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Addition of partly reduced bovine serum albumin to a metmyoglobin-fortified washed cod system gives reduced formation of lipid oxidation products and increased degradation of proteins
LWT - Food Science and Technology 44 (2011) 1005e1011
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Addition of partly reduced bovine serum albumin to a metmyoglobin-fortified washed cod system gives reduced formation of lipid oxidation products and increased degradation of proteins B. Egelandsdal*, L.P. Ren 1, P. Kathirvel 2, Y.S. Gong, M.L. Greaser, M.P. Richards Meat Science and Muscle Biology Laboratory, Department of Animal Sciences, University of WisconsineMadison, Madison, Wisconsin 53706-1284, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 March 2010 Received in revised form 18 August 2010 Accepted 2 November 2010
The aim of the investigation was to study the pro-oxidative system of washed cod with added horse metmyoglobin (metMb) regarding lipid and enzyme oxidation plus protein degradation. The changes were studied at pH 5.6 and 2 C for 47 h. Bovine serum albumin (BSA) with 0.2 or 3.5 thiol (reduced) groups per molecule was added as an antioxidant protein and as providers of free thiols. BSA with the higher amount of thiols decreased the formation of lipid peroxides and thiobarbituric acid reactive substances (TBARS) in the washed cod system. Metmyoglobin (metMb) addition slowed down the enzymatic release of proteins and protein fragments from insoluble washed cod proteins while the opposite was observed with added reduced BSA. Addition of metMb strongly inactivated the thiol proteases cathepsin B þ L and effectively oxidized the low molecular weight thiols in the system while reduced BSA slowed down inactivation of the proteases. The endogenous proteases degraded BSA to a fragment of 41.7 kDa. Formation of this fragment was reduced when metMb was added. BSA (not reduced) did not influence TBARS formation in the washed cod system with metMb, but increased the formation of lipid peroxides after 47 h compared to the system without BSA. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Bovine serum albumin Cathepsin Washed cod Metmyoglobin Oxidation
1. Introduction Polyunsaturated fatty acids (PUFAs) in phospholipids or triglycerides closely mixed with hemeproteins and other proteins form a pro-oxidative system. PUFAs are highly relevant components in muscle foods since more unsaturated products are wanted. Pro-oxidative conditions may affect enzyme activity (Miura, Muraoka, & Ogiso, 1995) in a complex manner that justify better understanding of each pro-oxidative system and their enzymes. The iron of heme/hemin in met- or hemoglobin, with added H2O2,
* Corresponding author. Permanent address: Department of Chemistry, Biotechnology and Food Science, University of Life Science, 1432-Ås, Norway. Tel.: þ47 64965858; fax: þ47 64965901 E-mail addresses: [email protected] (B. Egelandsdal), [email protected] (L.P. Ren), [email protected] (P. Kathirvel), [email protected] (Y.S. Gong), [email protected] (M.L. Greaser), [email protected] (M.P. Richards). 1 Permanent address: College of Animal Science and Technology, China Agricultural University, Beijing 100193, China. 2 Permanent address: Nova Scotia Agricultural College, Tree Fruit Bioproducts Research Laboratory, Department of Environmental Sciences, Truro, Nova Scotia, Canada B2N 5E3. 0023-6438/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2010.11.005
will lead to oxidation of amino acids’ side chains (Egawa, Shimada, & Ishimura, 2000; King & Winfield, 1963; Østdal, Søgaard, Bendixen, & Andersen, 2001), loss in enzyme activity (Moreau, Castilho, Ferreira, & Carvalho-Alves, 1998) or increased enzyme activity (Park, Xiong, Alderton, & Ooizumi, 2006). Park et al. (2006) who examined three different pro-oxidative systems concluded that the systems’ effects on enzyme activity were unique for each oxidizing system tested. Since enzymes may change the texture and influence flavour formation of foods, further studies of enzyme activities under pro-oxidative conditions seem justified. In this investigation, washed cod muscle added metmyoglobin, has been used for studying the formation of lipid and protein oxidation products and in particular the effect of oxidation on washed cod proteases. Lipid oxidation could be initiated in washed cod within hours after hemoglobin was added (Richards & Hultin, 2000). The washed cod can also be used to evaluate the effects of added antioxidants and pro-oxidants on lipid oxidation (SanchezAlonso, Borderias, Larsson, & Undeland, 2007), and the effect of pro-oxidants on endogenous proteases present in the lipid bilayers (Egelandsdal, Ren, Gong, Greaser, & Richards, 2010). The washed cod can be prepared so that the amount of soluble proteins can be reduced below 1 g/kg of total protein (Egelandsdal et al., 2010).
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Abbreviation BSA bovine serum albumin DTT Dithiothreitol WCOD washed cod system SUP supernatant SED sediment DTNB 5, 50 -dithiobis (2-nitrobenzoic acid) TCA trichloroacetic acid TBARS Thiobarbituric Acid Reactive Substances SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis MALDI-TOF-TOF Matrix-assisted Laser Desorption/ Ionization-time of Flight-time of Flight EDTA Ethylenediaminetetraacetic acid Chaps 3-cholamidopropyl)-dimetyl ammonio]-1 propanesulfonate (M)ANOVA multivariate analysis of variance metMb metmyoglobin hydrogen peroxide H2O2 TFA trifluoroacetic acid Tris tris (hydroxymethyl) aminomethane
Thus the pro-oxidative effect of the lipids in bilayers and the added heme protein on proteases can indirectly be studied for any soluble exogenous protein without substantial interference from endogenous proteins. The washed cod has to our knowledge, not been used to study the effect that heme protein plus lipid peroxides may have on fish cathepsins and then on the degradation of an added (exogenous) protein. Cathepsins are thiol proteases and since at least some prooxidative systems are highly aggressive towards thiol groups (Xiong, Park, & Ooizumi, 2009), it was hypothesized that adding thiols could prolong degradation by exchanging thiol groups to proteases. In this investigation bovine serum albumin (BSA) was added to the washed cod system. This protein is regarded as an important antioxidant protein (Halliwell, Gutteridge, & Cross, 1992). The mechanism behind its antioxidative properties is, however, still debated (Okazaki et al., 2008). Okazaki et al. (2008) pointed the importance of sulphur containing amino acids with respect to antioxidative properties. The aim of this work was to study the effects of two different BSA molecules, one carrying more thiols than the other through reduction, on the pro-oxidative conditions created essentially by marine phospholipids and metMb. Inactivation of cathepsin B þ L and lipid degradation was emphasized. 2. Materials and methods 2.1. Fish raw material Atlantic cod (Gadus morhua) was from a local fish market that overnight shipped the fillets from the Boston area (USA). The fishes were approx. 48e72 h post mortem. Minced fillets (2 batches used for duplications) were frozen vacuum-packed after a standard washing procedure described by Richards, Dettmann, and Grunwald (2005). After thawing, 1 part fish mince was added to 10 parts 0.05 M sodium phosphate buffer (pH 6.3) followed by centrifugation at 13,000e16,200g for 20 min. This additional washing was carried out three times to reduce the amount of soluble protein to less than 0.2% (w/w) of total protein content as
