Factors affecting solubilisation and oxidation of proteins during equine metmyoglobin-mediated lipid oxidation in extensively washed cod muscle

Food Chemistry 122 (2010) 1102–1110

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Factors affecting solubilisation and oxidation of proteins during equine metmyoglobin-mediated lipid oxidation in extensively washed cod muscle B. Egelandsdal a,*, L.P. Ren b, Y.S. Gong c, M. Greaser c, M.P. Richards c a

Department of Chemistry, Biotechnology and Food Science, University of Life Science, 1432-Ås, Norway College of Animal Science and Technology, China Agricultural University, Beijing 100193, China c Meat Science and Muscle Biology Laboratory, Department of Animal Sciences, University of Wisconsin-Madison, Madison, WI 53706-1284, USA b

a r t i c l e

i n f o

Article history: Received 22 November 2009 Received in revised form 18 March 2010 Accepted 23 March 2010

Keywords: Oxidation Cod Metmyoglobin Solubility Thiols Cathepsins

a b s t r a c t Lipid and protein changes in extensively washed cod muscle were studied at pH 5.6 and 2 °C for 47 h in the absence and presence of horse metmyoglobin (metMb). MetMb significantly increased lipid peroxides and TBARS formation. MetMb addition reduced solubility of proteins, such as myosin heavy chain and its fragments, and slowed down the enzymatic release of soluble proteins and protein fragments, e.g. fragments from collagen. Changes in thiols were monitored during storage as an indicator of protein oxidation. Added metMb reduced the number of soluble thiols as observed after 1.5 h using fluorescent labelling of formed disulfides and at later times for both soluble and insoluble proteins when determined by colorimetric measurements. Loss in the activity of the thiol-dependent proteases cathepsin L + B was observed with metMb addition. MetMb also became less soluble during the incubation period and a small amount of metMb was degraded by the still remaining protease activity. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction A low salt, washed cod matrix has been used for several years (Richards & Hultin, 2000) due to it being an efficient, and relatively inexpensive, source of highly unsaturated (marine) fatty acids (present as phospholipids). The standard washing (one part minced fish to three parts buffer, repeated three times) reduces anti- and pro-oxidants of various nature. Washing reduces the majority of low salt soluble proteins to 1.5% of their initial value leaving all salt soluble (meaning soluble at higher ionic strength than 0.05 mM NaH2PO4/Na2HPO4 at pH 6.3) and hydrophobic membrane proteins with the lipid bilayers. The model is useful with respect to studying the catalytic effect of hemiproteins, and their hemins, on oxidation of lipids. In fact this oxidation substrate has been used to reveal that lipid oxidation was promoted by hemeproteins and increased more by haemoglobins than myoglobins (Richards, Dettmann, & Grunwald, 2005). The actual structure of myoglobins will also affect the rate of oxidation, as expressed in the different wild varieties and mutants of hemeproteins (Grunwald & Richards, 2006a). The washed cod model is also an efficient model for evaluating antioxidants (Raghavan & Hultin, 2006). In the washed cod matrix, lipid peroxides are usually present in small amounts when myoglobins (or haemoglobins) are added (at * Corresponding author. Tel.: +47 64965858; fax: +47 64960333. E-mail address: [email protected] (B. Egelandsdal). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.03.092

40 lM) and thereafter they increase linearly after a certain lag period when 2 °C and pH 5.7–6.3 are used for inducing oxidation (Grunwald & Richards, 2006b). During two days, as a standard, rancid odour becomes apparent. Protein oxidation will also take place when free radicals are generated. Disulfide and carbonyl formation have been reported for the washed cod model or more precisely a similar model by Soyer and Hultin (2000) and Srinivasan and Hultin (1997). Srinivasan and Hultin (1997) used chelated (ATP) ferric iron, hydrogen peroxide, and ascorbate at pH 6.8 to induce a significant reduction in thiol oxidation within 2 h in the washed cod model. These authors did not study changes in thiols beyond 24 h. Soyer and Hultin (2000) observed disulfide formation after 2 h at pH 6.8 in sarcoplasmic reticulum (SR) from cod, but where chelated iron (ADP)-ascorbate was used to promote lipid oxidation. Initially, disulfide formation and lipid peroxide formation had comparable rate of formation. Their lowest value for accessible –SH groups in their SR model was 20 mmol/kg fish protein obtained after 2 h of incubation. Cysteine proteases require thiols to be active since their –SH is involved in the catalytic step that makes them active. Such cysteine proteases are caspases, calpains and cathepsins. All these different proteases are regarded as decisive in various steps defining food end-quality (for example in dry cured hams). Cathepsin activities (B and L) affect protein degradation and water-binding in white fish muscle. The cathepsins B, L and H are cysteine peptidases. Cathepsin L is capable of hydrolysing the major muscle

B. Egelandsdal et al. / Food Chemistry 122 (2010) 1102–1110

structural proteins, such as connectin, nebulin, myosin, collagen, a-actinin, and troponins T and I. Cathepsin B hydrolysed connectin, nebulin, and myosin at a low rate (Yamashita & Konagaya, 1991). Cathepsins B, L, L-like and calpain in fish muscle are very difficult to remove through washing and the former are also very stable during frozen storage (Jiang & Chen, 1998). Cathepsin B and L activities are preserved (to 80%) during surimi production. A broad pH range (5–6.8) for protein digestion by cathepsins is generally indicated, however, recent literature state that the lower pH values were more optimal for cathepsin L (Hu, Morioka, & Itoh, 2007). Recently, the importance of managing both degradation of proteins and lipid oxidation has been pointed at with respect to waterbinding in meat, tenderization of meat and flavour formation (Lund, Lametch, Hviid, Jensen, & Skibsted, 2008b; Medina, Gonzalez, Iglesias, & Hedges, 2009; Pan, Peng, Zhou, Yu, & Bao, 2008). However, scientific studies of the interaction of lipid and protein oxidation and protein degradation are still few. The aim of the present study was: (1) to describe the composition of the washed cod model in term of the major soluble and insoluble proteins present; (2) to monitor the changes in the proteins under strongly oxidising conditions produced by metMb and lipid peroxides; (3) to provide background knowledge regarding the use of the washed cod model as a general protein oxidation model. The hypothesis was that the model would be important for several quality aspects where protein and lipid oxidation take place.

