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Myoglobin and haemoglobin-mediated lipid oxidation in washed muscle: Observations on crosslinking, ferryl formation, porphyrin degradation, and haemin loss rate
Food Chemistry 167 (2015) 258–263
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Myoglobin and haemoglobin-mediated lipid oxidation in washed muscle: Observations on crosslinking, ferryl formation, porphyrin degradation, and haemin loss rate Sung Ki Lee a, Nantawat Tatiyaborworntham b, Eric W. Grunwald b, Mark P. Richards b,⇑ a b
Kangwon National University, Department of Animal Products & Food Science, Meat Science Laboratory, KNU Ave 1, Chuncheon, Gangwon 200-701, South Korea Department of Animal Sciences, Meat Science and Muscle Biology Laboratory, University of Wisconsin–Madison, Madison, WI 53706, United States
a r t i c l e
i n f o
Article history: Received 26 March 2014 Received in revised form 23 May 2014 Accepted 24 June 2014 Available online 2 July 2014 Keywords: Lipid oxidation Haem pigments Rancidity Hypervalent species Fish Beef
a b s t r a c t Reduced trout haemoglobin (Hb) is a mixture of oxy- and deoxy-Hb at pH 6.3. Addition of oxy/deoxyHb to washed muscle resulted in detectable ferryl Hb while adding bovine oxyHb, trout metHb, or bovine metHb did not. Trout metHb promoted lipid oxidation more rapidly than bovine metHb, attributable to lower haemin affinity in fish Hbs. Protoporphyrin IX degradation was prevalent during trout and bovine Hb-mediated lipid oxidation. Caffeic acid prevented porphyrin degradation and lipid oxidation. Crosslinked myoglobin (Mb) promoted lipid oxidation more effectively than metMb. Fish metMb released haemin more readily than mammalian metMb at pH 5.5. These studies suggest haemin dissociation from metHb causes formation of free radicals that degrade protoporphyrin and cause lipid oxidation, and appreciable quantities of deoxyHb are needed to generate ferryl Hb oxidant. Crosslinking appears to facilitate Mb-mediated lipid oxidation in washed muscle yet haemin release can occur from fish metMb at low pH. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Lipid oxidation mediated by myoglobin (Mb) and haemoglobin (Hb) has received much attention due to the ability of these proteins to cause quality deterioration (e.g. off-odours and offflavours) in muscle foods (Kanner, 1994). Mb and Hb have also been implicated in various disease states and pathologies (Reeder, Svistunenko, Cooper, & Wilson, 2004). There are numerous forms and derivatives of Mb and Hb that have the potential to promote lipid oxidation (Everse & Hsia, 1997). These include (i) haemin that dissociates from metMb/metHb, (ii) perferryl radical Mb/Hb, (iii) ferryl Mb/Hb, (iv) crosslinked Mb/Hb, and (v) iron atoms that are released upon degradation of the porphyrin moiety (Potor et al., 2013; Richards, 2013). Haem and haemin are the terms used to describe the protoporphyrin IX moiety containing a Fe2+ and Fe3+ atom, respectively. Perferryl Mb/Hb and ferryl Mb/Hb indicates the iron atom within the porphyrin that is bound to the globin is in the +4 state.
⇑ Corresponding author. Address: Meat Science and Muscle Biology Laboratory, 1805 Linden Dr. Madison, WI 53706, United States. Tel.: +1 608 262 1792. E-mail address: [email protected] (M.P. Richards). http://dx.doi.org/10.1016/j.foodchem.2014.06.098 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
The addition of Mb and Hb to washed muscle has been used to assess various aspects of Mb and Hb-mediated lipid oxidation. One finding was that enhancing iron release from Mb, using Mb mutants susceptible to porphyrin degradation, decreased lipid oxidation rates in washed cod (Grunwald & Richards, 2006). This suggested that released iron does not contribute to Mb-mediated lipid oxidation. Another finding was that Hb promoted lipid oxidation much more effectively compared to Mb in washed muscle which was attributed to the much lower haemin affinity of Hb (Thiansilakul, Benjakul, Park, & Richards, 2012). Assessment of the other oxidative forms and derivatives of Mb and Hb requires further attention to have a more complete understanding of Hb and Mb-mediated lipid oxidation. The purpose of this work was to better understand the primary mechanism of Mb and Hb-mediated lipid oxidation using the washed muscle model system. The tools employed include the use of acid–acetone to quantify degradation of the protoporphyrin IX moiety, sodium sulfide to detect ferryl Mb/Hb formation during storage, a haemin capturing reagent to measure haemin dissociation from Mb/Hb, and investigating crosslinked Mb as a reactant in washed cod muscle. The use of fish and mammalian Mbs and Hbs were also examined to further understand how the types of Hb and Mb in different muscle foods can differentially influence lipid oxidation reactions.
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2. Materials and methods
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has high optical density at 600 nm and is used to measure haemin loss rate (Hargrove et al., 1994).
2.1. Chemicals 2.8. Storage studies of added Hb and caffeic acid in washed cod muscle Horse heart myoglobin, potassium ferricyanide, streptomycin sulphate, sodium sulphide nonahydrate, and thiobarbituric acid were obtained from Sigma–Aldrich (St. Louis, MO). All other chemicals were reagent grade. 2.2. Preparation of trout and bovine Hb Bovine and trout haemoglobin were prepared from freshly drawn blood in heparin anticoagulant (30 Units/ml blood) as described previously (Sannaveerappa, Undeland, & Sandberg, 2007). 2.3. Preparation of metHb and metMb Four mol of potassium ferricyanide were added per mol of Hb (on a haem basis) or Mb and mixed. After incubation on ice for 1–2 h, ferricyanide was removed using 10 DG desalting columns (Bio-Rad, Hercules, CA). The millimolar extinction coefficients of 179 mM 1 cm 1 at 405 nm for metHb (haem basis) and 188 mM 1 cm 1 at 408 nm for metMb were used (Antoni & Brunoni, 1971). 2.4. Preparation of ferryl Hb and ferryl Hb treated with Na2S Trout metHb (25 lM on haem basis) was reacted with H2O2 (75 lM) in 50 mM sodium phosphate buffer (pH 7.4) at room temperature for 0.5 min. Then Na2S was added (2 mM final concentration) and immediately scanned for optical density from 700 to 400 nm using a Shimadzu double-beam absorbance spectrophotometer. A peak near 620 nm of the Na2S treated samples is representative of ferryl Hb (Arduini, Eddy, & Hochstein, 1990). 2.5. Detection of ferryl Hb in washed cod muscle Washed cod (pH 6.3) containing added Hb (0.5 g sample) was centrifuged at 16,100g for 10 min at 4 °C in a polypropylene microfuge tube at each time point of interest. One hundred microlitres of the supernatant were then mixed with 900 lL of 50 mM phosphate buffer (pH 7.4), scanned for optical density from 700 to 400 nm, followed by addition of 70 lL Na2S (2 mM final concentration) and scanned immediately after mixing. A peak near 620 nm of the Na2S treated samples is representative of ferryl Hb (Arduini et al., 1990). Sampling of supernatants from washed cod containing added Hbs was done at 1, 15, 25, 39, 48, 71, 113, 182, 234, and 279 h. A millimolar extinction coefficient of 10.5 mM 1 cm 1 at 620 nm was used to quantify the concentration of ferryl Hb present on a haem basis. 2.6. Preparation of crosslinked horse Mb and Mb from carp dark muscle Crosslinked horse Mb was provided by Professor Yoichi Osawa (University of Michigan). A millimolar extinction coefficient of 76 mM 1 cm 1 at 406 nm was used to quantify crosslinked Mb concentration (Vuletich, Osawa, & Aviram, 2000). Bighead carp Mb was prepared as described previously (Thiansilakul et al., 2012). 2.7. Measurement of haemin loss from metMbs MetMb (10 lM) was incubated with apoH64Y/V68F (40 lM) at 4 °C for 72 h. Haemin released from metMb is transferred to apo to form holoH64Y/V68F (containing bound haemin). HoloH64Y/V68F