verified by protein concentration measurements. Washing at the higher pH, as opposed to at pH 5.6, eased resolubilization. 2.2. Metmyoglobin purification Horse metmyoglobin (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 3410 ¼ 188 (Antonini & Brunori, 1971). 2.3. Purification and partial reduction of bovine serum albumin Standard bovine serum albumin (Sigma A3294, BSA; 50 mg/ml; 0.2 thiol/molecule) was purified similarly to metMb. In addition, BSA was dissolved in 0.25 M Dithiothreitol (DTT) and incubated for 15 min at room temperature before chromatographed on a DG 10 column to separate DTT from BSA. Separation was verified as lack of distinct redness by using freshly prepared nitroprusside (1 volume part) to 1 volume-part sample and 2 volume-parts saturated sodium bicarbonate. The concentration of BSA was determined using absorption at 279 nm (1 mg/ml BSA gives an absorbance of 0.659). The reduced BSA was kept under nitrogen until used (within 3 h). The partially reduced BSA had 3.5 thiols for each BSA molecule. 2.4. The washed cod (WCOD) system The washed fish system contained 86 2 mg/g of fish protein in phosphate buffer (0.05 M) at pH 5.6 to which metMb (40 mmol/kg; 0.68 mg/ml) and BSA (85 mmol/kg; 5.6 mg/ml) were added, if used. The moisture content was 91e90% w/w. These concentrations were chosen based on previous experiments by Richards and Hultin (2001) and Østdal et al. (2001), respectively. The details of the WCOD system are given by Richards and Hultin (2001) and Egelandsdal et al. (2010). metMb was added as a concentrated solution. The systems were monitored for 47 h after metMb addition. Samples were withdrawn at different time points and used as such or separated into supernatant (SUP) and sediment (SED) by centrifugation in an Eppendorf centrifuge at 16,200g for 20 min in microfuge tubes. This was done in order to study the soluble proteins essentially free of interference from the myofibrillar fish proteins that still dominated the system (weight ratio BSA:washed cod proteins was w15). 2.5. Determination of protein content Modified Lowry’s method (Markwell, Haas, Bieber, & Tolber, 1978) was used with an added heating step (85 C, 15 min) before addition of the Folin-Ciocalteu phenol reagent. The heating step was used to aid solubilization of otherwise insoluble proteins. For electrophoresis (see below), the protein concentration was determined with Quant Kit (Amersham Biosciences, England). Three replicates (N ¼ 3) were used for both the methods. 2.6. Free thiol determination in supernatant and total system Thiols in supernatants were measured (N ¼ 3) using the DTNB (5, 50 -dithiobis (2-nitrobenzoic acid), Sigma, USA) reaction with thiols (Hu, 1994) followed by precipitation of proteins in 80% v/v methanol and measurement of absorbance of SUP at 412 nm. Possible absorbance at 412 nm from still soluble metMb/hemin after the methanol precipitation was measured and corrected. No correction for myoglobin absorbance was necessary for the total (not centrifuged) system since the fish protein: myoglobin ratio
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and iron (II) chloride were added to the samples (N ¼ 2e3) and the absorbance was measured at 500 nm (Shimadzu, UV-2401) after 20 min of incubation at 25 C. A standard curve was constructed using cumene hydroperoxide and the concentration of lipid peroxide in the sample was expressed as mmol of lipid peroxides/kg of sediment. 2.8. Determination of thiobarbituric acid reactive substances (TBARS)
Fig. 1. Effect of addition of thiol-reduced and not thiol-reduced BSA on the formation of lipid peroxides in washed cod with and without added metmyoglobin. metMb addition was 40 mmol/kg and BSA addition was 85 mmol/kg. The measurements were made on proteins sedimented with their lipids at 16,200g for 20 min. The sediments contained 110e190 mg protein/g. Experimental points (standard error bars) with different letters indicate significant (p < 0.05) differences for samples added metMb. The symbols: * ¼ p < 0.05; ** ¼ p < 0.01; *** ¼ p < 0.001 compare data only in the vertical direction; the first symbol refers to the nearest datapoint. :WCOD þ Mb; A WCOD þ Mb þ REDBSA3.5; ,; WCOD þ REDBSA3.5; > WCOD þ Mb þ NOT REDBSA; 6 WCOD þ NOT REDBSA.
was much higher in the sediment. Reduced glutathione (1 mM) was used for generating a standard curve. 2.7. Determination of lipid peroxides Cold chloroform: methanol (1:1; 5 ml) was added to a 0.3 g muscle sample (sediment). Homogenization and centrifugation to obtain the lower chloroform layer were carried out as described by Undeland, Hultin, and Richards (2002). Ammonium thiocyanate
TBARS was determined (N ¼ 2e3) according to the modified method of Buege and Aust (1978). On the day of analysis, a solution of 50% trichloroacetic acid (TCA) with 13 g/L thiobarbituric acid (TBA) was prepared by mixing and heating at 65 C to dissolve the solutes. TCAeTBA reagent (1.2 ml) was then added to each sample (0.1 g) and the sample was mixed via inversion and heated at 65 C for 60 min and 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 as mmol of TBARS/kg of sediment. 2.9. Sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE was run (N ¼ 4) in accordance with the method described by Fritz, Swartz, and Greaser (1989) and Egelandsdal et al. (2010). Insoluble fish proteins were dissolved directly in the sample buffer at concentrations <5 mg/ml. Supernatants were precipitated with 80% (v/v) ice-cold methanol and centrifuged at 16,200g for 20 min before being dissolved in sample buffer. Ten percent (w/v) polyacrylamide gels were used. The gels were run in a Mighty Small II (SE 250) Slab Gel Unit-Amersham (Pharmacia Biotech., 800 Centennial Avenue, Piscataway, NJ) at 4 C and stained with Coomassie Brilliant Blue R-250. Pre-stained molecular weight markers used were a BCA marker protein kit (Thermo Fisher Scientific Inc.). Destained gels were scanned on an HPScanjet 3970 scanner at resolution 300 dpi. The images were thereafter transformed to 8 bits *.tif images before quantitative calculations were made. Lanes on gels were identified using ImageQuant version TL v2005 (Amersham Biosciences, England). Baselines for intensity profiles were identified manually and subtracted. 2.10. Mass spectroscopic determination of proteins following trypsin digestion
Fig. 2. Effect of addition of thiol-reduced and not thiol-reduced BSA on the formation of thiobarbituric acid reactive substances (TBARS) in washed cod with and without added metmyoglobin. metMb was added at 40 mmol/kg washed cod; BSA addition was at 85 mmol/kg. The measurements were made on proteins sedimented with their lipids (sediments) at 16,200g for 20 min. Experimental points (standard error bars) with different letters indicate significant (p < 0.05) differences for samples added metMb. The symbols: * ¼ p < 0.05; ** ¼ p < 0.01; *** ¼ p < 0.001 compare data only in the vertical direction; the first symbol refers to the nearest datapoint. : WCOD þ Mb; A WCOD þ Mb þ REDBSA3.5; , WCOD þ REDBSA3.5; > WCOD þ Mb þ NOT REDBSA; Δ WCOD þ NOT REDBSA.
The preparation of protein from SDS-PAGE gels is described by Egelandsdal et al. (2010). Trypsination was carried out with trypsin (Promega) overnight at 33 C. The peptides were extracted in 0.2% TFA, dried, reconstituted in 30 ml of Millipore water/0.1%TFA and desalted through a ZipTip-C18 column (Millipore) before spotting directly onto the Opti-TOFÔ 384 Well plate (Applied Biosystems, Foster City, CA) and re-crystallized with 0.50 ml of matrix [10 mg/ml a-Cyano-4-hydroxycinnamic acid in acetonitrile/H2O/TFA (60%:40%:0.2% v/v)]. Peptide Map Fingerprint result-dependent MS/MS analysis was performed on a 4800 Matrix-Assisted Laser Desorption/Ionization-Time of Flight-Time of Flight (MALDI-TOFTOF) mass spectrometer (Applied Biosystems, Foster City, CA). Raw data was deconvoluted using GPS ExplorerÔ 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).
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Table 1 Protein concentrations in supernatants obtained after centrifugation of WCOD for 20 min at 16,200g 20 min. Concentrations were determined by modified Lowry. System
Conc. (mg/ml) 0e2 h
WCOD WCOD WCOD WCOD
þ þ þ þ
NOT REDBSA Mb þ NOT REDBSA REDBSA3.5 Mb þ REDBSA3.5
6.04 6.71 5.88 6.53
15e18 h 0.30a,* 0.18a 0.44a 0.49a
6.97 7.64 6.05 6.57
0.65b 0.45b 0.13a 0.44a
21e22 h 7.45 7.66 7.62 7.35
0.77b 0.22b 0.73b 0.13b
46e47 h 7.48 7.34 8.06 7.45
0.44b 0.29b 0.65b 0.49b
*Different letters as superscript mean significantly (p< 0.05) different measurements to be compared row by row.
2.11. Determination of cathepsin B þ L activity
3. Results
One gram of the WCOD system was homogenized on ice, and the sample extract (supernatant) was incubated with Z-Phe-Arg 4methoxy-methylcoumarin hydrochloride as substrate as described by Egelandsdal et al. (2010). Chloroacetate was added to stop the enzymatic reaction. The emitted light of the samples (N ¼ 2e4) was 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. A calibration curve using the standard 7-amino-4methylcoumarin was prepared and the amount of formed product was determined from the standard curve. DTT was not used in the assay.