2. Materials and methods 2.1. Fish raw material Fillets (three batches) of Atlantic cod (Gadus morhua) were used after a standard washing procedure described by Richards et al. (2005). Th resulting product, called WCOD-3X was stored in cryovac bags in 50 g quantities at 80 °C. The results reported here reflect experiments carried out on WCOD-3X kept frozen at 80 °C for 2–3 months. A second type of raw material (WCOD-6X) was prepared by thawing one part of WCOD-3X to which 10 parts 0.05 M sodium phosphate buffer at pH 6.3 was added following centrifugation at 13,000–16,200g for 20 min. This procedure was repeated three times. 2.2. Metmyoglobin purification Horse metMb (Sigma M1882) was purified on a 10 DG column (Bio Rad Cat 732-2010) according to the instructions provided with the column. The concentration of metMb eluted from the column was determined spectroscopically using the millimolar extinction coefficient e410 = 188 (Antonini & Brunori, 1971). 2.3. The washed cod model (WCOD) The total content of fish proteins was 93–84 mg/g with 0.30– 0.71 mg/g or 0.06–0.28 mg/g of soluble protein in WCOD-3X and WCOD-6X, respectively. Phospholipid content was 1.2–1.5 mg/g. The concentration of myoglobin was 0.69 mg/ml. The water content was 91–90%. All experiments were carried out at pH 5.6, in 0.05 M phosphatebuffer and at 2 °C (kept on ice). This mixture was examined through 47 h. Zero time samples were in fact about 1–2 h after metMb addition due to the time needed for preparing the samples for the experiment and initiating analytical measurement. Supernatants (SU) were defined by centrifugation at 16,200 g for 20 min in an Eppendorf microfuge (at 4 °C). Samples were

1103

removed during incubation, and frozen in microtubes (air in headspace) at 80 °C until analysed. 2.4. Protein concentration determination Three ml of 0.04% CuSO4 in 2% Na2CO3, 0.4% NaOH, 0.16% sodium tartrate plus 1% SDS was added to 1 ml of the protein solution/suspension. The system was heated for 15 min at 85 °C followed by 45 min incubation at room temperature. Then 0.3 ml of 10 times diluted Folin Ciocalteu reagent was added. Incubation for 45 more min was carried out before the absorbance was read at 660 nm. The method was largely in accordance with Markwell, Haas, Bieber, and Tolber (1978), and it was used for determination of protein concentration of supernatants (SU), sediments (SE) and intact WCOD-3X and WCOD-6X. 2.5. Inhibition of proteases SIGMA’s protease inhibitor cocktail (P8340) was added to WCOD-6X (10 ll to 1 g WCOD-6X). The cocktail contained 4-(2aminoethyl) benzenesulfonyl fluoride, pepstatin A, E-64, bestatin, leupeptin, and aprotinin but no metal chelators, for the inhibition of serine, cysteine, aspartic proteases and aminopeptidases. 2.6. Free thiol determinations of supernatants and mixture Supernatants: Thiols were quantified using the DTNB (5,50 dithiobis (2 nitrobenzoic acid) from SIGMA) reaction with thiols. Reaction time was 30 min in the dark. The proteins were thereafter precipitated in 80% methanol. Centrifugation was carried out (16,200 g for 20 min in Eppendorf microfuge) before the absorbance of the supernatant was read at 412 nm (Hu, 1994). The presence of absorbance at 412 nm from metMb/hemin in the supernatants following methanol precipitation was quantified using a reference sample without DTNB addition. Mixture (the un-centrifuged sample): an incubation time of 1 h in the dark was used before methanol precipitation and centrifugation. No correction for myoglobin absorbance was necessary for the sediments or intact mixture since the fish protein: myoglobin was much higher for the intact system. Reduced glutathione (1 mM) was used for standard curve. 2.7. Visible spectra of supernatants The visible spectra (400–700 nm) were obtained at room temperature using disposable cuvettes. The supernatants were diluted 10 times using phosphate buffer (0.05 M phosphate buffer at pH 5.6). A Shimadzu (UV-2401) spectrophotometer was used for recording the spectra at a resolution of 1 nm. 2.8. Peroxides on sediment Five millilitres of cold chloroform: methanol (1:1) was added to 0.3 g muscle sample (sediment). Homogenisation and centrifugation to obtain the lower chloroform layer were carried out as described by Undeland, Hultin, and Richards (2002). Ammonium thiocyanate and iron(II) chloride were added and after 20 min (22 °C) the absorbance was read at 500 nm (Shimadzu, UV-2401). A standard curve was constructed using cumene hydroperoxide and the concentration of lipid peroxide in the sample was expressed as lmol of lipid peroxides/kg of sediment. 2.9. TBARS on sediment TBARS (Thiobarbituric Acid Reactive Substances) were determined according to the modified method of Buege and Aust