Washed cod (pH 6.3) was prepared as described previously (Sannaveerappa et al., 2007). Added Hb and Mb were examined at 44–58 lM (haem basis). HCl or NaOH (1 N) were used to adjust the pH when necessary. Final pH values of 6.3 for Hb trials and 5.7 for Mb trials were used. Streptomycin sulphate (200 ppm) was added as an antimicrobial. Final moisture content was adjusted to 90%. Caffeic acid was examined as an antioxidant at 100 lM. 2.9. Measurement of intact protoporphyrin IX during storage The amount of intact protoporphyrin IX extracted from washed cod containing added Hb using acidic acetone (Hornsey, 1956) was quantified spectrophotometrically according to Thiansilakul, Benjakul, and Richards (2010) with slight modifications. Protoporphyrin IX in washed muscle samples (0.5 g) was extracted with 4.5 mL acid acetone reagent (acetone:H2O:12 M HCl = 90:8:2, by volume) using a glass rod for 1.5 min. The mixture was incubated at room temperature in the absence of light for 1 h followed by centrifugation at 2000g for 10 min (4 °C). The acetone phase was scanned from 350 to 700 nm against blank (0.45 mL of milliQ water instead of the washed cod muscle containing added Hb). The difference between the absorbance values at 640 (A640) and 700 nm (A700) was used for calculating the concentration of protoporphyrin IX in the samples using a millimolar extinction coefficient (4.80 mM 1 cm 1). 2.10. Thiobarbituric acid reactive substances (TBARS) TBARS were determined according to the modified method of Buege and Aust (1978). On the day of analysis, a solution of 50% trichloroacetic acid (TCA) with 1.3% thiobarbituric acid (TBA) was prepared by mixing and heating on a hot plate (setting 5) until soluble. Then, 80–120 mg of sample was added to 1.2 ml of the reagent. After heating at 65 °C for 60 min, the samples were cooled at 4 °C for 60 min. Samples were then centrifuged at 16,000g for 5 min. Optical density in the supernatant was measured at 532 nm minus 650 nm. A standard curve was constructed using tetraethoxypropane and concentrations of TBARS in samples were expressed as lmol TBARS/kg washed muscle. 2.11. Statistical evaluations A MIXED procedure of the SAS system was used to analyse data from the storage studies (Littell, Henry, & Ammerman, 1998). Means were separated using the p-diff test. For each treatment, two or three separate reactions were examined at each time point during storage. Since a subsample was removed from each reaction vessel at each time point, repeated measures were used. Significance was determined at p < 0.05. 3. Results 3.1. Effect of caffeic acid on lipid oxidation and protoporphyrin IX degradation in washed cod containing added Hb Caffeic acid effectively inhibited trout Hb-mediated lipid oxidation during iced storage (p < 0.05) at pH 6.3 (Fig. 1A). In the absence of caffeic acid, Hb-mediated lipid oxidation reached maximal levels of 140 lmol/kg tissue based on TBARS at 20 h of storage, whereas in the presence of caffeic acid TBARS values were
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trout Hb trout Hb + CA
TBARS (µmol/kg tissue)
140 120 100 80 60 40 20 0
0
10
20
30
40
50
60
70
30 40 50 Ice storage (hours)
60
70
Ice storage (hours)
120 100
Protoporphyrin
80 60 40 20 0
0
10
20
Fig. 1. (A) Trout Hb mediated-lipid oxidation in washed cod with and without added caffeic acid; (B) concentrations of extracted protoporphyrin IX from washed cod containing added trout Hb with and without added caffeic acid during iced storage (pH 6.3). Hb concentration in washed cod was 58.3 lM (haem basis). Caffeic acid concentration was 100 lM.
negligible at 45 h and increased slightly by 69 h of storage. Caffeic acid also effectively inhibited porphyrin degradation during trout Hb-mediated lipid oxidation (Fig. 1B). In the absence of caffeic acid, porphyrin degradation during Hb-mediated lipid oxidation was mostly complete by hour 20, whereas in the presence of caffeic acid no detectable porphyrin degradation was observed during the entire storage period. 3.2. Assessment of ferryl Hb formation during storage of washed cod containing added Hb Na2S can be used to detect ferryl Mb and ferryl Hb due to formation of a large peak at 620 nm upon reaction of ferryl protein species with Na2S (Arduini et al., 1990). Trout ferryl Hb was generated by adding 3-fold H2O2 to trout metHb and incubating for 0.5 min followed by addition of Na2S (Fig. 2A). Reacting metHb with H2O2 for increasing time periods prior to Na2S addition decreased the optical density at 620 nm (data not shown). This may be due to time-dependent autoreduction of ferryl Hb to metHb and the ability of ferryl Hb to react with H2O2 equivalents (Alayash, Patel, & Cashon, 2001). Trout MetHb was also treated with Na2S in the absence of H2O2 to assess any nonspecific formation of the chromophore at 620 nm; no optical density at 620 nm was observed (Fig. 2A). Formation of ferryl Hb after addition of reduced trout Hb to washed muscle was investigated during iced storage. Ferryl trout Hb was detected at a single time point (25 h storage) during the
Fig. 2. (A) Spectra of trout metHb treated with 3-fold H2O2 (or no H2O2) for 0.5 min and then reacted with Na2S and scanned to quantify ferryl Hb; (B) Detection of ferryl Hb at hour 25 of iced storage (pH 6.3) based on treating the supernatant of the washed cod-trout Hb mixture with Na2S (10 dilution of supernatant); Inset: TBARS values in washed cod containing added trout Hb at time points in which ferryl Hb measurements were done during iced storage. Hb concentration in washed cod was 44 lM (haem basis).
exponential phase of TBARS formation based on the peak observed at 620 nm after reaction with Na2S (Fig. 2B). Ferryl trout Hb was not detected in the supernatants at any other time points of storage that were assayed (1, 15, 39, 49, and 71 h). After 71 h there was no detectable Hb in the supernatant based on optical density in the Soret region. Formation of ferryl Hb did not occur after addition of trout metHb to washed muscle during storage. Ferryl bovine Hb was not detected at any time points during iced storage of washed cod containing added bovine oxyHb or metHb (assayed at 1, 15, 25, 39, 49, 71, 113, 182, and 279 h).
3.3. Lipid oxidation and destruction of protoporphyrin IX due to trout metHb and bovine metHb in washed cod Lipid oxidation due to trout metHb was more rapid compared to bovine metHb (p < 0.05). TBARS reached maximal values at 20 h in washed cod containing trout metHb whereas TBARS values were still negligible in washed cod containing bovine metHb at 20 h (Fig. 3A). The final extent of lipid oxidation was similar when comparing trout and bovine metHb with bovine metHb reaching maximal TBARS values near 70 h of storage. MetHbs were examined so that the differing autooxidation rates of trout and bovine Hb did not confound the lipid oxidation kinetics. Protoporphyrin IX destruction of the trout metHb in washed cod was more rapid (p < 0.05) compared to the bovine metHb (Fig. 3B). The protoporphryin moiety of trout Hb was barely detectable at 42 h of storage while nearly all the bovine protoporphryin
261
Ice storage (hours)
Optical density (600-700 nm)
TBARS (µmol/kg itssue)
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Storage at 4°C (hours)
Protoporphyrin
Fig. 5. Haemin loss from bighead carp metMb and horse metMb at pH 5.5. Reactions contained 10 lM metMb, 40 lM apoH64Y/V68F, 150 mM BisTris (pH 5.5) and sucrose (400 lM). The blank reactions contained all components except metMb.
Mb was more rapid (p < 0.05) and extensive compared to metMb (Fig. 4). At 20 h of storage TBARS values reached 50 lmol/kg in crosslinked Mb compared to negligible values in metMb at 20 h. TBARS values remained below 10 lmol/kg during the entire storage period for metMb. Ice storage (hours)
Fig. 3. (A) TBARS values in washed cod containing added trout metHb or bovine metHb during iced storage (pH 6.3); (B) concentrations of extracted protoporphyrin IX from washed cod containing added trout or bovine metHb during iced storage (pH 6.3). Hb concentration in washed cod was 44 lM (haem basis).
IX was still detected at 42 h of storage. Approximately half of the protoporphyrin in bovine Hb was destroyed by 65 h of storage.
