15

During the method optimization, the acidified solutions wereleft to stir on a magnetic stirrer at room temperature for varioustime periods, ranging from 2 min to 3 h. The best recoverypercentages were found when stirring for 10 min after theaddition of HCl and subsequently readjusting the pH with 6 M

GuHCl in 1 M citric acid buffer (pH 4.5) for the 4-DPS assay(data not shown). Although the low pH of the BH−HClmixture is likely to protect the reduced thiols fromreoxidation,15 oxygen inclusion because of prolonged andvigorous stirring may induce further oxidation after thereducing agent has been deactivated.22

The method for disulfide reduction was adjusted on the basisof all of the above-mentioned considerations. Optimal recoverypercentages in the range from 25 to 200 μM GSSG wereobtained when carrying out the reduction as follows: 50 μL of1-octanol and 100 μL of freshly prepared 30% (w/v) sodiumborohydride in 1 M NaOH were added to 3 mL of sample. Themixture was incubated at 50 °C for 30 min with occasionalstirring. After incubation, the excess of borohydride wasremoved by adding 1.35 mL of 6 M HCl, followed by stirringfor 10 min. This procedure was applied for the reduction ofvarious concentrations of GSSG. The quantified thiols andrecovery percentages are presented in Table 1.

No significant differences between recovery percentages werefound among the GSSG concentrations (p = 0.642), indicatingthat the reduction procedure is precise for concentrationsranging from 25 to 200 μM disulfides and resulted in anapproximate recovery of 86%. According to AOAC Interna-tional, the expected recovery is from 80−90 to 107−110% inthe concentration range of the quantified thiols used in thepresent study; therefore, the observed accuracy is acceptable.28

Quantification of Disulfides in MAP Ground Beef with4-DPS. MAP ground beef obtained from trials 1 and 2 weresubjected to thiol and disulfide quantification by the 4-DPSassay at various storage times. Caution should be made whencomparing the meat from trials 1 and 2 for several reasons: (1)the batches of meat were not obtained from the same animal;(2) ground beef often comes from different muscles; thus, anyobserved difference in thiol or disulfide concentrations betweenthe two trials could be ascribed to differences in the types ofmuscles used in each batch of meat; and (3) there aredifferences in time between slaughter and packaging and theexact gas composition (unknown for trial 1).The significant decrease in free thiols in the meat from trial 1

as well as trial 2 (Tables 2 and 3, respectively) shows that thioloxidation (ca. 30 and 33% loss of thiols, respectively) tookplace during storage, which is in agreement with previousstudies.3,29 After reduction of the proteins from the beef

Table 1. Recovery of GSH after Reduction of GSSG in 6 MGuHCl and 100 mM Tris Buffer (pH 8.0), with 30% (w/v)Sodium Borohydride in 1 M NaOH at 50 °C for 30 min,Followed by Acidification with 6 M HCl (FinalConcentration of 1.8 M) and Stirring for 10 mina

GSSG (μM) thiols expected (μM) thiols quantified (μM) recovery (%)

0 (blank) 0 3.8 ± 1.625 50 42.9 ± 3.6 85.8 ± 7.150 100 82.7 ± 4.7 82.7 ± 4.775 150 129.1 ± 2.6 86.1 ± 1.7100 200 175.3 ± 1.2 87.7 ± 0.6125 250 211.5 ± 8.6 84.6 ± 3.4150 300 262.7 ± 5.6 87.6 ± 1.9175 350 304.7 ± 6.5 87.1 ± 1.9200 400 350.1 ± 8.1 87.5 ± 2.0

aRecovery percentages are expressed as the ratio quantified toexpected thiols (described in eq 2).



samples from trial 1 with borohydride, the amount of totalthiols was significantly higher than the amount of free thiols atall sampling days, indicating the formation of disulfides. Nosignificant changes in total thiol levels were found during the 9days of storage (Table 2). This suggests that all thiols lostduring 9 days of storage could be fully reduced by borohydrideand were, hence, lost because of reversible thiol oxidation(most likely disulfide formation).When subjecting the beef from trial 2 to borohydride

reduction, no significant difference was found between the freeand total thiol level on day 0, indicating that the initial level ofoxidation in this meat was minimal. After 3 days of storage, theconcentration of free thiols was significantly lower than at day 0

(Table 3) (p < 0.001), confirming thiol oxidation duringstorage. Reduction of the beef proteins resulted in asignificantly higher concentration of total thiols than free thiolsat day 3 (p < 0.001), proving the formation of disulfides.However, the concentration of total thiols significantlydecreased from day 0 to day 3 (p < 0.001), showing thatirreversible thiol oxidation had taken place concomitantly to thedisulfide formation. Although the amount of total thiols stayedconstant between days 3 and 12, there was a significantdecrease of free thiols between days 3 and 12, indicating thatpredominantly protein disulfides were formed in this storageperiod. With subtraction of the free thiol concentration on day0 with the free thiol concentration on day 12, a loss in free