3.1. Effect of reduced BSA addition on metMb-mediated lipid oxidation in washed cod system
2.12. Statistics The statistical program Minitab, version 15, was used. The changes in the thiols, PV and TBARS were analyzed using a generalized linear model using time ranges, myoglobin and their interaction as factors. For comparisons Tukey’s test (significance level 0.05) was used. Protein concentration data was analyzed using oneway analysis of variance (ANOVA) and Tukey’s test for comparisons. Significant linear trends were determined by the routine Regression in Minitab. Fifty: Fifty MANOVA: This statistical method was carried out as described by Langsrud (2002) and accessed through www. langsrud.com. 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.
The addition of reduced BSA to washed cod containing metMb reduced the formation of lipid peroxides (Fig. 1) and TBARS (Fig. 2). At approximately 15 h, the level of lipid peroxides in the WCOD þ Mb þ REDBSA3.5 system remained low compared to WCOD þ Mb and WCOD þ Mb þ NOT REDBSA systems (Fig. 1). A significant (p < 0.05) increase in lipid peroxides with time was observed during storage in all the systems with added metMb. A significant (p ¼ 0.004) difference in lipid peroxides between the metMb systems with reduced and not reduced BSA at 47 h was observed. Both these systems were also significantly different from WCOD þ Mb after 47 h storage (Fig. 1). TBARS increased with time for all systems containing metMb (Fig. 2). The magnitude of TBARS was always lower for the WCOD þ Mb þ REDBSA3.5 system compared to the WCOD þ Mb and WCOD þ Mb þ NOT REDBSA systems. 3.2. Effect of BSA addition on the amount of soluble proteins in the washed cod system The protein concentration of the supernatants increased with time for all systems reported in Table 1. The increase in soluble proteins with time was the least (the change was 0.63 mg/ml from 0 to 47 h) for the system WCOD þ Mb þ NOT REDBSA, while the largest increase (the changes was 2.18 mg/ml from 0 to 47 h) in soluble proteins with time was observed for the system WCOD þ REDBSA3.5. The difference in initial protein content largely reflected if myoglobin was added or not. This increase in soluble protein with time was less for the WCOD þ Mb þ REDBSA3.5 system compared to the WCOD þ REDBSA3.5 system (p ¼ 0.042; t-test). The system WCOD þ Mb had no change (i.e. 0.03 mg/ml) in soluble proteins within the same time frame (not shown in Table 1). Soluble proteins tended to increase the most from 0 to 47 h for the system WCOD þ REDBSA3.5 compared to WCOD þ NOT REDBSA, but the systems were not significantly different (p ¼ 0.12; t-test). 3.3. Effect of added BSA on the oxidation of thiols by metMb
Fig. 3. Effect of storage time on accessible thiols (in mM) of soluble proteins in the supernatant obtained after centrifugation of the washed cod system (pH 5.6) at 16200g for 20 min. The data points were fitted to linear models; sign of slope and its significance (p-value) is indicated on the right hand side. : WCOD þ Mb; A WCOD þ Mb þ REDBSA3.5; , WCOD þ REDBSA3.5; > WCOD þ Mb þ NOT REDBSA; 6 WCOD þ NOT REDBSA.
Fig. 3 shows the changes in the thiol content of the supernatants. The WCOD þ REDBSA3.5 system had a high thiol content that increased significantly with time (Fig. 3). Even for the WCOD þ NOT REDBSA system, the number of thiols tended to increase with time. The WCOD þ Mb and WCOD þ Mb þ NOT REDBSA all had low values for thiols measured after 1.5e2.0 h. The thiol content of WCOD þ Mb was extensively reduced already after 1.5 h with only a tendency for further reduction with time. The thiol content of WCOD þ Mb þ REDBSA3.5 decreased significantly with observation time. The WCOD þ Mb þ REDBSA3.5 system oxidized to the same thiol content as the WCOD þ Mb þ NOT REDBSA system after 46e48 h.
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Fig. 4. Panel a: Effect of time on accessible thiols of two intact WCOD systems (pH 5.6). The standard deviation bars for the WCOD-Mb system include batch deviations. : WCOD þ Mb; effect of time, p ¼ 0.025; , WCOD þ REDBSA3.5; effect of time, p ¼ 0.53. Panel b: The change in thiols for different intact systems after 47 h storage. Different letters indicate significant (p < 0.05) differences.
Metmyoglobin addition also reduced the thiol content of the intact (not centrifuged) systems with storage time (Fig. 4a) but the reduction was low compared to the reduction observed for the supernatant (soluble) proteins. After 47 h, thiol contents of all systems were constrained between WCOD þ REDBSA3.5 and WCOD þ Mb (Fig. 4b). 3.4. Cathepsin B þ L activity in WCOD systems after BSA addition The activity of cathepsin B þ L was maintained by the addition of reduced BSA during the observation period (Table 2). The WCOD þ REDBSA3.5 system had higher enzyme activity than the WCOD þ Mb and WCOD þ Mb þ REDBSA3.5 systems. BSA samples that carried intermediate amounts of thiols, gave systems with intermediate enzyme activities for cathepsin B þ L (not shown). No difference in enzyme activities could be observed for the WCOD þ Mb þ NOT REDBSA and WCOD þ Mb þ REDBSA3.5 systems at 47 h (Table 2). 3.5. Effect of storage time on the protein pattern of SDS-PAGE gels SDS-PAGE gels run on intact (not centrifuged) WCOD systems did not reveal a significant effect of metMb addition on fish proteins (Table 3). A main effect (29.6%; p < 0.002) of storage time on ice was observed, indicating more degradation of myosin HC with time and an increase in proteins with MW below 44 kDa. The interaction effect (time myoglobin) was also significant (p ¼ 0.03). The proteins of the supernatant of WCOD þ REDBSA3.5 was dominated by BSA (Fig. 5), but influenced by metMb addition
when all area volumes minus metMb were used for the statistical test (Table 3). Systematic differences with or without metMb addition were observed for area volumes 6 (p ¼ 0.03) and 1 (p ¼ 0.04; normalized densimetric profiles were used for statistical calculations). WCOD þ REDBSA3.5 contained less of area volume 1 and more of area volume 6 compared to WCOD þ Mb þ REDBSA3.5. The ratio between area volumes 6 and 5 increased steadily with storage time for WCOD þ REDBSA3.5. However, for the system WCOD þ Mb þ REDBSA3.5 the increase in area volume 6 with time was not significant (p ¼ 0.37). Area volume 1 was not attempted identified. Due to its high molecular weight it seemed to be a degraded molecule from a higher molecular weight (>240 kDa) component. There was a tendency (p ¼ 0.07) for more degraded components from collagen and myosin HC (area volumes 3 and 4) in systems with only reduced BSA added. The protein contained in area volume 6 was a BSA fragment as determined by MALDI-TOF-TOF after trypsin digestion (Table 4). This fragment had a molecular weight of 41.7 1.0 kDa estimated from identified peptides and the molecular weight standards used for electrophoresis. 4. Discussion At pH 5.6 the reducing power of thiol groups should be limited since pKa of thiol groups is typically above pH 8.0, still lipid peroxides and TBARS formation were slowed down by the addition of reduced BSA3.5. Since also proteolytic activity was
Table 2 Effect of addition of reduced and not reduced BSA on the activity of cathepsin B þ L in WCOD þ Mb systems. Time
1.5 h þ 3.5 he
System
WCOD þ Mb
Relative cathepsin B þ L activityd (30 C)
0.27 0.05
a
43 h þ 3.5 h WCOD þ Mb þ REDBSA 3.5 0.70 0.06b
WCOD þ RED BSA3.5 0.83 0.03b
WCOD þ Mb þ NOT REDBSA 0.46 0.09c
WCOD þ Mb 0.18 0.05
a
WCOD þ Mb þ REDBSA3.5 0.23 0.06a
WCOD þ REDBSA3.5 0.80 0.20b
Different letters in superscript (aec) indicate significantly different figures (p<0.05). d WCOD’s enzyme activity of 5.4 nmol substrate/gram protein min defines unit activity. e The activity of cathepsin B þ L as measured in WCOD after 1.5 h incubation plus a preparation period of 3.5 h; Statistics: Tukey’s test, p < 0.05.