3.4. Lipid oxidation due to metMb and crosslinked Mb in washed cod muscle
TBARS (µmol/kg itssue)
Enhanced lipid oxidation was previously observed in lipoproteins due to a crosslinked form of Mb (Vuletich et al., 2000). It was thus of interest to evaluate crosslinked Mb in washed cod muscle compared to metMb. Lipid oxidation due to crosslinked
Ice storage (hours) Fig. 4. TBARS values in washed cod containing added metMb or cross-linked Mb during iced storage. pH was 5.7 and Mb concentration was 44 lM.
3.5. Haemin loss from horse metMb compared to bighead carp metMb Fish myoglobins have been reported to be at least 2.5-fold more sensitive to autooxidation compared to mammalian Mbs (Livingston & Brown, 1981). This suggests structural differences between the different Mbs which may also affect haemin affinity. ApoH64Y/V68F was used to measure haemin dissociation from horse metMb compared to bighead carp metMb. Haemin dissociation from the fish metMb was up to 3.3-fold greater compared to the mammalian metMb during storage at pH 5.5 and 4 °C (Fig. 5). Haemin loss was negligible from both haem proteins at pH 6.0 and 4 °C (data not shown). 4. Discussion Haemin release appeared to be a critical step in the progression of Hb-mediated lipid oxidation in washed cod muscle at pH 6.3. This was based on the more rapid lipid oxidation due to trout metHb compared to bovine metHb. Trout metHb has a 55-fold higher haemin loss rate compared to bovine metHb at pH 6.3 (Aranda et al., 2009). This is consistent with rapid lipid oxidation by the Mb mutant H97A which has low haemin affinity compared to wild type Mb and V68T (Grunwald & Richards, 2006). In addition, apohemopexin, which strongly binds and inactivates haemin that dissociates from metHb, was shown to inhibit Hb-mediated lipid oxidation in washed cod muscle (Grunwald & Richards, 2012). Dissociated haemin can react with preformed lipid hydroperoxides (LOOH) in cellular membranes to produce lipid radicals that abstract hydrogen atom from polyunsaturated fatty acid, causing formation of LOOH and secondary lipid oxidation products (Van der Zee, Barr, & Mason, 1996). Those lipid radicals also have the potential to degrade the protoporphyrin moiety (Nagababu & Rifkind, 2004). Extensive protoporphyrin degradation during Hb-mediated lipid oxidation was observed in our studies based
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on loss of detectable porphyrin after acid–acetone extraction. This suggests free radicals that form due to haemin-mediated decomposition of LOOH not only abstract hydrogen atoms from PUFAs but also degrade the prophyrin ring during storage in washed cod. Since bovine metHb has a relatively higher haemin affinity than trout metHb, it was considered that porphyrin degradation might be less or nonexistent with the bovine metHb in washed cod. The idea being that a mechanism not associated with haemin-mediated lipid oxidation and prophyrin degradation could take place with Hbs that have elevated haemin affinity. Yet, extensive lipid oxidation and degradation of the prophyrin moiety of bovine Hb in washed cod was observed during storage. These results are consistent with haemin released from the globin causing lipid oxidation and porphyrin degradation when considering both trout and bovine Hb; just that the onset of lipid oxidation was more rapid with the fish Hb due to its more rapid rate of haemin dissociation while the extents of lipid oxidation due to trout and bovine metHb were similar. Caffeic acid strongly inhibited trout Hb-mediated lipid oxidation and porphyrin destruction during storage in washed cod muscle. It has been noted that caffeic acid accelerates metHb formation (Wallace & Caughey, 1975). Thus the antioxidant mechanism of caffeic acid is unlikely to involve the ability of caffeic acid to keep Hb reduced as oxyHb and deoxyHb. The free radical scavenging ability of caffeic acid may have contributed to the antioxidant effect, but the relatively low caffeic acid concentration (100 lM) should be noted. The ability of caffeic acid to bind to Hb, inhibit haemin loss from metHb, and alter Hb partitioning in washed muscle has been noted as possible antioxidant mechanisms (Park, Sannaveerappa, Undeland, & Richards, 2013). The ability of caffeic acid to reduce tocopherol radicals, regenerating tocopherol has also been noted as a possible antioxidant mechanism in washed cod muscle (Larsson & Undeland, 2010). Perferryl Hb (and perferrylMb) describes the hypervalent iron protein radical that can initiate lipid oxidation via hydrogen atom abstraction from PUFA. Perferryl Hb/Mb undergoes a one-electron reduction to form ferryl Hb/Mb. Ferryl Mb has a relatively long half-life (30 s) and cleaves preformed protein and lipid hydroperoxides to generate peroxyl radicals that can propagate lipid oxidation via hydrogen abstraction (Carlsen, Moller, & Skibsted, 2005). We found that adding reduced trout Hb to washed muscle resulted in ferryl Hb formation but not when trout metHb was added. This suggested that as trout Hb auotooxidized in washed muscle, the resulting superoxide radicals were converted to H2O2 (Balagopalakrishna, Manoharan, Abugo, & Rifkind, 1996; Halliwell & Gutteridge, 1999). H2O2 can then react with metHb to generate ferryl Hb (Giulivi & Davies, 1994). DeoxyHb can also react with H2O2 to generate ferryl Hb (Alayash et al., 2001). More than half of reduced trout Hb exists as deoxyHb at pH 6.3 with the remainder as oxyHb (Binotti et al., 1971). The fact that ferryl Hb was not detected when trout metHb was added to washed muscle can be attributed to the absence of any O2 ligand in the distal haem pocket, thereby preventing generation of superoxide radical and H2O2. In addition, H2O2 was more reactive with trout deoxyHb compared to trout oxyHb and metHb (Gabbianelli, Zolese, Bertoli, & Falcioni, 2004). Collectively, these results suggest deoxyHb facilitates ferryl Hb formation. Ferryl Hb was not detected at any time point during storage when bovine oxyHb or bovine metHb were added to washed muscle. This is in contrast to ferryl Hb formation when trout oxy/deoxy Hb was added to washed muscle. The inability of bovine oxyHb to be converted to ferryl Hb is likely partly due to the relatively slow autooxidation of bovine oxyHb in washed muscle so that a critical concentration of H2O2 is not achieved during storage to react with metHb (Aranda et al., 2009). Also, there will be little to no bovine deoxyHb to react with H2O2 compared to when reduced trout Hb is added to washed cod at pH 6.3. It is also possible that H2O2 is
more accessible to the haem pocket of trout Hb compared to bovine Hb. Increased peroxidase activity using H2O2 and guaiacol was noted in trout Hb compared to human (also mammalian) Hb (Fedeli et al., 2001). Thus, ferryl Hb may contribute to lipid oxidation progress in washed muscle due to added trout oxy/deoxy Hb but this is unlikely to be the case when bovine oxyHb, bovine metHb or trout metHb is added. It has been shown that Hb promotes lipid oxidation much more effectively than Mb in washed muscle which was attributed to the lower haemin affinity of Hb (Thiansilakul et al., 2012). TBARS values were elevated 14-fold at day 1 of iced storage in unwashed cod containing added Hb compared to added Mb at an equivalent level of haem (Castell & Bishop, 1969). Nevertheless, some lipid oxidation due to added Mb occurred in both washed and unwashed muscle during storage and the mechanism involved is of interest. Perferryl Mb is noted to undergo rearrangement at acidic conditions to form crosslinked Mb (Reeder, Svistunenko, Sharpe, & Wilson, 2002). We compared the ability of horse metMb to promote lipid oxidation in washed cod at pH 5.7 compared to crosslinked horse Mb at equivalent concentration of protoporphyrin and found that crosslinked Mb was a potent catalyst of lipid oxidation compared to metMb (Fig. 4). Crosslinked Mb was also found to promote lipid oxidation more effectively than metMb in pure phospholipids and low-density lipoproteins at pH 7.4 (Vuletich et al., 2000). The crosslinking appears to be due to a tyrosine residue forming a covalent bond with the porphyrin moiety, inducing a peroxidase activity that can readily oxidise lipids. Thus, the major form of Mb that oxidises lipids may be crosslinked Mb rather than perferryl Mb or ferryl Mb species, recognising that perferryl Mb is the likely precursor to generate crosslinked Mb. It is interesting to note that crosslinked Mb was not found to promote lipid oxidation in linoleic acid micelles at pH 5.5–6.5 (Baron, Skibsted, & Andersen, 1997). It should be kept in mind that Mb can also release haemin under appropriate conditions of pH, temperature, and the type of Mb that is examined. It was previously shown at 37 °C that haemin loss from sperm whale metMb was 142-fold faster at pH 5.0 compared to pH 7.0 (Hargrove et al., 1994). In the present work, Bighead carp metMb released haemin at pH 5.5 and 4 °C around 3-fold more rapidly compared to horse metMb (Fig. 5). Neither Mb released haemin at pH 6.0 and 4 °C. The ability of the fish Mb to more readily release haemin at pH 5.5 and 4 °C may be related to the lack of a clearly defined D-helix in fish Mbs (Birnbaum, Evans, Przybylska, & Rose, 1994). The D-helix which is clearly present in mammalian Mbs may stabilize the prophyrin moiety within the globin of the protein. Removal of the D-helix from mammalian Mb using sitedirected mutagenesis increased haemin loss rate (Whitaker et al., 1995). In addition, there are 17 amino acids that form contacts with the porphyrin moiety in Mbs (Hargrove, Wilkinson, & Olson, 1996). It turns out that tuna Mb contains a different amino acid at four of these sites (E14, G4, G5, and H15) compared to mammalian Mbs. Differing contacts of the amino acids at these sites with the porphyrin moiety in fish versus mammalian Mbs may explain the lower haemin affinity in the fish Mb at pH 5.5. Converting the alanine at site E14 in bovine Mb to glycine decreased haemin affinity 45-fold which was attributed to enhanced access of solvent to the proximal histidine (Cai & Richards, 2012). Yellowfin tuna Mb contains lysine at E14 which is further away from the porhphyrin ring (by around 0.3 Å) compared to mammalian Mbs that contain alanine at E14 (Birnbaum et al., 1994).