Table 2. Thiol and Disulfide Quantification with 4-DPS in Ground Beef from Trial 1, Stored under High-Oxygen Atmosphere at4 °C for up to 9 Daysa

storage time free thiols (nmol/mg of protein) total thiols (nmol/mg of protein) disulfides (nmol/mg of protein)

day 0 37.6 ± 0.5 a 51.7 ± 0.8 a 7.0 ± 0.6 aday 4 30.0 ± 1.1 b 49.2 ± 1.5 a 9.6 ± 1.1 bday 9 26.5 ± 1.7 c 49.4 ± 1.4 a 11.5 ± 1.4 c

aResults are shown as the mean ± standard deviation (SD) of three independent replicates. Different letters (a−c) within a column denote statisticaldifferences among storage times (p < 0.05).

Table 3. Thiol and Disulfide Quantification with 4-DPS in Ground Beef from Trial 2, Stored under High-Oxygen Atmosphere at4 °C during 12 Daysa

storage time free thiols (nmol/mg of protein) total thiols (nmol/mg of protein) disulfides (nmol/mg of protein)

day 0 58.3 ± 1.6 a 61.8 ± 0.2 a 1.8 ± 0.9 aday 3 44.4 ± 1.2 b 55.1 ± 1.2 b 5.3 ± 1.1 bday 6 42.4 ± 0.8 bc 54.4 ± 1.6 b 6.0 ± 1.1 bday 9 40.4 ± 2.4 bc 53.1 ± 2.2 b 6.4 ± 2.1 bday 12 39.2 ± 0.8 c 52.1 ± 1.6 b 6.4 ± 1.0 b

aResults are shown as the mean ± SD of three independent replicates. Different letters (a−c) within a column denote statistical differences amongstorage times (p < 0.05).

Figure 3. SDS−PAGE gel of non-reduced ground beef from trial 1 stored under high-oxygen atmosphere at 4 °C for 9 days. The gel shows allsamples in triplicate (A−C). CL-MHC, cross-linked myosin heavy chain; MHC, myosin heavy chain.



thiols of 19 nmol/mg of protein was obtained. Similarly, theloss in total thiols over 12 days of storage was only 10 nmol/mgof protein, which means that, over the full duration of storage,about half of the thiols was lost because of irreversible oxidationand the other half was lost because of reversible oxidation. Theconsequence of thiol oxidation in meat has previously beenascribed mainly to disulfide formation;3,29 however, thioloxidation is very complex, and numerous oxidation productsmay be formed, such as sulfenic acid, sulfinic acid, sulfonic acid,and thiosulfinates.10 The present study shows that other thioloxidation products than disulfides may be formed in meatduring storage. While the formation of sulfonic acid isirreversible, sulfenic acid is extremely reactive and may reactfurther to create compounds, such as disulfides or sulfonic acid.Because of the efficient reducing capacity of BH, it is likely thatsulfenic acids are also reduced to thiols by this reducing agent;thus, it is unknown whether a part of the quantified total thiolsis in fact derived from the reduction of sulfenic acid, thusoverestimating the amount of disulfides. However, theapproach based on BH and 4-DPS has previously been usedfor the quantification of the thiol−disulfide redox status in cellswithout considering a potential sulfenic acid formation.30 Theformation of sulfenic acids in living cells has been shown to berather widespread in some cases,31 but it has never beendetermined in meat samples during storage.Comparison between SDS−PAGE and 4-DPS for the

Quantification of Disulfides in MAP Ground Beef. Thequantification of thiols and disulfides in the ground beef fromtrial 1 with 4-DPS was compared to the conventional cross-linking analysis by SDS−PAGE. MHC has previously beenfound to be the meat protein that is most susceptible tooxidation, and SDS−PAGE analysis has been widely used tostudy disulfide cross-link formation in meat and meatproducts.3,8,13,29 The non-reduced samples showed an increaseof the CL-MHC band intensity on the SDS gel as the storagetime increased (Figure 3), whereas no CL-MHC was seen inthe reduced samples (data not shown).The degrees of disulfide formation in the ground beef

according to the 4-DPS assay and SDS−PAGE are compared inTable 4. With both methods, a significant increase of disulfideswas found in the ground beef during 9 days of storage underhigh-oxygen atmosphere (p < 0.001 and p = 0.005 for 4-DPSand SDS−PAGE, respectively). A Pearson correlation of 0.913