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(1978) using trichloroacetic acid (TCA), thiobarbituric acid (TBA) and heating at 65 °C to dissolve solutes. The samples were centrifuged at 1600g for 20 min (Eppendorf centrifuge 5411D). Absorbance of supernatants was read at 532 nm (Shimadzu, UV-2401). A standard curve was constructed using tetraethoxypropane and concentrations of TBARS in samples were expressed in lmol of TBARS/kg of sediment. 2.10. Electrophoresis One dimensional sodium dodecyl polyacrylamide electrophoresis (1D SDS–PAGE) was run in accordance with the method developed by Fritz, Swartz, and Greaser (1989). Supernatants (min 0.25 ml) were concentrated by precipitated in 80% (v/v) ice-cold methanol and redissolved in sample buffer but not heat treated. Ten percent (w/v) polyacrylamide was used for gels. The gels were run in a Mighty Small II (SE 250) Slab Gel UnitAmersham (Pharmacia Biotech., 800 Centennial Avenue, Piscataway, NJ 08855) at 4 °C and Coomassie Blue stained. A BCA marker protein kit (Thermo Fisher Scientific Inc.) was used with the lower molecular weights adjusted through the use of a regression model that combined the marker protein with other low molecular weight protein. Destained gels were scanned on an HPScanjet 3970 scanner at resolution 300dpi. The images were thereafter transformed to 8 bits *.tif images before quantitative calculations were made. 2.11. Fluorescence labelling of oxidised thiols The labelling was adapted from Pretzer and Wiktorowicz (2008). N-Ethyl Maleimide (NEM) solution was used to block free thiols at any time. Formed disulfides during incubation were reduced and labelled with BODIPY-N-(2aminoethyl)maleimide (Invitrogen) before electrophoresis was carried out. The gels were imaged using Molecular Imager FX Pro (Biorad Laboratories) equipped with PDQuest 2D analysis software (Biorad laboratories) for image capture. The images were recorded as 16 bits *.tif images. Excitation was at 532 nm with emission pass filter for light above 555 nm. After fluorescence imaging, the gels were Coomassie Blue stained and treated as described above. 2.12. Qualitative calculations on 1D SDS–PAGE gels The lanes of gels were identified using ImageQuant version TL versions 2005 and 2007 (GE Healthcare). Baselines for intensity profiles were identified manually and subtracted. Thereafter the gels obtained from insoluble fish proteins were divided into 12 (13 when myoglobin was added) regions/bands (Table 1) and the density of these area volumes was determined. The sum of area volumes, minus Mb, was normalised to one, before subjected to statistical analysis. The gels obtained for soluble proteins were divided into 17 regions/bands (see below) and treated similarly. 2.13. Mass spectroscopic determination of proteins following trypsin digestion Washing, alkylation of thiols and trypsination of selected protein in gel were carried out according to proteomics protocols (Katayama, Nagasu, & Oda, 2001). The peptides were reconstituted and desalted through a ZipTip-C18 column (Millipore) before spotted directly onto the Opti-TOF™ 384 Well plate (Applied Biosystems, Foster City, CA) and re-crystallized with 0.50 ll of matrix [10 mg/ml a-Cyano-4hydroxycinnamic acid in acetonitrile/H2O/ TFA (60%:40%:0.2%)]. Peptide Map Fingerprint result-dependent MS/MS analysis was performed on a 4800 Matrix-Assisted Laser Desorption/Ionisation-Time of Flight-Time of Flight (MALDI TOFTOF) mass spectrometer (Applied Biosystems, Foster City, CA).

Peptide fingerprint was generated by scanning the 700–4000 Da mass range. Raw data was deconvoluted using GPS Explorer™ software and submitted for peptide mapping and MS/MS ion search analysis against non-redundant NCBI database (# 031309) with an in-house licensed Mascot search engine (Matrix Science, London, UK). The search results were ranked according to ions scores through the Mascot server from Matrix Science Ltd. (Boston, USA). Some peptide fractions were analysed by nano-LC–MS/MS using 1100 series LC/MSD Trap SL spectrometer (Agilent, Palo Alto, CA). An Agilent 1100 series HPLC delivered solvents A: 0.1% (v/v) formic acid in water, and B: 95% (v/v) acetonitrile, 0.1% (v/v) formic acid at either 20 ll/min, to load sample, or 0.22 ll/min, to elute peptides directly into the nano-electrospray. As peptides eluted from the HPLC-column/electrospray source, MS/MS spectra were collected over four channels from 300 to 2200 m/z. Raw data was converted to mgf file format using Data Analysis Software (Agilent). Resulting mgf files were used to search the NCBI database. 2.14. Determination of cathepsin L + B activity One gramme of the WCOD model was homogenised on ice for 1 min in 5 ml extraction buffer (50 mM natriumacetate, 0.1 M NaCl, 0.2% w/v[3-cholamidopropyl)-dimethyl ammonio]-1 propanesulfonate (Chaps); pH 5.0) using Tissue Tearor model 98537-395 from Biospec Products Inc. (Bartlesville, USA) at 32,000 rpm for 1 min. The system was thereafter stored on ice for 30 min and centrifuged for 30 min at 16,200g at 4 °C. Aliquots of 100 ll sample extract (supernatant) was added with 0.47 ml sample buffer (0.2 M Natriumacetate, 2 mM EDTA, 0.05% Chaps; pH 6.0). Thereafter 0.63 ml of 30 lM of substrate Z-Phe-Arg 4-methoxy-methylcoumarin hydrochloride was added. Incubation was for 25 min at 30 °C. Then 2.3 ml of chloroacetate was added to stop the enzymatic reaction; the reaction was started 3.5 h after thawing of the fish sample. Dithiotreitol was not used in the assay. All reactions were carried out in the dark. The samples were measured with a QuantaMaster Model C-60/2000 Spectrofluorimeter (Photon Technologies International, Birmingham, NJ, USA). Excitation was at 380 nm. Emitted light (photon counting) was read at 460 nm. Four replicates were read. A calibration curve using the standard Z-Phe-Arg 4-methoxy-methylcoumarin hydrochloride was prepared and the amount of formed product was determined from the standard curve. 2.15. Statistics The statistical programme Minitab, version 15, was used. The changes in thiols were analysed using a generalised linear model with factors: time and myoglobin and their interaction factor. For comparisons, Tukeys test (significance level 0.05) was used. Protein concentration data was analysed using one-way analysis of variance (ANOVA) and Tukeys test for comparisons. Significant linear trends were determined by the routine Regression in Minitab. Fifty: Fifty MANOVA (Langsrud, 2002): This statistical method was used to calculate significant difference between densitometric scans from gels caused my myoglobin addition or storage time on ice. The method uses principal component analysis to reduce the dimensionality of the data with a final test based on classical test statistics and their distributions. 3. Results 3.1. Effect of metMb addition on lipid oxidation progress in the washed cod model The lipid oxidation progress of this model was monitored over 47 h using lipid peroxides (PV) and TBARS. MetMb effectively

B. Egelandsdal et al. / Food Chemistry 122 (2010) 1102–1110 Table 1 SDS–PAGE pattern of sedimented washed cod.

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not observed for the WCOD+Mb system (Table 2). Adding protease inhibitor cocktail to the WCOD-6X system slowed down the release of proteins into the supernatant (Table 2). The average rate of appearance of soluble proteins/peptides in WCOD was calculated as 73 mg/kg total protein per hour.

3.3. Effect of storage time on protein pattern of SDS–PAGE gels

a

The numbers refer to identifications made for quantitative calculations. Mean and st.dev. c Early post mortem data in parenthesis (Connell & Howgate, 1959). d Contained largely degraded myosin heavy chain (MHC; area volume dissected for MS determination is indicated by arrow) as identified from MALDI. e Identification from Thys, Blank, Coughlin, and Schachat (2001), MLC = myosin light chain. b

promoted lipid oxidation based on increased PV and TBARS values with time compared to the control (WCOD-6X) (Fig. 1). Significant changes in PV and TBARS values were observed after 13 h incubation when metMb was added. After a lag time, PV increased linearly with time. TBARS showed kinetics of lipid oxidation similar to formation of lipid peroxides.