5. Conclusion Trout metHb promoted lipid oxidation more effectively than bovine metHb which can be explained by the lower haemin affinity
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of the fish Hb. Dissociated haemin from metHb decomposes preformed lipid hydroperoxides to free radicals that oxidise lipids and degrade the porphyrin ring. Degradation of the porphyrin moiety of Hb was observed during storage based on loss of extractable porphyrin using acid acetone. Thus, haemin dissociation from the globins appears to be the primary mechanism of Hb-mediated lipid oxidation in washed muscle at pH 6.3. At the same time, it should be recognised that appreciable amounts of deoxyHb (present in reduced trout Hb at pH 6.3) resulted in formation of ferryl Hb as an additional oxidant. Although Hb promotes lipid oxidation more effectively than Mb, crosslinked Mb oxidised lipids extensively compared to metMb at pH 5.7. The primary mechanism of lipid oxidation by Mb can change depending on various factors that include pH, temperature, and whether the Mb is mammalian, avian, or fish. As an example, lowering pH to 5.5 at 4 °C will increase haemin loss from a fish Mb while haemin will remain more associated with the globin in a mammalian Mb at this pH value and thus favour reactivity of the crosslinked form. Future work should further characterise the ability of ferryl Hb and crosslinked Mb to incur lipid oxidation amongst the pool of other oxidants that are present. Acknowledgements This project was supported by the Kangwon National University, South Korea. The project was also funded through a Grant from the Improving Food Quality Foundational Program (Award No. 2014-67016-21648) of the National Institute of Food and Agriculture, U.S. Department of Agriculture. We thank Professor Osawa at the University of Michigan for providing crosslinked horse Mb (NIH Grant GM077430). We also thank Dr. Sung Yong Park for haemin loss measurements and Dr. Yaowapa Thiansilakul for preparing bighead carp myoglobin. References Alayash, A. I., Patel, R. P., & Cashon, R. E. (2001). Redox reactions of hemoglobin and myoglobin: biological and toxicological implications. Antioxidants & Redox Signaling, 3(2), 313–327. Antoni, E., & Brunoni, M. (1971). Hemoglobin and Myoglobin in their Reactions with Ligands. Amsterdam, The Netherlands: North-Holland Publ. Co. Aranda, R., IV, He, C., Worley, C. E., Levin, E., Li, R., Olson, J. S., et al. (2009). Structural analysis of fish versus mammalian hemoglobins: effects of the heme pocket environment on autooxidation and hemin loss rates. Proteins, 75, 217–230. Arduini, A., Eddy, L., & Hochstein, P. (1990). Detection of ferryl myoglobin in the isolated ischemic rat heart. Free Radical Biology & Medicine, 9, 511–513. Balagopalakrishna, C., Manoharan, P. T., Abugo, O. O., & Rifkind, J. M. (1996). Production of superoxide from hemoglobin-bound oxygen under hypoxic conditions. Biochemistry, 35, 6393–6398. Baron, C. P., Skibsted, L. H., & Andersen, H. J. (1997). Prooxidative activity of myoglobin species in linoleic acid emulsions. Journal of Agriculture and Food Chemistry, 45, 1704–1710. Binotti, I., Giovenco, S., Giardina, B., Antonini, E., Brunori, M., & Wyman, J. (1971). Studies on the functional properties of fish hemoglobins. II. The oxygen equilibrium of the isolated hemoglobin components from trout blood. Archives of Biochemistry and Biophysics, 142, 274–280. Birnbaum, G. I., Evans, S. V., Przybylska, M., & Rose, D. R. (1994). 1.70 A resolution structure of myoglobin from yellowfin tuna. An example of a myoglobin lacking the D helix. Acta Crystallographica. Section D, Biological Crystallography, 50(Pt 3), 283–289. Buege, J. A., & Aust, S. D. (1978). Microsomal lipid peroxidation. In S. Fleischer & L. Packer (Eds.), Methods in Enzymology (pp. 302–310). New York: Academic Press. Vol. 52. Cai, H., & Richards, M. P. (2012). Site E14 in hemoglobins and myoglobins: A key residue that affects hemin loss and lipid oxidation capacity. Journal of Agriculture and Food Chemistry, 60, 7729–7734. Carlsen, C. U., Moller, J. K. S., & Skibsted, L. H. (2005). Heme-iron in lipid oxidation. Coordination Chemistry Reviews, 249, 485–498.