was found between the two methods, but the concentration ofdisulfides found by the 4-DPS assay was considerably higherthan that by SDS−PAGE. This can be explained by the fact thattwo proteins may be cross-linked by several disulfide bridges;consequently, cleaving these disulfide bridges leads to morethan two newly formed thiol groups. From the three-dimensional protein structure of MHC, it can be seen thatseveral cysteine residues are transversely positioned in the tailof MHC; thus, it is likely that several intermolecular disulfidesare formed between two MHC molecules. Furthermore, thiolsmay be lost to disulfide formation intramolecularly in MHC,without causing a cross-link between two proteins, that is,without generating the CL-MHC band that appears in theSDS−PAGE gel. Additionally, it is worth noting that severelyCL-MHC polymers are probably too large to enter the SDSgel,12 leading to an underestimation of CL-MHC. This is aproblem that is actually one of the many drawbacks with the gelelectrophoresis methods. Finally, even though MHC has beenfound to be the meat protein that is most prone to thioloxidation,32 disulfide formation in other meat proteins may alsocontribute to the values obtained with the 4-DPS assay, and asmentioned above, the potential presence of sulfenic acid maycontribute to an overestimation of disulfides by the 4-DPSassay. In conclusion, SDS−PAGE analysis provides informationabout intermolecular disulfide formation in MHC formoderately oxidized samples, while the 4-DPS assay providesa more accurate and quantitative assessment of protein thiolsand intra- and intermolecular disulfides and, thus, offers a goodtool to investigate the thiol−disulfide reactions in meat andpotentially also in meat products.In the present study, we have shown that both reversible and

irreversible thiol oxidation takes place in meat during storage.Disulfide formation is often the consequence of thiol oxidation,as observed by a thiol loss, but our results clearly show thatdisulfide formation in meat during storage is not the onlyconsequence of thiol oxidation. This is an importantobservation in relation to the further development of strategiesto avoid or repair thiol oxidation.

■ AUTHOR INFORMATIONCorresponding Author*Telephone: +45-35333547. Fax: +45-35333344. E-mail:[email protected] authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThe authors thank the Institute for Agricultural and FisheriesResearch (ILVO) for granting the project entitled “ProteinOxidation in Meat Products: Effects of Natural BioactiveCompounds from Side Flows on Quality and Health Aspects”.The authors also thank the Danish Council for IndependentResearch|Technology and Production within the DanishAgency for Science, Technology and Innovation for grantingthe project entitled “Antioxidant Mechanisms of NaturalPhenolic Compounds against Protein Cross-Link Formationin Meat and Meat Systems” (11-117033).

■ ABBREVIATIONS USED4-DPS, 4,4′-dithiodipyridine; 4-TP, 4-thiopyridone; BH,sodium borohydride; BSA, bovine serum albumine; CL-MHC, cross-linked myosin heavy chain; DTNB, 5,5′-dithiobis-

Table 4. Degrees of Cross-Linking in Ground Beef Obtainedfrom Trial 1 during 9 Days of Storage under High-OxygenAtmosphere at 4 °C, Measured with 4-DPS and SDS−PAGEa

4-DPS assay SDS−PAGE

storagetime

disulfides/total thiolsday 0(%)

CL-MHCnon‑red/MHCred, day 0(%)

day 0 13.6 ± 0.4 a 3.1 ± 1.6 aday 4 18.6 ± 0.9 b 6.9 ± 1.0 bday 9 22.2 ± 0.3 c 8.5 ± 1.2 b

aDegrees of cross-linking are expressed as the level of cross-linking(disulfides and CL-MHC for 4-DPS and SDS−PAGE, respectively)divided by the reference level of reduced protein on day 0 (total thiolsand MHCred for 4-DPS and SDS−PAGE, respectively). Results areshown as the mean ± SD of three independent replicates. CL-MHC,cross-linked myosin heavy chain; MHC, myosin heavy chain. Differentletters (a−c) within a column denote statistical differences amongstorage times (p < 0.05).



mailto:[email protected]

(2-nitrobenzoic acid); DTT, dithiothreitol; GuHCl, guanidinehydrochloride; GSH, reduced glutathione; GSSG, oxidizedglutathione; HiOx, high-oxygen; LDS, lithium dodecyl sulfate;MAP, modified atmosphere packaging; MHC, myosin heavychain; NTB, 2-nitro-5-thiobenzoate; RSH, thiol; RSSR,disulfide; SDS, sodium dodecyl sulfate; SDS−PAGE, sodiumdodecyl sulfate−polyacrylamide gel electrophoresis; Tris,tris(hydroxymethyl)-aminomethane

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