SDS–PAGE gels run with WCOD-6X and WCOD-3X sediments revealed an effect of storage time at 2 °C (p < 0.001). The largest change caused by storage was that the relative density of area volume SE4 (Table 1) increased with time. This area contained degraded myosin heavy chain. The proteins in the supernatants of WCOD and WCOD+Mb were also examined using SDS–PAGE (Fig. 2A). The soluble proteins had another band pattern compared to WCOD (not centrifuged) and WCOD sediments. Frequently there were, at zero time, five distinct area volumes SU1, SU2, SU7 and SU8 and SU12 (Fig. 2A). These labelled area volumes were not all individual proteins. SU12 was the most dense area in three (out of four) WCOD-6X replicates and the second most dense in the 4th replicate where SU8 was the most dense area volume. WCOD-3X gave either SU2 or SU8 as dominant initial area volume (results not shown). A significant (p < 0.021) effect of storage time at 2 °C was observed for the soluble proteins as well; the most pronounced change with time was a relative reduction of proteins in SU8. Proteins identified in this region were actin (actinfamily) and creatine kinase (Table 3).

3.4. Effect of Mb addition on protein patterns of SDS–PAGE gels 3.2. Effect of storage time on the soluble proteins in the washed cod model The changes in soluble proteins with time were monitored in the washed cod model (Table 2). After the 6th wash little soluble protein remained. A significant and steady increase in soluble proteins with time was noted in the WCOD systems, while this was

Fig. 1. Changes in lipid peroxides (PV) and TBARS induced by added metmyoglobin to WCOD-6X. The measurements were made on proteins sedimented with their lipids (sediments) at 16,200g  20 min. The sediments contained 105–189 mg/g protein. The letters indicate significant differences (p < 0.05). All systems devoid of Mb carry letter a.

The proteins of WCOD+Mb systems were added to each lane of the SDS–PAGE gels, ignoring the presence of metMb. The concentration of metMb in applied sample buffer could therefore reach 5–17 mg/ml or even higher concentrations for the 1.5 h sample from WCOD-6X+Mb. At such high metMb concentrations a myoglobin dimer was observed (Fig. 2). This was observed when the concentration of myoglobin in the sample buffer was above 5 mg/ml independent of pre-preparation and type of sample buffer used. MALDI-TOF-TOF identified the protein as myoglobin and no peptide origination from any other protein could be identified in the database searched (Table 3). There was no significant change in the ratio between the dimer and the monomer myoglobin with time (1.5–47 h) for the two systems examined. Normalised SDS–PAGE densitometric scans (on SU gel samples), with the myoglobin monomers and dimers removed prior to normalisation and statistical analysis, gave a significant effect (p < 0.002) of myoglobin addition. WCOD-6X (four batches) gave significantly (p < 0.05) less intensity for area SU2 (Fig. 2) and sometimes for area volume SU1. SU1 contained degraded myosin HC and presumed degraded collagen (Table 3). Area volume SU1 was significantly (p = 0.049) less dense when myoglobin was added also for WCOD-3X batches. Area volume SU4 and SU5 were significantly less dense when metMb was added to WCOD-6X. Only SU5 was attempted to be identified (Table 3); it appeared be a fragment from connective tissue. Area SU7 (dominant protein was enolase) and SU8 (actin and creatine kinase; Table 3) appeared to be more resistant to myoglobin induced precipitation/removal from the supernatant as no significant changes in area volumes were found with Mb addition. Significantly (p = 0.047) less protein in area SU12 was found when Mb was added, but only for WCOD-6X. For WCOD-6X significantly (p = 0.003) more SU17 was detected when Mb was

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Table 2 Different chemical and biochemical changes induced with incubation time on ice for systems with and without myoglobin addition. Measurements

Systemntime (h)

0–2

15–18

46–47

WCOD-3X WCOD-3X+Mb WCOD-6X WCOD-6X-PICB WCOD-6X+Mb

0.70 ± 0.01 1.36 ± 0.03a 0.12 ± 0.02a 0.15 ± 0.01a 0.86 ± 0.04a

n.m. n.m. 0.19 ± 0.01b n.m. 1.01 ± 0.06a

0.77 ± 0.05 1.23 ± 0.04a 0.28 ± 0.01c 0.15 ± 0.01a 0.89 ± 0.06a

0.91 ± 0.03b 1.22 ± 0.03a 0.43 ± 0.02d 0.16 ± 0.01a 0.89 ± 0.06a

Thiol content (mmol/l)-SUC

WCOD-6X WCOD-6X+Mb WCOD-6X WCOD-6X+Mb WCOD-6X WCOD-6X+Mb

0.014 ± 0.001a 0.041 ± 0.001e 375 ± 60a 48 ± 7d 63.7 ± 0.7a 62.4 ± 1.7a

0.053 ± 0.003b 0.014 ± 0.012f 1395 ± 76b 11 ± 12e 74.0 ± 1.2b 56.2 ± 0.3a

0.061 ± 0.004c 0.017 ± 0.011f 1100 ± 71b 19 ± 10e 87.5 ± 1.2c 62.1 ± 0.3a

0.129 ± 0.007d 0.010 ± 0.001f 1536 ± 92c 51 ± 11f 138.8 ± 1.9d 83.6 ± 1.4b,c

Absorbance (410 nm)

WCOD-6X WCOD-6X+Mb

0.009 ± 0.001a 0.686 ± 0.028a

0.007 ± 0.003a 0.625 ± 0.066a

0.007 ± 0.003a 0.619 ± 0.044a

0.006 ± 0.001a 0.451 ± 0.044b

Cathepsin B + L activity (30 °C) (nmol g protein1 min1)D

WCOD-6X WCOD-6X+Mb

5.42 ± 0.11a 1.03 ± 0.11b

Thiol content (mmol/kg protein)

A

21–23

Solubility (mg/ml)

Thiol content (mmol/kg protein)-SU

a

n.m. n.m

a

n.m. n.m

4.41 ± 0.06c 0.71 ± 0.06d

Numbers with different letters in a row were significantly different (Tukey’s test) p < 0.05. The protein concentrations (modified Lowry) were determined in the supernatant after centrifugation at 16,200g  20 min. Time intervals are given as the samples were not removed at exactly the same times. A n.m. = not measured. B PIC = protease inhibitor cocktail. C SU means supernatant. D 3.5 h from thawing to incubation with substrate.