WCOD þ Mb þ NOT REDBSA 0.22 0.02a
1010
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Table 3 Changes in the gel pattern of insoluble and soluble proteins promoted by storage time (at 2 C) and added myoglobin (0.7 mg/g). Type of protein
Insoluble Soluble
Mb:protein weight ratio
0.007e0.008 0.11
Explained variance (%) of effects Storage
Myoglobin
Storage myoglobin
29.2(p < 0.002) 25.8(p ¼ 0.054)
n.s.a 22.4 (p ¼ 0.0013)
6.8 (p ¼ 0.03) n.s.a
# of lanes
System
16 16
WCOD Mb þ REDBSA3.5 WCOD Mb þ REDBSA3.5
Ten percent (w/v) SDS-PAGE gels were used. The data were analyzed by 50:50 MANOVA (Langsrud, 2002) without standardization. The normalized protein area volumes of added Mb were removed prior to statistical analysis. a n.s. ¼ not significant.
affected, it appeared that the quality variables aimed at in these investigations were affected by the addition of reduced BSA. Untreated (not reduced) BSA had no similar effect and the peroxide measurements even suggested that this type of BSA would act as a pro-oxidant at the longest incubation time. Spectral measurement (0e2 h, not shown) showed a tendency for myoglobin (reduced form) to be present in the system containing REDBSA3.5. However, after 47 h the absorbance at 410 nm was significantly (p < 0.05) higher for the WCOD þ Mb þ REDBSA3.5 compared to the WCOD þ Mb system (not shown). This observation suggested less degradation of the hemin when reduced BSA was added. The less oxidative conditions provided by adding reduced BSA to the washed cod system also promoted proteolysis of BSA since the endogenous proteases present in the washed cod system were kept active for a longer period. Also the washed cod protein appeared more degraded when reduced BSA was added. Enzymes have been reported to become quickly inactivated (Spanier & Bird, 1982) by metMb addition. However, reduced BSA can protect against inactivation of thiol proteases in two manners: 1) by providing thiols for general reduction, and specifically, for exchange, to thiol proteases and 2) by acting as a general radical scavenging protein and thereby remove free radicals that otherwise may inactive the enzymes. Cathepsin B þ L remained active longer with the addition of reduced BSA as supported by the fact that substantially less of the BSA fragment was detected when Mb was added to the WCOD þ REDBSA3.5 system. Reduced BSA therefore postponed inactivation of thiol proteases. The data reported here, however, cannot identify for certain that BSA has been degraded by cathepsin B (or L). Cathepsin B is not a very selective thiol protease but has been reported to: 1)
cut at arginineearginine; 2) have a preference for valine or threonine next to arginine; 3) have a preference for valine and alanine next to lysine (Gosalia, Salisbury, Ellman, & Diamond, 2005) or 4) cleave at methionineeglycine, threonineeleucine or leucineethreonine (Hughes, O’Neill, Sweeney & Healy, 1999). Cathepsin L has been reported to have preference for arginineephenylalanine, arginineeleucine or lysineeleucine (Gosalia et al., 2005). The data above (Fig. 5), nevertheless, confirmed that the reduced BSA3.5 system stimulated degradation compared to the BSAeMb system that could be in accordance with cathepsin activity. The commercial BSA powder contained 0.2 0.02 mol of thiols/ mol BSA. Complete reduction of BSA’s 17 disulfides is possible, but gives a highly unstable protein as reported by Lee and Hirose (1992). The reduction procedure used in this study made it possible to handle the concentrated BSA solution without gel formation/aggregation both before and after reduction. The addition of partially reduced BSA to WCOD provided 0.40 mmol thiols/ kg of the system. The extensively washed fish had 70 mmol/kg proteins of accessible thiols. Since the protein content of the WCOD system used here was 89 mg/g, it is obvious that the thiol content of WCOD was dominated (>90%) by the thiols originating from the insoluble fish proteins independent of whether reduced or not reduced BSA was used. Accordingly, a large potential antioxidant capacity in WCOD was present as thiols. Some of these thiols would be released through degradation when metMb was not added. However, the design used allowed only for interpretation of the importance of the minor fraction of thiols coming from BSA plus the increase in soluble thiols associated with the ongoing protein degradation. To what extent the antioxidant capacity of cysteine
Table 4 The amino acid sequence of bovine serum albumin (Bos taurus, giê30794280).
Amino acids coloured fair grey were identified as peptides using MALDI-TOF-TOF. The framed part indicates the fragment from BSA identified. Possible fragmentation sites that would extend the fragment to the observed molecular weight are indicated by squares.
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Fig. 5. SDS-PAGE gels (10% w/v) of WCOD þ REDBSA3.5 supernatants prepared by centrifugation at 16200g for 20 min. From left: L1: Standards with molecular weights indicated in kDa; L2: WCOD þ Mb þ REDBSA3.5 1.5 h; L3: WCOD þ REDBSA3.5 1.5 h; L4: WCOD þ Mb þ REDBSA3.5 15 h; L5: WCOD þ REDBSA3.5 15 h; L6: WCOD þ Mb þ REDBSA3.5 22 h; L7: WCOD þ REDBSA3.5 22 h; L8: WCOD þ Mb þ REDBSA3.5 46 h; L9: WCOD þ REDBSA3.5 46 h; L10: Standards as for L1). The supernatants were precipitated in 80% methanol, dried and re-suspended in sample buffer to give solutions containing approx. 5 mg/ml. The figure shows how different area volumes among soluble proteins were identified before statistical calculations were done. The area volumes were identified as follows: 1) Not identified w240 kDa; 2) Myosin HC; 3) and 4) Degraded components from myosin and collagen (identified by Egelandsdal et al., 2010); 5) BSA 69 kDa; 6) BSA fragment 41.7 1.0 kDa; 7) Tropomyosin; 8) Troponin I; 9) Myoglobin.
depended on the fish protein being soluble or not, could not be elucidated. 5. Conclusion Partly reduced BSA added to a washed cod system reduced the formation of lipid peroxides values and TBARS compared to using untreated (not reduced) BSA. Reduced BSA led to increased degradation of insoluble fish proteins and extended the activity of cathepsin B þ L compared to system where both untreated (not reduced) BSA and metMb were added. metMb prevented the release of thiols as soluble proteins or as peptides and also the actual degradation of BSA in the washed cod system. Acknowledgement The Fulbright Foundation, The University of Life Science and The Research Council of Norway (grant no. 184846) and The College of Agricultural and Life Sciences, UW-Madison, HATCH project PRJ28VY are thanked for financial support. Tine and Nortura are also thanked for their financial support. The funding source(s) has only been involved in the decision to submit the paper for publication. 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 (pp. 44). Amsterdam: North-Holland Publishing.