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Methods in Enzymology, 231, 490–496. Grunwald, E. W., & Richards, M. P. (2006). Studies with myoglobin variants indicate that released hemin is the primary promoter of lipid oxidation in washed fish muscle. Journal of Agriculture and Food Chemistry, 54, 4452–4460. Grunwald, E. W., & Richards, M. P. (2012). Effects of hemopexin on hemin and hemoglobin-mediated lipid oxidation in washed fish muscle. LWT – Food Science and Technology, 46(2), 412–418. Halliwell, B., & Gutteridge, J. M. C. (1999). Free Radicals in Biology and Medicine (3rd ed.). New York: Oxford University Press. Hargrove, M. S., Singleton, E. W., Quillin, M. L., Ortiz, L. A., Phillips, G. N., Jr., Olson, J. S., et al. (1994). His 64(E7) Tyr apomyoglobin as a reagent for measuring rates of hemin dissociation. Journal of Biological Chemistry, 269, 4207–4214. Hargrove, M. S., Wilkinson, A. J., & Olson, J. S. (1996). Structural factors governing hemin dissociation from metmyoglobin. Biochemistry, 35, 11300–11309. Hornsey, H. C. (1956). The color of cooked cured pork. I.- Estimation of the nitric oxide-haem pigments. Journal of the Science of Food and Agriculture, 7, 534–540. Kanner, J. (1994). Oxidative processes in meat and meat products: quality implications. Meat Science, 36, 169–189. Larsson, K. J., & Undeland, I. K. (2010). Effect of caffeic acid on haemoglobinmediated lipid and protein oxidation in washed cod mince during ice and frozen storage. Journal of the Science of Food and Agriculture, 90(14), 2531–2540. Littell, R. C., Henry, P. R., & Ammerman, C. B. (1998). Statistical analysis of repeated measures data using SAS procedures. Journal of Animal Science, 76, 1216–1231. Livingston, D. J., & Brown, D. W. (1981). The chemistry of myoglobin and its reactions. Food Technology, 35, 244–252. Nagababu, E., & Rifkind, J. M. (2004). Heme degradation by reactive oxygen species. Antioxidants & Redox Signaling, 6(6), 967–978. Park, S. Y., Sannaveerappa, T., Undeland, I., & Richards, M. P. (2013). Novel interactions of caffeic acid with different hemoglobins: effects on discoloration and lipid oxidation in different washed muscles. Meat Science, 95, 110–117. Potor, L., Banyai, E., Becs, G., Soares, M. P., Balla, G., Balla, J., et al. (2013). Atherogenesis may involve the prooxidant and proinflammatory effects of ferryl hemoglobin. Oxididative Medicine and Cellular Longevity, 2013, 676425. Reeder, B. J., Svistunenko, D. A., Cooper, C. E., & Wilson, M. T. (2004). The radical and redox chemistry of myoglobin and hemoglobin: from in vitro studies to human pathology. Antioxidants & Redox Signaling, 6, 954–966. Reeder, B. J., Svistunenko, D. A., Sharpe, M. S., & Wilson, M. T. (2002). Characteristics and mechanism of formation of peroxide-induced heme to protein cross-linking in myoglobin. Biochemistry, 41, 367–375. Richards, M. P. (2013). Redox reactions of myoglobin. Antioxidants & Redox Signaling, 18, 2342–2351. Sannaveerappa, T., Undeland, I., & Sandberg, A. S. (2007). Evaluation of occasional non-response of a washed cod mince model to hemoglobin (Hb) mediated oxidation. Journal of Agriculture and Food Chemistry, 55, 4429–4435. Thiansilakul, Y., Benjakul, S., Park, S. Y., & Richards, M. P. (2012). Characteristics of myoglobin and hemoglobin-mediated lipid oxidation in washed mince from bighead carp (Hypothalimichthys nobilis). Food Chemistry, 132, 892–900. Thiansilakul, Y., Benjakul, S., & Richards, M. P. (2010). Changes in heme proteins and lipids associated with off-odour of seabass (Lates calcarifer) and red tilapia (Oreochromis mossambicus O. niloticus) during iced storage. Food Chemistry, 121, 1109–1119. Van der Zee, J., Barr, D. P., & Mason, R. P. (1996). ESR spin trapping investigation of radical formation from the reaction between hematin and tert-butyl hydroperoxide. Free Radical Biology & Medicine, 20, 199–206. Vuletich, J. L., Osawa, Y., & Aviram, M. (2000). 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Myoglobin and haemoglobin-mediated lipid oxidation in washed muscle: Observations on crosslinking, ferryl formation, porphyrin degradation, and haemin loss rate Sung Ki Lee a, Nantawat Tatiyaborworntham b, Eric W. Grunwald b, Mark P. Richards b,⇑ a b
Kangwon National University, Department of Animal Products & Food Science, Meat Science Laboratory, KNU Ave 1, Chuncheon, Gangwon 200-701, South Korea Department of Animal Sciences, Meat Science and Muscle Biology Laboratory, University of Wisconsin–Madison, Madison, WI 53706, United States
a r t i c l e
i n f o
Article history: Received 26 March 2014 Received in revised form 23 May 2014 Accepted 24 June 2014 Available online 2 July 2014 Keywords: Lipid oxidation Haem pigments Rancidity Hypervalent species Fish Beef
a b s t r a c t Reduced trout haemoglobin (Hb) is a mixture of oxy- and deoxy-Hb at pH 6.3. Addition of oxy/deoxyHb to washed muscle resulted in detectable ferryl Hb while adding bovine oxyHb, trout metHb, or bovine metHb did not. Trout metHb promoted lipid oxidation more rapidly than bovine metHb, attributable to lower haemin affinity in fish Hbs. Protoporphyrin IX degradation was prevalent during trout and bovine Hb-mediated lipid oxidation. Caffeic acid prevented porphyrin degradation and lipid oxidation. Crosslinked myoglobin (Mb) promoted lipid oxidation more effectively than metMb. Fish metMb released haemin more readily than mammalian metMb at pH 5.5. These studies suggest haemin dissociation from metHb causes formation of free radicals that degrade protoporphyrin and cause lipid oxidation, and appreciable quantities of deoxyHb are needed to generate ferryl Hb oxidant. Crosslinking appears to facilitate Mb-mediated lipid oxidation in washed muscle yet haemin release can occur from fish metMb at low pH. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Lipid oxidation mediated by myoglobin (Mb) and haemoglobin (Hb) has received much attention due to the ability of these proteins to cause quality deterioration (e.g. off-odours and offflavours) in muscle foods (Kanner, 1994). Mb and Hb have also been implicated in various disease states and pathologies (Reeder, Svistunenko, Cooper, & Wilson, 2004). There are numerous forms and derivatives of Mb and Hb that have the potential to promote lipid oxidation (Everse & Hsia, 1997). These include (i) haemin that dissociates from metMb/metHb, (ii) perferryl radical Mb/Hb, (iii) ferryl Mb/Hb, (iv) crosslinked Mb/Hb, and (v) iron atoms that are released upon degradation of the porphyrin moiety (Potor et al., 2013; Richards, 2013). Haem and haemin are the terms used to describe the protoporphyrin IX moiety containing a Fe2+ and Fe3+ atom, respectively. Perferryl Mb/Hb and ferryl Mb/Hb indicates the iron atom within the porphyrin that is bound to the globin is in the +4 state.
⇑ Corresponding author. Address: Meat Science and Muscle Biology Laboratory, 1805 Linden Dr. Madison, WI 53706, United States. Tel.: +1 608 262 1792. E-mail address: [email protected] (M.P. Richards). http://dx.doi.org/10.1016/j.foodchem.2014.06.098 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
The addition of Mb and Hb to washed muscle has been used to assess various aspects of Mb and Hb-mediated lipid oxidation. One finding was that enhancing iron release from Mb, using Mb mutants susceptible to porphyrin degradation, decreased lipid oxidation rates in washed cod (Grunwald & Richards, 2006). This suggested that released iron does not contribute to Mb-mediated lipid oxidation. Another finding was that Hb promoted lipid oxidation much more effectively compared to Mb in washed muscle which was attributed to the much lower haemin affinity of Hb (Thiansilakul, Benjakul, Park, & Richards, 2012). Assessment of the other oxidative forms and derivatives of Mb and Hb requires further attention to have a more complete understanding of Hb and Mb-mediated lipid oxidation. The purpose of this work was to better understand the primary mechanism of Mb and Hb-mediated lipid oxidation using the washed muscle model system. The tools employed include the use of acid–acetone to quantify degradation of the protoporphyrin IX moiety, sodium sulfide to detect ferryl Mb/Hb formation during storage, a haemin capturing reagent to measure haemin dissociation from Mb/Hb, and investigating crosslinked Mb as a reactant in washed cod muscle. The use of fish and mammalian Mbs and Hbs were also examined to further understand how the types of Hb and Mb in different muscle foods can differentially influence lipid oxidation reactions.
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2. Materials and methods
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has high optical density at 600 nm and is used to measure haemin loss rate (Hargrove et al., 1994).