Fig. 2. Panel A: SDS–PAGE gels (10% w/v) of WCOD-6X supernatants prepared by centrifugation at 16,200g for 20 min. The figure shows how the different area volumes among soluble proteins were numbered before statistical calculations were done. Lane 1: standards with MW in kDa; Lanes 2–5: WCOD-6X 1.5, 14, 22 and 47 h, respectively; Lanes 6–9; WCOD-6X+Mb 1.5, 14, 22 and 47 h, respectively. The supernatants were precipitated in 80% (v/v) methanol, dried and re-suspended in sample buffer to give solutions containing approx. 3 mg/ml when the presence of Mb was ignored in the WCOD+Mb samples. Applied fish proteins were 30 lg. SU3, SU4, SU6, SU9, SU15 and SU16, SU17B were not attempted identified, the remaining area volumes were identified with MS techniques and their identification is given in Table 3. Panel B: relationship between the absorbance of supernatants at 410 nm and the density of the globin monomer area measured on SDS–PAGE gels. The data are reported relative to the absorbance and density measured after 1.5 h. Target line is drawn. Determination coefficient R2 = 0.876.

added. The upper part of that area volume (SU17A) was identified as a Mb fragment (Table 3). 3.5. Effect of storage time and metMb addition on thiols in the washed cod model The number of accessible thiols in the supernatant of WCOD-6X increased with time (p < 0.001) (Table 2). For WCOD-6X+Mb the changes were smaller and no significant, linear change in thiols (mmol/l) with time was found. However, a significant reduction in thiols was found after 15–18 h (Table 2). The difference in thiols between WCOD-6X and WCOD-6X+Mb was highly significant (p < 0.001) after 47 h. An increase (p = 0.03) in –SH with time for WCOD-6X was also observed when the number of thiols in the supernatant was divided by protein concentration (Table 2). Differences in disulfides induced by added metMb

after 1.5 h are shown in Fig. 3 (lanes 1 and 2). Oxidation induced disulfides measured by the fluorescence intensity to the intensity of the Coomassie stain, was more extensive for SU7, SU8 and SU12 in WCOD-3X+Mb compared to WCOD-3X. The number of accessible thiols increased (p < 0.05) linearly with time for WCOD-6X both for the total system and the sediments. The reduction in thiols in the presence of Mb was significant (p < 0.001) after 47 h also when the un-centrifuged system was examined. The amount of accessible thiols of soluble proteins, no Mb added, was several times higher than those of the insoluble (SE) proteins. 3.6. Effect of metMb and storage on spectral changes The changes in Mb absorbance during 47 h are given in Table 2. A significant reduction in Mb absorbance was obtained after 47 h

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Table 3 Identification of proteins after tryptic digestion from gels followed by MALDI-TOF-TOF or LC–MS/MS (LC) and search in databases. Only significant identifications were reported. Area volume

Description

Species

NCBI no in database

Mascot score

SU17AFig. 2a SU14-Fig. 2 SE12-Table 1

(Degraded) Myoglobin Myoglobin Myosin LC 2

Equus caballus Equus caballus Theragra calcogramma

gi| 7546624

130

gi| 7546624 gi| 1463956

SE11-Table 1

Troponin I (fast)

Gadus morhua

SU12-Fig. 2 SU10-Fig. 2

Troponin I (fast) Myoglobin (dimer)

SU8-Fig. 2 SU8-Fig. 2 SU7-Fig. 2

a b c d

% Seq. covered

MW (kDa)

pI

2(3)

20

7.36

517 338

8(10) 9

86 60

gi| 20258048

123

2(4)

21

As above Equus caballus

As above gi| 7546624

127 415

2(8) 8(9)

60 71

Creatin kinase

Gadus morhau

gi| 13274539

LC

1 (5)c

22.1

Unnamed protein product (actinfamily) Enolase 3–1

Mus musculus

gi| 12852068

LC

5(6)

19.3

Salmo salar

gi| 213511756

129

1(9)

40

Tetraodon nigrovirdis

gi| 47208602

146

3(9)

19

16.9 (14.5 ± 0.5)b 16.9 19.0 (18.0 ± 0.4) 18.9 (18.8 ± 0.3) As above 16.9 (34.0 ± 0.1) 28.9 (40.3 ± 1.4)d 41.9 (40.3 ± 1.4) 47.2 (47.9 ± 0.9) 53.6 (59 ± 5) 128.5 (115 ± 6) 74.1 (115 ± 6) 128.5 (131 ± 5) 128.5 (198 ± 7)

d

Peptides matched

SU5-Fig. 2

Unnamed protein product

SU2-Fig. 2

Myosin heavy chain

Theragra calcogramma

gi| 1777305

LC

5(5)

7.4

SU2-Fig. 2

Collagen 1a1

Oncorhynchus mykiss

gi|185133699

LC

1(1)

3.4

SE4-Table 1

(Degraded) Myosin HC

Theragra calcogramma

gi| 1777305

83

1(19)

21

SU1-Fig. 2

Myosin HC

Theragra calcogramma

gi| 1777305

500

6(32)

33

7.36 4.67 8.74 As above 7.36 – – 6.81 6.04 – – 5.23 5.23

WCOD-6X+Mb system. Calculated MW from SDS–PAGE gels. The higher number for Salmo salar (39.5 kDa). Fibronectin type III domain (from connective tissue).