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Buege, J. A., & Aust, S. D. (1978). Microsomal lipid peroxidation. Methods in Enzymology, 52, 302e310. Egawa, T., Shimada, H., & Ishimura, Y. (2000). Formation of compound I in the reaction of native myoglobins with hydrogen peroxide. Journal of Biological Chemistry, 275, 34858e34866. Egelandsdal, B., Ren, L. P., Gong, Y. S., Greaser, M. L., & Richards, M. P. (2010). Factors affecting solubilization and oxidation of proteins during equine metmyoglobinmediated lipid oxidation in extensively washed cod muscle. Food Chemistry, 122, 1102e1110. 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, 205e210. Gosalia, D. N., Salisbury, C. M., Ellman, J. A., & Diamond, S. L. (2005). High troughput substrate specificity profiling of serine and cysteine proteases using solutionephase fluorogenic peptide microarrays. Molecular and Cellular Proteomics, 4, 625e636. Halliwell, B., Gutteridge, J. M., & Cross, C. E. (1992). Free radicals, antioxidants and human diseases: where are we now. Journal of Laboratory and Clinical Medicine, 119, 568e620. Hu, M. (1994). Measurements of protein thiol groups and glutathione in plasma. Methods in Enzymology, 233, 380e385. Hughes, M. C., O’Neill, E. E., Sweeney, P. L. H., & Healy, A. (1999). Proteolysis of bovine F-actin by cathepsin B. Food Chemistry, 64, 525e530. King, N. K., & Winfield, M. E. (1963). The mechanism of metmyoglobin oxidation. Journal of Biological Chemistry, 238, 1520e1528. Langsrud, Ø (2002). 50:50 multivariate analysis of variance for collinear responses. The Statistician, 51, 305e317. Lee, J. Y., & Hirose, M. (1992). Effects of disulfide reduction on the emulsifying properties of bovine serum albumin. Biotechnology and Biochemistry, 56, 1810e1813. 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, 206e210. Miura, T., Muraoka, S., & Ogiso, T. (1995). Adriamycin-Fe(3þ)-induced inactivation of enzymes in erythrocyte membranes during lipid peroxidation. Research Communication Molecular Pathology Pharmacology, 87, 133e143. Moreau, V. H., Castilho, R. F., Ferreira, S. T., & Carvalho-Alves, P. C. (1998). Oxidative damage to sarcoplasmic reticulum Ca2þ-ATPase at submicromolar iron concentrations: evidence for metal-catalyzed oxidation. Free Radical Biology & Medicine, 25, 554e560. Okazaki, T., Okudaira, N., Ishii, N., Yotsuyanagi, H., Nagai, T., Tokudome, S., et al. (2008). Comparison of the antioxidant activity of albumin from various animal species. Zoological Science, 25, 172e177. Park, D., Xiong, Y. L., Alderton, A. L., & Ooizumi, T. (2006). Biochemical changes in myofibrillar protein isolates exposed to three oxidizing systems. Journal of Agricultural and Food Chemistry, 54, 4445e4451. 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, 10231e10238. 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, 3141e3147. Richards, M. P., & Hultin, H. O. (2001). Rancidity development in a fish model system as affected by phospholipids. Journal of Food Lipids, 8, 215e230. Sanchez-Alonso, I., Borderias, J., Larsson, K., & Undeland, I. (2007). Inhibition of hemoglobin-mediated oxidation of regular and lipid-fortified washed cod mince by a white grape dietary fiber. Journal of Agricultural and Food Chemistry, 55, 5299e5305. 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, 313e320. 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, 6847e6853. Xiong, Y. L., Park, D., & Ooizumi, T. (2009). Variation in the cross-linking pattern of porcine myofibrillar protein exposed to three oxidative environments. Journal of Agricultural and Food Chemistry, 57, 153e159. 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, 757e766.
Web reference http://www.langsrud.com/stat/ffmanova.htm (Downloadable 12th August 2010).
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LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Addition of partly reduced bovine serum albumin to a metmyoglobin-fortified washed cod system gives reduced formation of lipid oxidation products and increased degradation of proteins B. Egelandsdal*, L.P. Ren 1, P. Kathirvel 2, Y.S. Gong, M.L. Greaser, M.P. Richards Meat Science and Muscle Biology Laboratory, Department of Animal Sciences, University of WisconsineMadison, Madison, Wisconsin 53706-1284, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 March 2010 Received in revised form 18 August 2010 Accepted 2 November 2010
The aim of the investigation was to study the pro-oxidative system of washed cod with added horse metmyoglobin (metMb) regarding lipid and enzyme oxidation plus protein degradation. The changes were studied at pH 5.6 and 2 C for 47 h. Bovine serum albumin (BSA) with 0.2 or 3.5 thiol (reduced) groups per molecule was added as an antioxidant protein and as providers of free thiols. BSA with the higher amount of thiols decreased the formation of lipid peroxides and thiobarbituric acid reactive substances (TBARS) in the washed cod system. Metmyoglobin (metMb) addition slowed down the enzymatic release of proteins and protein fragments from insoluble washed cod proteins while the opposite was observed with added reduced BSA. Addition of metMb strongly inactivated the thiol proteases cathepsin B þ L and effectively oxidized the low molecular weight thiols in the system while reduced BSA slowed down inactivation of the proteases. The endogenous proteases degraded BSA to a fragment of 41.7 kDa. Formation of this fragment was reduced when metMb was added. BSA (not reduced) did not influence TBARS formation in the washed cod system with metMb, but increased the formation of lipid peroxides after 47 h compared to the system without BSA. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Bovine serum albumin Cathepsin Washed cod Metmyoglobin Oxidation
1. Introduction Polyunsaturated fatty acids (PUFAs) in phospholipids or triglycerides closely mixed with hemeproteins and other proteins form a pro-oxidative system. PUFAs are highly relevant components in muscle foods since more unsaturated products are wanted. Pro-oxidative conditions may affect enzyme activity (Miura, Muraoka, & Ogiso, 1995) in a complex manner that justify better understanding of each pro-oxidative system and their enzymes. The iron of heme/hemin in met- or hemoglobin, with added H2O2,
* Corresponding author. Permanent address: Department of Chemistry, Biotechnology and Food Science, University of Life Science, 1432-Ås, Norway. Tel.: þ47 64965858; fax: þ47 64965901 E-mail addresses: [email protected] (B. Egelandsdal), [email protected] (L.P. Ren), [email protected] (P. Kathirvel), [email protected] (Y.S. Gong), [email protected] (M.L. Greaser), [email protected] (M.P. Richards). 1 Permanent address: College of Animal Science and Technology, China Agricultural University, Beijing 100193, China. 2 Permanent address: Nova Scotia Agricultural College, Tree Fruit Bioproducts Research Laboratory, Department of Environmental Sciences, Truro, Nova Scotia, Canada B2N 5E3. 0023-6438/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2010.11.005
will lead to oxidation of amino acids’ side chains (Egawa, Shimada, & Ishimura, 2000; King & Winfield, 1963; Østdal, Søgaard, Bendixen, & Andersen, 2001), loss in enzyme activity (Moreau, Castilho, Ferreira, & Carvalho-Alves, 1998) or increased enzyme activity (Park, Xiong, Alderton, & Ooizumi, 2006). Park et al. (2006) who examined three different pro-oxidative systems concluded that the systems’ effects on enzyme activity were unique for each oxidizing system tested. Since enzymes may change the texture and influence flavour formation of foods, further studies of enzyme activities under pro-oxidative conditions seem justified. In this investigation, washed cod muscle added metmyoglobin, has been used for studying the formation of lipid and protein oxidation products and in particular the effect of oxidation on washed cod proteases. Lipid oxidation could be initiated in washed cod within hours after hemoglobin was added (Richards & Hultin, 2000). The washed cod can also be used to evaluate the effects of added antioxidants and pro-oxidants on lipid oxidation (SanchezAlonso, Borderias, Larsson, & Undeland, 2007), and the effect of pro-oxidants on endogenous proteases present in the lipid bilayers (Egelandsdal, Ren, Gong, Greaser, & Richards, 2010). The washed cod can be prepared so that the amount of soluble proteins can be reduced below 1 g/kg of total protein (Egelandsdal et al., 2010).