2.1. Chemicals 2.8. Storage studies of added Hb and caffeic acid in washed cod muscle Horse heart myoglobin, potassium ferricyanide, streptomycin sulphate, sodium sulphide nonahydrate, and thiobarbituric acid were obtained from Sigma–Aldrich (St. Louis, MO). All other chemicals were reagent grade. 2.2. Preparation of trout and bovine Hb Bovine and trout haemoglobin were prepared from freshly drawn blood in heparin anticoagulant (30 Units/ml blood) as described previously (Sannaveerappa, Undeland, & Sandberg, 2007). 2.3. Preparation of metHb and metMb Four mol of potassium ferricyanide were added per mol of Hb (on a haem basis) or Mb and mixed. After incubation on ice for 1–2 h, ferricyanide was removed using 10 DG desalting columns (Bio-Rad, Hercules, CA). The millimolar extinction coefficients of 179 mM 1 cm 1 at 405 nm for metHb (haem basis) and 188 mM 1 cm 1 at 408 nm for metMb were used (Antoni & Brunoni, 1971). 2.4. Preparation of ferryl Hb and ferryl Hb treated with Na2S Trout metHb (25 lM on haem basis) was reacted with H2O2 (75 lM) in 50 mM sodium phosphate buffer (pH 7.4) at room temperature for 0.5 min. Then Na2S was added (2 mM final concentration) and immediately scanned for optical density from 700 to 400 nm using a Shimadzu double-beam absorbance spectrophotometer. A peak near 620 nm of the Na2S treated samples is representative of ferryl Hb (Arduini, Eddy, & Hochstein, 1990). 2.5. Detection of ferryl Hb in washed cod muscle Washed cod (pH 6.3) containing added Hb (0.5 g sample) was centrifuged at 16,100g for 10 min at 4 °C in a polypropylene microfuge tube at each time point of interest. One hundred microlitres of the supernatant were then mixed with 900 lL of 50 mM phosphate buffer (pH 7.4), scanned for optical density from 700 to 400 nm, followed by addition of 70 lL Na2S (2 mM final concentration) and scanned immediately after mixing. A peak near 620 nm of the Na2S treated samples is representative of ferryl Hb (Arduini et al., 1990). Sampling of supernatants from washed cod containing added Hbs was done at 1, 15, 25, 39, 48, 71, 113, 182, 234, and 279 h. A millimolar extinction coefficient of 10.5 mM 1 cm 1 at 620 nm was used to quantify the concentration of ferryl Hb present on a haem basis. 2.6. Preparation of crosslinked horse Mb and Mb from carp dark muscle Crosslinked horse Mb was provided by Professor Yoichi Osawa (University of Michigan). A millimolar extinction coefficient of 76 mM 1 cm 1 at 406 nm was used to quantify crosslinked Mb concentration (Vuletich, Osawa, & Aviram, 2000). Bighead carp Mb was prepared as described previously (Thiansilakul et al., 2012). 2.7. Measurement of haemin loss from metMbs MetMb (10 lM) was incubated with apoH64Y/V68F (40 lM) at 4 °C for 72 h. Haemin released from metMb is transferred to apo to form holoH64Y/V68F (containing bound haemin). HoloH64Y/V68F
Washed cod (pH 6.3) was prepared as described previously (Sannaveerappa et al., 2007). Added Hb and Mb were examined at 44–58 lM (haem basis). HCl or NaOH (1 N) were used to adjust the pH when necessary. Final pH values of 6.3 for Hb trials and 5.7 for Mb trials were used. Streptomycin sulphate (200 ppm) was added as an antimicrobial. Final moisture content was adjusted to 90%. Caffeic acid was examined as an antioxidant at 100 lM. 2.9. Measurement of intact protoporphyrin IX during storage The amount of intact protoporphyrin IX extracted from washed cod containing added Hb using acidic acetone (Hornsey, 1956) was quantified spectrophotometrically according to Thiansilakul, Benjakul, and Richards (2010) with slight modifications. Protoporphyrin IX in washed muscle samples (0.5 g) was extracted with 4.5 mL acid acetone reagent (acetone:H2O:12 M HCl = 90:8:2, by volume) using a glass rod for 1.5 min. The mixture was incubated at room temperature in the absence of light for 1 h followed by centrifugation at 2000g for 10 min (4 °C). The acetone phase was scanned from 350 to 700 nm against blank (0.45 mL of milliQ water instead of the washed cod muscle containing added Hb). The difference between the absorbance values at 640 (A640) and 700 nm (A700) was used for calculating the concentration of protoporphyrin IX in the samples using a millimolar extinction coefficient (4.80 mM 1 cm 1). 2.10. Thiobarbituric acid reactive substances (TBARS) TBARS were determined according to the modified method of Buege and Aust (1978). On the day of analysis, a solution of 50% trichloroacetic acid (TCA) with 1.3% thiobarbituric acid (TBA) was prepared by mixing and heating on a hot plate (setting 5) until soluble. Then, 80–120 mg of sample was added to 1.2 ml of the reagent. After heating at 65 °C for 60 min, the samples were cooled at 4 °C for 60 min. Samples were then centrifuged at 16,000g for 5 min. Optical density in the supernatant was measured at 532 nm minus 650 nm. A standard curve was constructed using tetraethoxypropane and concentrations of TBARS in samples were expressed as lmol TBARS/kg washed muscle. 2.11. Statistical evaluations A MIXED procedure of the SAS system was used to analyse data from the storage studies (Littell, Henry, & Ammerman, 1998). Means were separated using the p-diff test. For each treatment, two or three separate reactions were examined at each time point during storage. Since a subsample was removed from each reaction vessel at each time point, repeated measures were used. Significance was determined at p < 0.05. 3. Results 3.1. Effect of caffeic acid on lipid oxidation and protoporphyrin IX degradation in washed cod containing added Hb Caffeic acid effectively inhibited trout Hb-mediated lipid oxidation during iced storage (p < 0.05) at pH 6.3 (Fig. 1A). In the absence of caffeic acid, Hb-mediated lipid oxidation reached maximal levels of 140 lmol/kg tissue based on TBARS at 20 h of storage, whereas in the presence of caffeic acid TBARS values were
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trout Hb trout Hb + CA
TBARS (µmol/kg tissue)
140 120 100 80 60 40 20 0
0
10
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30
40
50
60
70
30 40 50 Ice storage (hours)
60
70
Ice storage (hours)
120 100
Protoporphyrin
80 60 40 20 0
0
10
20
Fig. 1. (A) Trout Hb mediated-lipid oxidation in washed cod with and without added caffeic acid; (B) concentrations of extracted protoporphyrin IX from washed cod containing added trout Hb with and without added caffeic acid during iced storage (pH 6.3). Hb concentration in washed cod was 58.3 lM (haem basis). Caffeic acid concentration was 100 lM.
negligible at 45 h and increased slightly by 69 h of storage. Caffeic acid also effectively inhibited porphyrin degradation during trout Hb-mediated lipid oxidation (Fig. 1B). In the absence of caffeic acid, porphyrin degradation during Hb-mediated lipid oxidation was mostly complete by hour 20, whereas in the presence of caffeic acid no detectable porphyrin degradation was observed during the entire storage period. 3.2. Assessment of ferryl Hb formation during storage of washed cod containing added Hb Na2S can be used to detect ferryl Mb and ferryl Hb due to formation of a large peak at 620 nm upon reaction of ferryl protein species with Na2S (Arduini et al., 1990). Trout ferryl Hb was generated by adding 3-fold H2O2 to trout metHb and incubating for 0.5 min followed by addition of Na2S (Fig. 2A). Reacting metHb with H2O2 for increasing time periods prior to Na2S addition decreased the optical density at 620 nm (data not shown). This may be due to time-dependent autoreduction of ferryl Hb to metHb and the ability of ferryl Hb to react with H2O2 equivalents (Alayash, Patel, & Cashon, 2001). Trout MetHb was also treated with Na2S in the absence of H2O2 to assess any nonspecific formation of the chromophore at 620 nm; no optical density at 620 nm was observed (Fig. 2A). Formation of ferryl Hb after addition of reduced trout Hb to washed muscle was investigated during iced storage. Ferryl trout Hb was detected at a single time point (25 h storage) during the
Fig. 2. (A) Spectra of trout metHb treated with 3-fold H2O2 (or no H2O2) for 0.5 min and then reacted with Na2S and scanned to quantify ferryl Hb; (B) Detection of ferryl Hb at hour 25 of iced storage (pH 6.3) based on treating the supernatant of the washed cod-trout Hb mixture with Na2S (10 dilution of supernatant); Inset: TBARS values in washed cod containing added trout Hb at time points in which ferryl Hb measurements were done during iced storage. Hb concentration in washed cod was 44 lM (haem basis).
exponential phase of TBARS formation based on the peak observed at 620 nm after reaction with Na2S (Fig. 2B). Ferryl trout Hb was not detected in the supernatants at any other time points of storage that were assayed (1, 15, 39, 49, and 71 h). After 71 h there was no detectable Hb in the supernatant based on optical density in the Soret region. Formation of ferryl Hb did not occur after addition of trout metHb to washed muscle during storage. Ferryl bovine Hb was not detected at any time points during iced storage of washed cod containing added bovine oxyHb or metHb (assayed at 1, 15, 25, 39, 49, 71, 113, 182, and 279 h).