(p < 0.05). The relationship between the absorbance of the supernatant at 410 nm and the density of the globin’s area volume on the SDS–PAGE gels was significant (Fig. 2B). But there was a systematic bias; when the absorbance of the supernatant fell, the density of the area volume of the globin fell relatively more from the target line (Fig. 2B). 3.7. Effect of metmyoglobin on cathepsin B + L activity Table 2 shows the changes in cathepsin L + B activity during 43 h. A significant reduction in enzyme activity was found when metMb was added to WCOD-6X. The cathepsin activity of WCOD-6X decreased during incubation time but remained much higher than for WCOD-6X+Mb. 4. Discussion 4.1. General Extensive washing (WCOD-6X) was carried out to reduce the amount of soluble protein to a very low concentration. In addition, the aim was to achieve a background of soluble fish protein that was stable over time. Such a model would be essentially free of interference from endogenous soluble proteins if an exogenous soluble protein was to be added. Our data suggested a more predictable variation in the amount of soluble proteins for WCOD-6X compared to WCOD-3X, but with some variation still with time in the amount of soluble proteins of WCOD-6X. 4.2. Effect of metMb addition on lipid oxidation progress in the washed cod model The kinetics of lipid oxidation of the WCOD model is defined by the amount of Mb, pH, type of Mb and ligand (Grunwald & Richards, 2006a, 2006b). The kinetics of formation of TBARS reported

Fig. 3. Fluorescence labelled thiols after reduction of formed disulfides; L1 is WCOD-3X 1.5 h; L2 is WCOD-3X+Mb1.5 h. Commassie labelled proteins: L3 is WCOD-3X 1.5 h; L4 is WCOD-3X+Mb 1.5 h; L5 is standards in kDa.

here were high, when compared with the results using WCOD3X, even when a correction was made for the fact that our determination of TBARS was on the sediments. The kinetics of formation of PV were more comparable, or even slow, when compared to previous studies (Grunwald & Richards, 2006b; Soyer & Hultin, 2000). However, the magnitude of our PV values was unusually high. Differences in individual fish, Mb type, the length of the pre-washing period, the storage time in the freezer (at 80 °C), washing proce-

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dure, the time given between the thawing/washing and the actual determination of amount (and type) of soluble protein (here about 3–4 h), could be decisive factors in determining the magnitudes for PV and TBARS. 4.3. Effect of storage time on insoluble and soluble proteins in the washed cod model The largest effect of storage time on insoluble proteins seemed to be generation of fragments from higher molecular weight proteins. Although only the fragments from myosin were identified with MS techniques, despite several attempts, it is likely that nebulin and titin also were degraded since high molecular weight bands were observed to disappear with time for some replicates. Storage generally increased solubilisation and degradation of proteins. Since a (substantial) reduction in the amount of soluble protein was observed with the addition of a protease inhibitor cocktail to WCOD-6X, it was assumed that proteolytic degradation contributed to solubilisation of proteins. 4.4. Effect of Mb on changes in the soluble protein of the washed cod model MetMb addition can affect the (re-)solubilisation of protein and protease activity as well as promoting precipitation (insolubilisation) of proteins through oxidation. Mb will also be able to reveal general proteins characteristics in the model in addition to maintaining its strong pro-oxidative effect through its heme-group. Lund, Luxford, Skibsted, and Davies (2008a) and Fredriksen, Lund, Andersen, and Skipsted (2008) reported that Mb/H2O2 systems will crosslink porcine myosin very quickly (<1 h) through disulfide formation. This could be the same as reporting reduced solubility for myosin in the presence of Mb. Chaijan, Benjakul, Viesssanguan, and Faustman (2007) reported that tuna oxyMb/ metMb will bind to fish (sardine and tuna) myosin apparently instantaneously at pH 6.5 and 4 °C without adding peroxides. The authors reported that 20% of added Mb would precipitate with myosin as soon as the measurement could be carried out. Our results suggested that myosin heavy chain and the larger (115 ± 6 kDa) degraded part of myosin heavy chain were removed from the soluble phase within 1.5 h. Since cod MHC and its degraded part have isoelectric points between 5.2 and 5.5, (degraded) myosin and Mb will have opposite charges at pH 5.6. This fact may aid their removal from the soluble phase and contribute to removal from the supernatant in addition to disulfide formation and other possible crosslinks. Labelling for oxidation induced disulfides also indicated that other proteins formed disulfides after 1.5 h using the combination of metMb and marine phospholipids. But some proteins stayed relatively more soluble than others during incubation with metMb and WCOD phospholipids (e.g. SU7 where enolase was identified). There is no clear-cut reason why enolase stayed soluble. Also its thiols appeared to oxidise as seen from Fig. 3. It should be pointed out that Mb always dominated among soluble proteins, in particular when the WCOD-6X model was used. The high concentration of Mb in WCOD+Mb systems may in itself reduce the solubility of proteins observed on gels. However, since systems WCOD-3X+Mb and WCOD-6X+Mb with different Mb: fish protein ratio both indicated reduced solubility of myosin HC and the degraded myosin HC, reduced solubility of these proteins with myoglobin addition seemed real. This is also supported by the fact that the total amount of protein in the supernatant never increased with time for the WCOD+Mb systems.

4.5. Effect of storage and Mb addition on thiol changes in the washed cod model Cysteine is presumed to be the most sensitive amino acid to oxidation in terms of fraction oxidised, and is thus a suitable indicator of early protein oxidation. Leblanc and Leblanc (1992) reported values between 28 and 56 mmol/kg for the thiol content of myofibrillar proteins: i.e. values approaching those reported here for the total system. The increase in thiol with time was interpreted to be due to degradation possibly modified by easier accessibility with time for the reactants of the assay for the WCOD system. Several proteins have been identified as released into the supernatant with time. All major proteins that were released into the supernatants contain cysteines, that is, myosin heavy chain and its fragments, the assumed degraded component from fish connective tissue (fibronectin-like, unnamed protein product), enolase, actin and troponin I have cysteines. The increase in thiols in the supernatant with time appears attributed largely to proteolytic degradation of the fish protein. The antioxidant activity of the supernatant therefore improved with time since the oxidation rate of thiols in WCOD-6X was slower than the release of thiols into the solution. The thiol content of the supernatant was unexpectedly high. A value this high can only be interpreted as a substantial release of low molecular weight peptides containing thiols. Some support is given from the disulfide labelling where one volume area containing a thiol compound of lower molecular weight than troponin I was observed on the gel (Fig. 3). Soyer and Hultin (2000), using inorganic iron, reported reduced number of accessible thiols with time. In H2O2 activated horse metMb systems, the free thiols of porcine myosin were reduced to approximately 1/10 of their initial value within 2 min at 22 °C using a Mb:myosin molar ratio of 7.7 and a Mb:H2O2 ratio equal to 1 (Lund et al., 2008a). Fredriksen et al. (2008) demonstrated that porcine myosin would crosslink through reducible crosslinks within 25 s around pH 5.5 when 75 lM H2O2 was available. Such a high value for LOOH was not obtained here until several hours (see Fig. 1) had passed. However, our myoglobin: LOOH, if comparable with myoglobin: H2O2, started low but ended at approx. 1:9 after 47 h; i.e. close to the system used by Lund et al. (2008a). The disulfide labelling supported thiol decrease at a much lower Mb:LOOH ratio. The DTNB labelling did not detect this, but may have been less sensitive at earlier times since the initial, soluble fish protein concentration was low for WCOD-6X. 4.6. Effect of storage and metMb addition on cathepsin B + L in the washed cod model In agreement with previous literature we found active thiol proteases in the washed cod despite extensive washing of the cod (Jiang & Chen, 1998). The presence of active proteases supported the hypothesis that proteins were released due to proteolytic degradation, and that metMb addition would reduce the rate of enzymatic degradation and release of fish protein into the supernatant. Spanier and Bird (1982) indicated that cathepsin B can be inhibited by Mb. However, Volle, Dutaud, and Ouali (1999) postulated that such inhibition were in most cases a methodological artifact and backed up this postulation by using a myoglobin to a fluorescent substrate ratio of 0.1–0.5 mg:0.5 lM 7-amino-4 methylcoumarylamide. However, for our assay we did not use more than 0.02 mg/ml of Mb in the assay. This is a Mb concentration where the impact on Mb/hemin absorbance of emitted light would be low (Volle et al., 1999). In addition we have used post-reaction addition of metMb to WCOD systems as control, and no change was observed in the emission spectrum. The fact that we did not