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Abbreviation BSA bovine serum albumin DTT Dithiothreitol WCOD washed cod system SUP supernatant SED sediment DTNB 5, 50 -dithiobis (2-nitrobenzoic acid) TCA trichloroacetic acid TBARS Thiobarbituric Acid Reactive Substances SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis MALDI-TOF-TOF Matrix-assisted Laser Desorption/ Ionization-time of Flight-time of Flight EDTA Ethylenediaminetetraacetic acid Chaps 3-cholamidopropyl)-dimetyl ammonio]-1 propanesulfonate (M)ANOVA multivariate analysis of variance metMb metmyoglobin hydrogen peroxide H2O2 TFA trifluoroacetic acid Tris tris (hydroxymethyl) aminomethane
Thus the pro-oxidative effect of the lipids in bilayers and the added heme protein on proteases can indirectly be studied for any soluble exogenous protein without substantial interference from endogenous proteins. The washed cod has to our knowledge, not been used to study the effect that heme protein plus lipid peroxides may have on fish cathepsins and then on the degradation of an added (exogenous) protein. Cathepsins are thiol proteases and since at least some prooxidative systems are highly aggressive towards thiol groups (Xiong, Park, & Ooizumi, 2009), it was hypothesized that adding thiols could prolong degradation by exchanging thiol groups to proteases. In this investigation bovine serum albumin (BSA) was added to the washed cod system. This protein is regarded as an important antioxidant protein (Halliwell, Gutteridge, & Cross, 1992). The mechanism behind its antioxidative properties is, however, still debated (Okazaki et al., 2008). Okazaki et al. (2008) pointed the importance of sulphur containing amino acids with respect to antioxidative properties. The aim of this work was to study the effects of two different BSA molecules, one carrying more thiols than the other through reduction, on the pro-oxidative conditions created essentially by marine phospholipids and metMb. Inactivation of cathepsin B þ L and lipid degradation was emphasized. 2. Materials and methods 2.1. Fish raw material Atlantic cod (Gadus morhua) was from a local fish market that overnight shipped the fillets from the Boston area (USA). The fishes were approx. 48e72 h post mortem. Minced fillets (2 batches used for duplications) were frozen vacuum-packed after a standard washing procedure described by Richards, Dettmann, and Grunwald (2005). After thawing, 1 part fish mince was added to 10 parts 0.05 M sodium phosphate buffer (pH 6.3) followed by centrifugation at 13,000e16,200g for 20 min. This additional washing was carried out three times to reduce the amount of soluble protein to less than 0.2% (w/w) of total protein content as
verified by protein concentration measurements. Washing at the higher pH, as opposed to at pH 5.6, eased resolubilization. 2.2. Metmyoglobin purification Horse metmyoglobin (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 3410 ¼ 188 (Antonini & Brunori, 1971). 2.3. Purification and partial reduction of bovine serum albumin Standard bovine serum albumin (Sigma A3294, BSA; 50 mg/ml; 0.2 thiol/molecule) was purified similarly to metMb. In addition, BSA was dissolved in 0.25 M Dithiothreitol (DTT) and incubated for 15 min at room temperature before chromatographed on a DG 10 column to separate DTT from BSA. Separation was verified as lack of distinct redness by using freshly prepared nitroprusside (1 volume part) to 1 volume-part sample and 2 volume-parts saturated sodium bicarbonate. The concentration of BSA was determined using absorption at 279 nm (1 mg/ml BSA gives an absorbance of 0.659). The reduced BSA was kept under nitrogen until used (within 3 h). The partially reduced BSA had 3.5 thiols for each BSA molecule. 2.4. The washed cod (WCOD) system The washed fish system contained 86 2 mg/g of fish protein in phosphate buffer (0.05 M) at pH 5.6 to which metMb (40 mmol/kg; 0.68 mg/ml) and BSA (85 mmol/kg; 5.6 mg/ml) were added, if used. The moisture content was 91e90% w/w. These concentrations were chosen based on previous experiments by Richards and Hultin (2001) and Østdal et al. (2001), respectively. The details of the WCOD system are given by Richards and Hultin (2001) and Egelandsdal et al. (2010). metMb was added as a concentrated solution. The systems were monitored for 47 h after metMb addition. Samples were withdrawn at different time points and used as such or separated into supernatant (SUP) and sediment (SED) by centrifugation in an Eppendorf centrifuge at 16,200g for 20 min in microfuge tubes. This was done in order to study the soluble proteins essentially free of interference from the myofibrillar fish proteins that still dominated the system (weight ratio BSA:washed cod proteins was w15). 2.5. Determination of protein content Modified Lowry’s method (Markwell, Haas, Bieber, & Tolber, 1978) was used with an added heating step (85 C, 15 min) before addition of the Folin-Ciocalteu phenol reagent. The heating step was used to aid solubilization of otherwise insoluble proteins. For electrophoresis (see below), the protein concentration was determined with Quant Kit (Amersham Biosciences, England). Three replicates (N ¼ 3) were used for both the methods. 2.6. Free thiol determination in supernatant and total system Thiols in supernatants were measured (N ¼ 3) using the DTNB (5, 50 -dithiobis (2-nitrobenzoic acid), Sigma, USA) reaction with thiols (Hu, 1994) followed by precipitation of proteins in 80% v/v methanol and measurement of absorbance of SUP at 412 nm. Possible absorbance at 412 nm from still soluble metMb/hemin after the methanol precipitation was measured and corrected. No correction for myoglobin absorbance was necessary for the total (not centrifuged) system since the fish protein: myoglobin ratio
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and iron (II) chloride were added to the samples (N ¼ 2e3) and the absorbance was measured at 500 nm (Shimadzu, UV-2401) after 20 min of incubation at 25 C. A standard curve was constructed using cumene hydroperoxide and the concentration of lipid peroxide in the sample was expressed as mmol of lipid peroxides/kg of sediment. 2.8. Determination of thiobarbituric acid reactive substances (TBARS)
Fig. 1. Effect of addition of thiol-reduced and not thiol-reduced BSA on the formation of lipid peroxides in washed cod with and without added metmyoglobin. metMb addition was 40 mmol/kg and BSA addition was 85 mmol/kg. The measurements were made on proteins sedimented with their lipids at 16,200g for 20 min. The sediments contained 110e190 mg protein/g. Experimental points (standard error bars) with different letters indicate significant (p < 0.05) differences for samples added metMb. The symbols: * ¼ p < 0.05; ** ¼ p < 0.01; *** ¼ p < 0.001 compare data only in the vertical direction; the first symbol refers to the nearest datapoint. :WCOD þ Mb; A WCOD þ Mb þ REDBSA3.5; ,; WCOD þ REDBSA3.5; > WCOD þ Mb þ NOT REDBSA; 6 WCOD þ NOT REDBSA.
was much higher in the sediment. Reduced glutathione (1 mM) was used for generating a standard curve. 2.7. Determination of lipid peroxides Cold chloroform: methanol (1:1; 5 ml) was added to a 0.3 g muscle sample (sediment). Homogenization and centrifugation to obtain the lower chloroform layer were carried out as described by Undeland, Hultin, and Richards (2002). Ammonium thiocyanate
TBARS was determined (N ¼ 2e3) according to the modified method of Buege and Aust (1978). On the day of analysis, a solution of 50% trichloroacetic acid (TCA) with 13 g/L thiobarbituric acid (TBA) was prepared by mixing and heating at 65 C to dissolve the solutes. TCAeTBA reagent (1.2 ml) was then added to each sample (0.1 g) and the sample was mixed via inversion and heated at 65 C for 60 min and 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 as mmol of TBARS/kg of sediment. 2.9. Sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE was run (N ¼ 4) in accordance with the method described by Fritz, Swartz, and Greaser (1989) and Egelandsdal et al. (2010). Insoluble fish proteins were dissolved directly in the sample buffer at concentrations <5 mg/ml. Supernatants were precipitated with 80% (v/v) ice-cold methanol and centrifuged at 16,200g for 20 min before being dissolved in sample buffer. Ten percent (w/v) polyacrylamide gels were used. The gels were run in a Mighty Small II (SE 250) Slab Gel Unit-Amersham (Pharmacia Biotech., 800 Centennial Avenue, Piscataway, NJ) at 4 C and stained with Coomassie Brilliant Blue R-250. Pre-stained molecular weight markers used were a BCA marker protein kit (Thermo Fisher Scientific Inc.). Destained gels were scanned on an HPScanjet 3970 scanner at resolution 300 dpi. The images were thereafter transformed to 8 bits *.tif images before quantitative calculations were made. Lanes on gels were identified using ImageQuant version TL v2005 (Amersham Biosciences, England). Baselines for intensity profiles were identified manually and subtracted. 2.10. Mass spectroscopic determination of proteins following trypsin digestion
Fig. 2. Effect of addition of thiol-reduced and not thiol-reduced BSA on the formation of thiobarbituric acid reactive substances (TBARS) in washed cod with and without added metmyoglobin. metMb was added at 40 mmol/kg washed cod; BSA addition was at 85 mmol/kg. The measurements were made on proteins sedimented with their lipids (sediments) at 16,200g for 20 min. Experimental points (standard error bars) with different letters indicate significant (p < 0.05) differences for samples added metMb. The symbols: * ¼ p < 0.05; ** ¼ p < 0.01; *** ¼ p < 0.001 compare data only in the vertical direction; the first symbol refers to the nearest datapoint. : WCOD þ Mb; A WCOD þ Mb þ REDBSA3.5; , WCOD þ REDBSA3.5; > WCOD þ Mb þ NOT REDBSA; Δ WCOD þ NOT REDBSA.
The preparation of protein from SDS-PAGE gels is described by Egelandsdal et al. (2010). Trypsination was carried out with trypsin (Promega) overnight at 33 C. The peptides were extracted in 0.2% TFA, dried, reconstituted in 30 ml of Millipore water/0.1%TFA and desalted through a ZipTip-C18 column (Millipore) before spotting directly onto the Opti-TOFÔ 384 Well plate (Applied Biosystems, Foster City, CA) and re-crystallized with 0.50 ml of matrix [10 mg/ml a-Cyano-4-hydroxycinnamic acid in acetonitrile/H2O/TFA (60%:40%:0.2% v/v)]. Peptide Map Fingerprint result-dependent MS/MS analysis was performed on a 4800 Matrix-Assisted Laser Desorption/Ionization-Time of Flight-Time of Flight (MALDI-TOFTOF) mass spectrometer (Applied Biosystems, Foster City, CA). Raw data was deconvoluted using GPS ExplorerÔ 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).