3.3. Lipid oxidation and destruction of protoporphyrin IX due to trout metHb and bovine metHb in washed cod Lipid oxidation due to trout metHb was more rapid compared to bovine metHb (p < 0.05). TBARS reached maximal values at 20 h in washed cod containing trout metHb whereas TBARS values were still negligible in washed cod containing bovine metHb at 20 h (Fig. 3A). The final extent of lipid oxidation was similar when comparing trout and bovine metHb with bovine metHb reaching maximal TBARS values near 70 h of storage. MetHbs were examined so that the differing autooxidation rates of trout and bovine Hb did not confound the lipid oxidation kinetics. Protoporphyrin IX destruction of the trout metHb in washed cod was more rapid (p < 0.05) compared to the bovine metHb (Fig. 3B). The protoporphryin moiety of trout Hb was barely detectable at 42 h of storage while nearly all the bovine protoporphryin
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Ice storage (hours)
Optical density (600-700 nm)
TBARS (µmol/kg itssue)
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Storage at 4°C (hours)
Protoporphyrin
Fig. 5. Haemin loss from bighead carp metMb and horse metMb at pH 5.5. Reactions contained 10 lM metMb, 40 lM apoH64Y/V68F, 150 mM BisTris (pH 5.5) and sucrose (400 lM). The blank reactions contained all components except metMb.
Mb was more rapid (p < 0.05) and extensive compared to metMb (Fig. 4). At 20 h of storage TBARS values reached 50 lmol/kg in crosslinked Mb compared to negligible values in metMb at 20 h. TBARS values remained below 10 lmol/kg during the entire storage period for metMb. Ice storage (hours)
Fig. 3. (A) TBARS values in washed cod containing added trout metHb or bovine metHb during iced storage (pH 6.3); (B) concentrations of extracted protoporphyrin IX from washed cod containing added trout or bovine metHb during iced storage (pH 6.3). Hb concentration in washed cod was 44 lM (haem basis).
IX was still detected at 42 h of storage. Approximately half of the protoporphyrin in bovine Hb was destroyed by 65 h of storage.
3.4. Lipid oxidation due to metMb and crosslinked Mb in washed cod muscle
TBARS (µmol/kg itssue)
Enhanced lipid oxidation was previously observed in lipoproteins due to a crosslinked form of Mb (Vuletich et al., 2000). It was thus of interest to evaluate crosslinked Mb in washed cod muscle compared to metMb. Lipid oxidation due to crosslinked
Ice storage (hours) Fig. 4. TBARS values in washed cod containing added metMb or cross-linked Mb during iced storage. pH was 5.7 and Mb concentration was 44 lM.
3.5. Haemin loss from horse metMb compared to bighead carp metMb Fish myoglobins have been reported to be at least 2.5-fold more sensitive to autooxidation compared to mammalian Mbs (Livingston & Brown, 1981). This suggests structural differences between the different Mbs which may also affect haemin affinity. ApoH64Y/V68F was used to measure haemin dissociation from horse metMb compared to bighead carp metMb. Haemin dissociation from the fish metMb was up to 3.3-fold greater compared to the mammalian metMb during storage at pH 5.5 and 4 °C (Fig. 5). Haemin loss was negligible from both haem proteins at pH 6.0 and 4 °C (data not shown). 4. Discussion Haemin release appeared to be a critical step in the progression of Hb-mediated lipid oxidation in washed cod muscle at pH 6.3. This was based on the more rapid lipid oxidation due to trout metHb compared to bovine metHb. Trout metHb has a 55-fold higher haemin loss rate compared to bovine metHb at pH 6.3 (Aranda et al., 2009). This is consistent with rapid lipid oxidation by the Mb mutant H97A which has low haemin affinity compared to wild type Mb and V68T (Grunwald & Richards, 2006). In addition, apohemopexin, which strongly binds and inactivates haemin that dissociates from metHb, was shown to inhibit Hb-mediated lipid oxidation in washed cod muscle (Grunwald & Richards, 2012). Dissociated haemin can react with preformed lipid hydroperoxides (LOOH) in cellular membranes to produce lipid radicals that abstract hydrogen atom from polyunsaturated fatty acid, causing formation of LOOH and secondary lipid oxidation products (Van der Zee, Barr, & Mason, 1996). Those lipid radicals also have the potential to degrade the protoporphyrin moiety (Nagababu & Rifkind, 2004). Extensive protoporphyrin degradation during Hb-mediated lipid oxidation was observed in our studies based
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on loss of detectable porphyrin after acid–acetone extraction. This suggests free radicals that form due to haemin-mediated decomposition of LOOH not only abstract hydrogen atoms from PUFAs but also degrade the prophyrin ring during storage in washed cod. Since bovine metHb has a relatively higher haemin affinity than trout metHb, it was considered that porphyrin degradation might be less or nonexistent with the bovine metHb in washed cod. The idea being that a mechanism not associated with haemin-mediated lipid oxidation and prophyrin degradation could take place with Hbs that have elevated haemin affinity. Yet, extensive lipid oxidation and degradation of the prophyrin moiety of bovine Hb in washed cod was observed during storage. These results are consistent with haemin released from the globin causing lipid oxidation and porphyrin degradation when considering both trout and bovine Hb; just that the onset of lipid oxidation was more rapid with the fish Hb due to its more rapid rate of haemin dissociation while the extents of lipid oxidation due to trout and bovine metHb were similar. Caffeic acid strongly inhibited trout Hb-mediated lipid oxidation and porphyrin destruction during storage in washed cod muscle. It has been noted that caffeic acid accelerates metHb formation (Wallace & Caughey, 1975). Thus the antioxidant mechanism of caffeic acid is unlikely to involve the ability of caffeic acid to keep Hb reduced as oxyHb and deoxyHb. The free radical scavenging ability of caffeic acid may have contributed to the antioxidant effect, but the relatively low caffeic acid concentration (100 lM) should be noted. The ability of caffeic acid to bind to Hb, inhibit haemin loss from metHb, and alter Hb partitioning in washed muscle has been noted as possible antioxidant mechanisms (Park, Sannaveerappa, Undeland, & Richards, 2013). The ability of caffeic acid to reduce tocopherol radicals, regenerating tocopherol has also been noted as a possible antioxidant mechanism in washed cod muscle (Larsson & Undeland, 2010). Perferryl Hb (and perferrylMb) describes the hypervalent iron protein radical that can initiate lipid oxidation via hydrogen atom abstraction from PUFA. Perferryl Hb/Mb undergoes a one-electron reduction to form ferryl Hb/Mb. Ferryl Mb has a relatively long half-life (30 s) and cleaves preformed protein and lipid hydroperoxides to generate peroxyl radicals that can propagate lipid oxidation via hydrogen abstraction (Carlsen, Moller, & Skibsted, 2005). We found that adding reduced trout Hb to washed muscle resulted in ferryl Hb formation but not when trout metHb was added. This suggested that as trout Hb auotooxidized in washed muscle, the resulting superoxide radicals were converted to H2O2 (Balagopalakrishna, Manoharan, Abugo, & Rifkind, 1996; Halliwell & Gutteridge, 1999). H2O2 can then react with metHb to generate ferryl Hb (Giulivi & Davies, 1994). DeoxyHb can also react with H2O2 to generate ferryl Hb (Alayash et al., 2001). More than half of reduced trout Hb exists as deoxyHb at pH 6.3 with the remainder as oxyHb (Binotti et al., 1971). The fact that ferryl Hb was not detected when trout metHb was added to washed muscle can be attributed to the absence of any O2 ligand in the distal haem pocket, thereby preventing generation of superoxide radical and H2O2. In addition, H2O2 was more reactive with trout deoxyHb compared to trout oxyHb and metHb (Gabbianelli, Zolese, Bertoli, & Falcioni, 2004). Collectively, these results suggest deoxyHb facilitates ferryl Hb formation. Ferryl Hb was not detected at any time point during storage when bovine oxyHb or bovine metHb were added to washed muscle. This is in contrast to ferryl Hb formation when trout oxy/deoxy Hb was added to washed muscle. The inability of bovine oxyHb to be converted to ferryl Hb is likely partly due to the relatively slow autooxidation of bovine oxyHb in washed muscle so that a critical concentration of H2O2 is not achieved during storage to react with metHb (Aranda et al., 2009). Also, there will be little to no bovine deoxyHb to react with H2O2 compared to when reduced trout Hb is added to washed cod at pH 6.3. It is also possible that H2O2 is