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observe a substantial increase in the cathepsin activity after 47 h compared to 1.5 h, attributable to the significant loss in myoglobin/hemin absorbance over the same time period, supported the interpretation that inactivation of cathepsins B + L in the washed cod model occurred. Rapid (<5 h) inactivation of enzymes through ferrylMb addition has been reported by Miura, Muraoka, and Ogiso (1995).

4.7. The fate of metMb in the washed cod model Several different phenomena seemed to affect metMb. These were: (1) early co-precipitation with other proteins; (2) precipitation of the globin, with or without its heme; (3) enzymatic degradation of the globin protein. Early co-precipitation of some of the metMb was predicted from the experience that the concentration measured in the supernatant was approximately 0.63 mg/ml after 1.5 h incubation at 2 °C despite that the target was always 0.69 mg/ml. Early precipitation of myoglobin is in agreement with other studies (Chaijan et al., 2007). A 40% reduction in the Soret band was observed after 46–48 h, but a significant reduction was found after 24 h for some preparations. The most likely reasons for the reduction in absorbance at 410 nm with time were: (1) precipitation of myoglobin and/or hemin (2) destruction of hemin in the supernatant. The fact that the protein concentration in the supernatant changed little with time favoured an interpretation where hemin was lost/precipitated and the apoprotein stayed soluble. However, the fact that there was an underestimation of globin on the gels with time, suggest that globin precipitated with time, in fact more extensively than would be expected if its precipitation was promoted only by hemin loss. In addition, some fish proteins were released into the supernatant with time also in the WCOD+Mb system. The fact that some proteolytic activity was measured in the WCOD-6X+Mb even after 47 h supported this interpretation. In addition, a fragment of metMb was found after 47 h. This fragment could be formed by Cathepsin B or L since metMb is a substrate for these enzymes through its amino acids phenylalanine and arginine at position 138 and 139, respectively. The fragment had molecular weight 15.6 kDa as estimated from MALDI-TOF-TOF and electrophoresis. Degradation of Mb would also contribute to loss of density originating from the globin (SU14).

4.8. Effect of Mb: protein ratio on protein patterns of SDS–PAGE gels It was not possible to observe any changes in the insoluble proteins using 1D SDS–PAGE gel under reducing conditions that could be related to Mb addition. This may be due to the complexity of this system where many proteins may be affected by metMb, but only to a small extent, since the ratio of fish protein to metMb was very high. When only the soluble proteins were studied, the ratio between fish protein and Mb were dramatically reduced, and this should favour the opportunity of observing changes in fish proteins promoted by metMb addition. Østdal, Søgaard, Bendixen, and Andersen (2001) observed the formation of a dimer (dityrosine crosslink) from equine myoglobin and H2O2 system using gel electrophoresis under reducing conditions. We found no evidence that the Mb dimer observed in our systems was attributed to the presence of peroxides that created Mb radicals since its formation was also observed in pure metMb solutions devoid of peroxides. The mechanism behind the dimer formation observed here was not known.