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Table 1 Protein concentrations in supernatants obtained after centrifugation of WCOD for 20 min at 16,200g 20 min. Concentrations were determined by modified Lowry. System
Conc. (mg/ml) 0e2 h
WCOD WCOD WCOD WCOD
þ þ þ þ
NOT REDBSA Mb þ NOT REDBSA REDBSA3.5 Mb þ REDBSA3.5
6.04 6.71 5.88 6.53
15e18 h 0.30a,* 0.18a 0.44a 0.49a
6.97 7.64 6.05 6.57
0.65b 0.45b 0.13a 0.44a
21e22 h 7.45 7.66 7.62 7.35
0.77b 0.22b 0.73b 0.13b
46e47 h 7.48 7.34 8.06 7.45
0.44b 0.29b 0.65b 0.49b
*Different letters as superscript mean significantly (p< 0.05) different measurements to be compared row by row.
2.11. Determination of cathepsin B þ L activity
3. Results
One gram of the WCOD system was homogenized on ice, and the sample extract (supernatant) was incubated with Z-Phe-Arg 4methoxy-methylcoumarin hydrochloride as substrate as described by Egelandsdal et al. (2010). Chloroacetate was added to stop the enzymatic reaction. The emitted light of the samples (N ¼ 2e4) was 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. A calibration curve using the standard 7-amino-4methylcoumarin was prepared and the amount of formed product was determined from the standard curve. DTT was not used in the assay.
3.1. Effect of reduced BSA addition on metMb-mediated lipid oxidation in washed cod system
2.12. Statistics The statistical program Minitab, version 15, was used. The changes in the thiols, PV and TBARS were analyzed using a generalized linear model using time ranges, myoglobin and their interaction as factors. For comparisons Tukey’s test (significance level 0.05) was used. Protein concentration data was analyzed using oneway analysis of variance (ANOVA) and Tukey’s test for comparisons. Significant linear trends were determined by the routine Regression in Minitab. Fifty: Fifty MANOVA: This statistical method was carried out as described by Langsrud (2002) and accessed through www. langsrud.com. 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.
The addition of reduced BSA to washed cod containing metMb reduced the formation of lipid peroxides (Fig. 1) and TBARS (Fig. 2). At approximately 15 h, the level of lipid peroxides in the WCOD þ Mb þ REDBSA3.5 system remained low compared to WCOD þ Mb and WCOD þ Mb þ NOT REDBSA systems (Fig. 1). A significant (p < 0.05) increase in lipid peroxides with time was observed during storage in all the systems with added metMb. A significant (p ¼ 0.004) difference in lipid peroxides between the metMb systems with reduced and not reduced BSA at 47 h was observed. Both these systems were also significantly different from WCOD þ Mb after 47 h storage (Fig. 1). TBARS increased with time for all systems containing metMb (Fig. 2). The magnitude of TBARS was always lower for the WCOD þ Mb þ REDBSA3.5 system compared to the WCOD þ Mb and WCOD þ Mb þ NOT REDBSA systems. 3.2. Effect of BSA addition on the amount of soluble proteins in the washed cod system The protein concentration of the supernatants increased with time for all systems reported in Table 1. The increase in soluble proteins with time was the least (the change was 0.63 mg/ml from 0 to 47 h) for the system WCOD þ Mb þ NOT REDBSA, while the largest increase (the changes was 2.18 mg/ml from 0 to 47 h) in soluble proteins with time was observed for the system WCOD þ REDBSA3.5. The difference in initial protein content largely reflected if myoglobin was added or not. This increase in soluble protein with time was less for the WCOD þ Mb þ REDBSA3.5 system compared to the WCOD þ REDBSA3.5 system (p ¼ 0.042; t-test). The system WCOD þ Mb had no change (i.e. 0.03 mg/ml) in soluble proteins within the same time frame (not shown in Table 1). Soluble proteins tended to increase the most from 0 to 47 h for the system WCOD þ REDBSA3.5 compared to WCOD þ NOT REDBSA, but the systems were not significantly different (p ¼ 0.12; t-test). 3.3. Effect of added BSA on the oxidation of thiols by metMb
Fig. 3. Effect of storage time on accessible thiols (in mM) of soluble proteins in the supernatant obtained after centrifugation of the washed cod system (pH 5.6) at 16200g for 20 min. The data points were fitted to linear models; sign of slope and its significance (p-value) is indicated on the right hand side. : WCOD þ Mb; A WCOD þ Mb þ REDBSA3.5; , WCOD þ REDBSA3.5; > WCOD þ Mb þ NOT REDBSA; 6 WCOD þ NOT REDBSA.
Fig. 3 shows the changes in the thiol content of the supernatants. The WCOD þ REDBSA3.5 system had a high thiol content that increased significantly with time (Fig. 3). Even for the WCOD þ NOT REDBSA system, the number of thiols tended to increase with time. The WCOD þ Mb and WCOD þ Mb þ NOT REDBSA all had low values for thiols measured after 1.5e2.0 h. The thiol content of WCOD þ Mb was extensively reduced already after 1.5 h with only a tendency for further reduction with time. The thiol content of WCOD þ Mb þ REDBSA3.5 decreased significantly with observation time. The WCOD þ Mb þ REDBSA3.5 system oxidized to the same thiol content as the WCOD þ Mb þ NOT REDBSA system after 46e48 h.
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Fig. 4. Panel a: Effect of time on accessible thiols of two intact WCOD systems (pH 5.6). The standard deviation bars for the WCOD-Mb system include batch deviations. : WCOD þ Mb; effect of time, p ¼ 0.025; , WCOD þ REDBSA3.5; effect of time, p ¼ 0.53. Panel b: The change in thiols for different intact systems after 47 h storage. Different letters indicate significant (p < 0.05) differences.
Metmyoglobin addition also reduced the thiol content of the intact (not centrifuged) systems with storage time (Fig. 4a) but the reduction was low compared to the reduction observed for the supernatant (soluble) proteins. After 47 h, thiol contents of all systems were constrained between WCOD þ REDBSA3.5 and WCOD þ Mb (Fig. 4b). 3.4. Cathepsin B þ L activity in WCOD systems after BSA addition The activity of cathepsin B þ L was maintained by the addition of reduced BSA during the observation period (Table 2). The WCOD þ REDBSA3.5 system had higher enzyme activity than the WCOD þ Mb and WCOD þ Mb þ REDBSA3.5 systems. BSA samples that carried intermediate amounts of thiols, gave systems with intermediate enzyme activities for cathepsin B þ L (not shown). No difference in enzyme activities could be observed for the WCOD þ Mb þ NOT REDBSA and WCOD þ Mb þ REDBSA3.5 systems at 47 h (Table 2). 3.5. Effect of storage time on the protein pattern of SDS-PAGE gels SDS-PAGE gels run on intact (not centrifuged) WCOD systems did not reveal a significant effect of metMb addition on fish proteins (Table 3). A main effect (29.6%; p < 0.002) of storage time on ice was observed, indicating more degradation of myosin HC with time and an increase in proteins with MW below 44 kDa. The interaction effect (time myoglobin) was also significant (p ¼ 0.03). The proteins of the supernatant of WCOD þ REDBSA3.5 was dominated by BSA (Fig. 5), but influenced by metMb addition
when all area volumes minus metMb were used for the statistical test (Table 3). Systematic differences with or without metMb addition were observed for area volumes 6 (p ¼ 0.03) and 1 (p ¼ 0.04; normalized densimetric profiles were used for statistical calculations). WCOD þ REDBSA3.5 contained less of area volume 1 and more of area volume 6 compared to WCOD þ Mb þ REDBSA3.5. The ratio between area volumes 6 and 5 increased steadily with storage time for WCOD þ REDBSA3.5. However, for the system WCOD þ Mb þ REDBSA3.5 the increase in area volume 6 with time was not significant (p ¼ 0.37). Area volume 1 was not attempted identified. Due to its high molecular weight it seemed to be a degraded molecule from a higher molecular weight (>240 kDa) component. There was a tendency (p ¼ 0.07) for more degraded components from collagen and myosin HC (area volumes 3 and 4) in systems with only reduced BSA added. The protein contained in area volume 6 was a BSA fragment as determined by MALDI-TOF-TOF after trypsin digestion (Table 4). This fragment had a molecular weight of 41.7 1.0 kDa estimated from identified peptides and the molecular weight standards used for electrophoresis. 4. Discussion At pH 5.6 the reducing power of thiol groups should be limited since pKa of thiol groups is typically above pH 8.0, still lipid peroxides and TBARS formation were slowed down by the addition of reduced BSA3.5. Since also proteolytic activity was
Table 2 Effect of addition of reduced and not reduced BSA on the activity of cathepsin B þ L in WCOD þ Mb systems. Time
1.5 h þ 3.5 he
System
WCOD þ Mb
Relative cathepsin B þ L activityd (30 C)
0.27 0.05
a
43 h þ 3.5 h WCOD þ Mb þ REDBSA 3.5 0.70 0.06b
WCOD þ RED BSA3.5 0.83 0.03b
WCOD þ Mb þ NOT REDBSA 0.46 0.09c
WCOD þ Mb 0.18 0.05
a
WCOD þ Mb þ REDBSA3.5 0.23 0.06a
WCOD þ REDBSA3.5 0.80 0.20b
Different letters in superscript (aec) indicate significantly different figures (p<0.05). d WCOD’s enzyme activity of 5.4 nmol substrate/gram protein min defines unit activity. e The activity of cathepsin B þ L as measured in WCOD after 1.5 h incubation plus a preparation period of 3.5 h; Statistics: Tukey’s test, p < 0.05.