more accessible to the haem pocket of trout Hb compared to bovine Hb. Increased peroxidase activity using H2O2 and guaiacol was noted in trout Hb compared to human (also mammalian) Hb (Fedeli et al., 2001). Thus, ferryl Hb may contribute to lipid oxidation progress in washed muscle due to added trout oxy/deoxy Hb but this is unlikely to be the case when bovine oxyHb, bovine metHb or trout metHb is added. It has been shown that Hb promotes lipid oxidation much more effectively than Mb in washed muscle which was attributed to the lower haemin affinity of Hb (Thiansilakul et al., 2012). TBARS values were elevated 14-fold at day 1 of iced storage in unwashed cod containing added Hb compared to added Mb at an equivalent level of haem (Castell & Bishop, 1969). Nevertheless, some lipid oxidation due to added Mb occurred in both washed and unwashed muscle during storage and the mechanism involved is of interest. Perferryl Mb is noted to undergo rearrangement at acidic conditions to form crosslinked Mb (Reeder, Svistunenko, Sharpe, & Wilson, 2002). We compared the ability of horse metMb to promote lipid oxidation in washed cod at pH 5.7 compared to crosslinked horse Mb at equivalent concentration of protoporphyrin and found that crosslinked Mb was a potent catalyst of lipid oxidation compared to metMb (Fig. 4). Crosslinked Mb was also found to promote lipid oxidation more effectively than metMb in pure phospholipids and low-density lipoproteins at pH 7.4 (Vuletich et al., 2000). The crosslinking appears to be due to a tyrosine residue forming a covalent bond with the porphyrin moiety, inducing a peroxidase activity that can readily oxidise lipids. Thus, the major form of Mb that oxidises lipids may be crosslinked Mb rather than perferryl Mb or ferryl Mb species, recognising that perferryl Mb is the likely precursor to generate crosslinked Mb. It is interesting to note that crosslinked Mb was not found to promote lipid oxidation in linoleic acid micelles at pH 5.5–6.5 (Baron, Skibsted, & Andersen, 1997). It should be kept in mind that Mb can also release haemin under appropriate conditions of pH, temperature, and the type of Mb that is examined. It was previously shown at 37 °C that haemin loss from sperm whale metMb was 142-fold faster at pH 5.0 compared to pH 7.0 (Hargrove et al., 1994). In the present work, Bighead carp metMb released haemin at pH 5.5 and 4 °C around 3-fold more rapidly compared to horse metMb (Fig. 5). Neither Mb released haemin at pH 6.0 and 4 °C. The ability of the fish Mb to more readily release haemin at pH 5.5 and 4 °C may be related to the lack of a clearly defined D-helix in fish Mbs (Birnbaum, Evans, Przybylska, & Rose, 1994). The D-helix which is clearly present in mammalian Mbs may stabilize the prophyrin moiety within the globin of the protein. Removal of the D-helix from mammalian Mb using sitedirected mutagenesis increased haemin loss rate (Whitaker et al., 1995). In addition, there are 17 amino acids that form contacts with the porphyrin moiety in Mbs (Hargrove, Wilkinson, & Olson, 1996). It turns out that tuna Mb contains a different amino acid at four of these sites (E14, G4, G5, and H15) compared to mammalian Mbs. Differing contacts of the amino acids at these sites with the porphyrin moiety in fish versus mammalian Mbs may explain the lower haemin affinity in the fish Mb at pH 5.5. Converting the alanine at site E14 in bovine Mb to glycine decreased haemin affinity 45-fold which was attributed to enhanced access of solvent to the proximal histidine (Cai & Richards, 2012). Yellowfin tuna Mb contains lysine at E14 which is further away from the porhphyrin ring (by around 0.3 Å) compared to mammalian Mbs that contain alanine at E14 (Birnbaum et al., 1994).
5. Conclusion Trout metHb promoted lipid oxidation more effectively than bovine metHb which can be explained by the lower haemin affinity
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of the fish Hb. Dissociated haemin from metHb decomposes preformed lipid hydroperoxides to free radicals that oxidise lipids and degrade the porphyrin ring. Degradation of the porphyrin moiety of Hb was observed during storage based on loss of extractable porphyrin using acid acetone. Thus, haemin dissociation from the globins appears to be the primary mechanism of Hb-mediated lipid oxidation in washed muscle at pH 6.3. At the same time, it should be recognised that appreciable amounts of deoxyHb (present in reduced trout Hb at pH 6.3) resulted in formation of ferryl Hb as an additional oxidant. Although Hb promotes lipid oxidation more effectively than Mb, crosslinked Mb oxidised lipids extensively compared to metMb at pH 5.7. The primary mechanism of lipid oxidation by Mb can change depending on various factors that include pH, temperature, and whether the Mb is mammalian, avian, or fish. As an example, lowering pH to 5.5 at 4 °C will increase haemin loss from a fish Mb while haemin will remain more associated with the globin in a mammalian Mb at this pH value and thus favour reactivity of the crosslinked form. Future work should further characterise the ability of ferryl Hb and crosslinked Mb to incur lipid oxidation amongst the pool of other oxidants that are present. Acknowledgements This project was supported by the Kangwon National University, South Korea. The project was also funded through a Grant from the Improving Food Quality Foundational Program (Award No. 2014-67016-21648) of the National Institute of Food and Agriculture, U.S. Department of Agriculture. We thank Professor Osawa at the University of Michigan for providing crosslinked horse Mb (NIH Grant GM077430). We also thank Dr. Sung Yong Park for haemin loss measurements and Dr. Yaowapa Thiansilakul for preparing bighead carp myoglobin. References Alayash, A. I., Patel, R. P., & Cashon, R. E. (2001). Redox reactions of hemoglobin and myoglobin: biological and toxicological implications. Antioxidants & Redox Signaling, 3(2), 313–327. Antoni, E., & Brunoni, M. (1971). Hemoglobin and Myoglobin in their Reactions with Ligands. Amsterdam, The Netherlands: North-Holland Publ. Co. Aranda, R., IV, He, C., Worley, C. E., Levin, E., Li, R., Olson, J. S., et al. (2009). Structural analysis of fish versus mammalian hemoglobins: effects of the heme pocket environment on autooxidation and hemin loss rates. Proteins, 75, 217–230. Arduini, A., Eddy, L., & Hochstein, P. (1990). Detection of ferryl myoglobin in the isolated ischemic rat heart. Free Radical Biology & Medicine, 9, 511–513. Balagopalakrishna, C., Manoharan, P. T., Abugo, O. O., & Rifkind, J. M. (1996). Production of superoxide from hemoglobin-bound oxygen under hypoxic conditions. Biochemistry, 35, 6393–6398. Baron, C. P., Skibsted, L. H., & Andersen, H. J. (1997). Prooxidative activity of myoglobin species in linoleic acid emulsions. Journal of Agriculture and Food Chemistry, 45, 1704–1710. Binotti, I., Giovenco, S., Giardina, B., Antonini, E., Brunori, M., & Wyman, J. (1971). Studies on the functional properties of fish hemoglobins. II. The oxygen equilibrium of the isolated hemoglobin components from trout blood. Archives of Biochemistry and Biophysics, 142, 274–280. Birnbaum, G. I., Evans, S. V., Przybylska, M., & Rose, D. R. (1994). 1.70 A resolution structure of myoglobin from yellowfin tuna. An example of a myoglobin lacking the D helix. Acta Crystallographica. Section D, Biological Crystallography, 50(Pt 3), 283–289. Buege, J. A., & Aust, S. D. (1978). Microsomal lipid peroxidation. In S. Fleischer & L. Packer (Eds.), Methods in Enzymology (pp. 302–310). New York: Academic Press. Vol. 52. Cai, H., & Richards, M. P. (2012). Site E14 in hemoglobins and myoglobins: A key residue that affects hemin loss and lipid oxidation capacity. Journal of Agriculture and Food Chemistry, 60, 7729–7734. Carlsen, C. U., Moller, J. K. S., & Skibsted, L. H. (2005). Heme-iron in lipid oxidation. Coordination Chemistry Reviews, 249, 485–498.
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