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4.9. Practical implications The washed cod system is an established accelerated model for oxidation and degradation of marine lipids. This investigation elucidates the impact on cathepsin activity and lipid oxidation under pro-oxidative conditions induced by hemins. Maintenance of enzyme activity is critical when dry cured hams are produced (Toldra, 2002). This investigation showed that the heme-catalysed pro-oxidative stress negatively impacted two flavour forming processes: decreased endogenous proteolysis and accelerated lipid oxidation. For fish, reduced proteolysis could have been advantageous if unaccompanied by lipid oxidation. Acknowledgements The Fulbright Foundation, The University of Life Science and The Research Council of Norway (grant No. 184846), Nortura, Tine, and The College of Agricultural and Life Sciences, UW-Madison, HATCH project are thanked PRJ28VY for financial support. The assistance from Eric Grunwald and the staff at Genetics-Biotechnology Center and The Biophysics Instrumental Facility (UW-Madison, WI) is also highly appreciated. John E. Wiktorowicz, University of Texas, suggestions for analytical improvements are highly appreciated. References Antonini, E., & Brunori, M. (1971). Hemoglobin and myoglobin in their reactions with ligands. In A. Neuberger & E. L. Tatum (Eds.). Frontiers of biology (Vol. 21, p. 44). Amsterdam: North-Holland Publishing Company. Buege, J. A., & Aust, S. D. (1978). Microsomal lipid peroxidation. Methods in Enzymology, 52, 302–310. Connell, J. J., & Howgate, P. F. (1959). Proteins of fish skeletal muscle. VI. Amino-acid composition of cod fibrillar proteins. Biochemical Journal, 71, 83–86. Chaijan, M., Benjakul, S., Viesssanguan, W., & Faustman, C. (2007). Interaction between fish myoglobin and myosin in vitro. Food Chemistry, 103(4), 1168–1175. Fredriksen, A. M., Lund, M. N., Andersen, M. L., & Skipsted, L. H. (2008). Oxidation of porcine myosin by hypervalent myoglobin: The role of thiol groups. Journal of Agricultural and Food Chemistry, 56(9), 3297–3303. Fritz, J. D., Swartz, D. R., & Greaser, M. L. (1989). Factors affecting polyacrylamide gel electrophoresis and electroblotting of high-molecular-weight myofibrillar proteins. Analytical Biochemistry, 180(2), 205–210. Grunwald, E., & Richards, M. P. (2006a). Studies with myoglobin variants indicate that released hemin is the primary promoter of lipid oxidation in washed fish model. Journal of Agricultural and Food Chemistry, 54(12), 4452–4460. Grunwald, E. W., & Richards, M. P. (2006b). Mechanisms of heme protein mediated lipid oxidation using hemomyoglobin and myoglobin variants in raw and heated washed muscle. Journal of Agricultural and Food Chemistry, 54(21), 8271–8280. Hu, M. (1994). Measurements of protein thiol groups and glutathione in plasma. Methods in Enzymology, 233, 380–385. Hu, Y., Morioka, K., & Itoh, Y. (2007). Existence of cathepsin L and its characterization in red bulleye surimi. Pakistan Journal of Biological Sciences, 10(1), 78–83. Jiang, S., & Chen, G. (1998). Effect of cathepsins B, L, L-like and calpain on the protein degradation of surimi. In Y. L. Xiong, C.-T. Ho, & F. Shahidi (Eds.), Quality attributes of muscle foods (pp. 393–405). New York: Kluwer Academic/Plenum Publishers. Katayama, H., Nagasu, T., & Oda, Y. (2001). Improvement of in-gel digestion protocol for peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Communication Mass Spectrometry, 15(16), 1416–1421. Langsrud, Ø. (2002). 50–50 Multivariate analysis of variance for collinear responses. The Statistician, 51(3), 305–317. Leblanc, E. L., & Leblanc, R. J. (1992). Determination of hydrophobicity and reactive groups in protein of cod (Gadus morhua) muscle during frozen storage. Food Chemistry, 43(1), 3–11. Lund, M. N., Luxford, C., Skibsted, L. H., & Davies, M. J. (2008a). Oxidation of myosin by haem protein radicals and protein cross-links. Biochemical Journal, 410(3), 565–574. Lund, M. N., Lametch, R., Hviid, M. S., Jensen, O. N., & Skibsted, L. H. (2008b). High oxygen packing atmosphere influences protein oxidation and tenderness of porcine longissimus dorsi during chill storage. Meat Science, 77(3), 295–303. Markwell, M. A. K., Haas, S. M., Bieber, L. L., & Tolber, N. E. (1978). A modification of the Lowry procedure to simplify protein determination in membrane and lipoproteins samples. Analytical Biochemistry, 87(1), 206–210.

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B. Egelandsdal et al. / Food Chemistry 122 (2010) 1102–1110

Medina, I., Gonzalez, M. J., Iglesias, J., & Hedges, N. D. (2009). Effect of hydroxycinnamic acids on lipid oxidation and protein changes as well as water holding capacity in frozen minced horse mackerel white muscle. Food Chemistry, 114(3), 881–888. Miura, T., Muraoka, S., & Ogiso, T. (1995). Relationship between protein damage and ferrylmyoglobin. Biochemistry and Molecular Biology International, 36(3), 587–593. Østdal, H., Søgaard, S. G., Bendixen, E., & Andersen, H. J. (2001). Protein radical in the reaction between H2O2-activated metmyoglobin and bovine serum albumin. Free radical Research, 35(6), 757–766. Pan, X., Peng, Z., Zhou, G., Yu, J., & Bao, E. (2008). Effect of heat stress on oxidative damage and protein functionality in broiler breast muscle. Zhongguo Nongye Kexue, 41(6), 1778–1785 [Journal written in Chinese]. Pretzer, E., & Wiktorowicz, J. E. (2008). Saturation fluorescence labeling for proteomic analyses. Analytical Biochemistry, 374(2), 250–262. Raghavan, S., & Hultin, H. O. (2006). Oxidative stability of a cod-canola oil model system: Effect of order addition of tocopherol and canola oil to washed, minced cod muscle. Journal of Aquatic Food Product Technology, 158(2), 37–45. Richards, M. P., & Hultin, H. O. (2000). Effect of pH on lipid oxidation using trout hemolysate as a catalyst: A possible role for deoxyhemoglobin. Journal of Agricultural and Food Chemistry, 48(8), 3141–3147. Richards, M. P., Dettmann, M. A., & Grunwald, E. W. (2005). Pro-oxidative characteristics of trout hemoglobin and myoglobin: A role for released heme in oxidation of lipids. Journal of Agricultural and Food Chemistry, 53(26), 10231–10238.

Soyer, A., & Hultin, H. O. (2000). Kinetics of oxidation of the lipids and proteins of cod sarcoplasmic reticulum. Journal of Agricultural and Food Chemistry, 48(6), 2127–2134. Spanier, A. M., & Bird, I. W. C. (1982). Endogenous cathepsin B inhibitor activity in normal and myopathic red and white skeletal muscle. Muscle and Nerve, 5(4), 313–320. Srinivasan, S., & Hultin, H. O. (1997). Chemical, physical, and functional properties of cod proteins modified by a nonenzymic free-radical-generating system. Journal of Agricultural and Food Chemistry, 45(2), 310–320. Thys, T. M., Blank, J. M., Coughlin, D. J., & Schachat, F. (2001). Longitudianal variation in muscle protein expression and contraction kinetics of largemouth bass axial muscle. Journal of Experimental Biology, 204(24), 4249–4257. Undeland, I., Hultin, H. O., & Richards, M. P. (2002). Added triacylglycerols do not hasten hemoglobin-mediated lipid oxidation in washed minced cod muscle. Journal of Agricultural and Food Chemistry, 50(23), 6847–6853. Toldra, F. (2002). Characterisation of proteolysis. In Dry cured meat products (pp. 113). Hoboken: Wiley-Blackwell. Volle, B., Dutaud, D., & Ouali, A. (1999). Myoglobin inhibition of most protease activities measured with fluorescent substrates is an artifact! Meat Science, 52(1), 81–87. Yamashita, M., & Konagaya, S. (1991). Hydrolytic action of salmon cathepsins B and L to muscle structural proteins in respect to muscle softening. Nippon Suisan Gakkaishi, 57(10), 1917–1922.