WCOD þ Mb þ NOT REDBSA 0.22 0.02a
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Table 3 Changes in the gel pattern of insoluble and soluble proteins promoted by storage time (at 2 C) and added myoglobin (0.7 mg/g). Type of protein
Insoluble Soluble
Mb:protein weight ratio
0.007e0.008 0.11
Explained variance (%) of effects Storage
Myoglobin
Storage myoglobin
29.2(p < 0.002) 25.8(p ¼ 0.054)
n.s.a 22.4 (p ¼ 0.0013)
6.8 (p ¼ 0.03) n.s.a
# of lanes
System
16 16
WCOD Mb þ REDBSA3.5 WCOD Mb þ REDBSA3.5
Ten percent (w/v) SDS-PAGE gels were used. The data were analyzed by 50:50 MANOVA (Langsrud, 2002) without standardization. The normalized protein area volumes of added Mb were removed prior to statistical analysis. a n.s. ¼ not significant.
affected, it appeared that the quality variables aimed at in these investigations were affected by the addition of reduced BSA. Untreated (not reduced) BSA had no similar effect and the peroxide measurements even suggested that this type of BSA would act as a pro-oxidant at the longest incubation time. Spectral measurement (0e2 h, not shown) showed a tendency for myoglobin (reduced form) to be present in the system containing REDBSA3.5. However, after 47 h the absorbance at 410 nm was significantly (p < 0.05) higher for the WCOD þ Mb þ REDBSA3.5 compared to the WCOD þ Mb system (not shown). This observation suggested less degradation of the hemin when reduced BSA was added. The less oxidative conditions provided by adding reduced BSA to the washed cod system also promoted proteolysis of BSA since the endogenous proteases present in the washed cod system were kept active for a longer period. Also the washed cod protein appeared more degraded when reduced BSA was added. Enzymes have been reported to become quickly inactivated (Spanier & Bird, 1982) by metMb addition. However, reduced BSA can protect against inactivation of thiol proteases in two manners: 1) by providing thiols for general reduction, and specifically, for exchange, to thiol proteases and 2) by acting as a general radical scavenging protein and thereby remove free radicals that otherwise may inactive the enzymes. Cathepsin B þ L remained active longer with the addition of reduced BSA as supported by the fact that substantially less of the BSA fragment was detected when Mb was added to the WCOD þ REDBSA3.5 system. Reduced BSA therefore postponed inactivation of thiol proteases. The data reported here, however, cannot identify for certain that BSA has been degraded by cathepsin B (or L). Cathepsin B is not a very selective thiol protease but has been reported to: 1)
cut at arginineearginine; 2) have a preference for valine or threonine next to arginine; 3) have a preference for valine and alanine next to lysine (Gosalia, Salisbury, Ellman, & Diamond, 2005) or 4) cleave at methionineeglycine, threonineeleucine or leucineethreonine (Hughes, O’Neill, Sweeney & Healy, 1999). Cathepsin L has been reported to have preference for arginineephenylalanine, arginineeleucine or lysineeleucine (Gosalia et al., 2005). The data above (Fig. 5), nevertheless, confirmed that the reduced BSA3.5 system stimulated degradation compared to the BSAeMb system that could be in accordance with cathepsin activity. The commercial BSA powder contained 0.2 0.02 mol of thiols/ mol BSA. Complete reduction of BSA’s 17 disulfides is possible, but gives a highly unstable protein as reported by Lee and Hirose (1992). The reduction procedure used in this study made it possible to handle the concentrated BSA solution without gel formation/aggregation both before and after reduction. The addition of partially reduced BSA to WCOD provided 0.40 mmol thiols/ kg of the system. The extensively washed fish had 70 mmol/kg proteins of accessible thiols. Since the protein content of the WCOD system used here was 89 mg/g, it is obvious that the thiol content of WCOD was dominated (>90%) by the thiols originating from the insoluble fish proteins independent of whether reduced or not reduced BSA was used. Accordingly, a large potential antioxidant capacity in WCOD was present as thiols. Some of these thiols would be released through degradation when metMb was not added. However, the design used allowed only for interpretation of the importance of the minor fraction of thiols coming from BSA plus the increase in soluble thiols associated with the ongoing protein degradation. To what extent the antioxidant capacity of cysteine
Table 4 The amino acid sequence of bovine serum albumin (Bos taurus, giê30794280).
Amino acids coloured fair grey were identified as peptides using MALDI-TOF-TOF. The framed part indicates the fragment from BSA identified. Possible fragmentation sites that would extend the fragment to the observed molecular weight are indicated by squares.
B. Egelandsdal et al. / LWT - Food Science and Technology 44 (2011) 1005e1011
Fig. 5. SDS-PAGE gels (10% w/v) of WCOD þ REDBSA3.5 supernatants prepared by centrifugation at 16200g for 20 min. From left: L1: Standards with molecular weights indicated in kDa; L2: WCOD þ Mb þ REDBSA3.5 1.5 h; L3: WCOD þ REDBSA3.5 1.5 h; L4: WCOD þ Mb þ REDBSA3.5 15 h; L5: WCOD þ REDBSA3.5 15 h; L6: WCOD þ Mb þ REDBSA3.5 22 h; L7: WCOD þ REDBSA3.5 22 h; L8: WCOD þ Mb þ REDBSA3.5 46 h; L9: WCOD þ REDBSA3.5 46 h; L10: Standards as for L1). The supernatants were precipitated in 80% methanol, dried and re-suspended in sample buffer to give solutions containing approx. 5 mg/ml. The figure shows how different area volumes among soluble proteins were identified before statistical calculations were done. The area volumes were identified as follows: 1) Not identified w240 kDa; 2) Myosin HC; 3) and 4) Degraded components from myosin and collagen (identified by Egelandsdal et al., 2010); 5) BSA 69 kDa; 6) BSA fragment 41.7 1.0 kDa; 7) Tropomyosin; 8) Troponin I; 9) Myoglobin.
depended on the fish protein being soluble or not, could not be elucidated. 5. Conclusion Partly reduced BSA added to a washed cod system reduced the formation of lipid peroxides values and TBARS compared to using untreated (not reduced) BSA. Reduced BSA led to increased degradation of insoluble fish proteins and extended the activity of cathepsin B þ L compared to system where both untreated (not reduced) BSA and metMb were added. metMb prevented the release of thiols as soluble proteins or as peptides and also the actual degradation of BSA in the washed cod system. Acknowledgement The Fulbright Foundation, The University of Life Science and The Research Council of Norway (grant no. 184846) and The College of Agricultural and Life Sciences, UW-Madison, HATCH project PRJ28VY are thanked for financial support. Tine and Nortura are also thanked for their financial support. The funding source(s) has only been involved in the decision to submit the paper for publication. 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 (pp. 44). Amsterdam: North-Holland Publishing.
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