Metmyoglobin reducing activity in the mitochondrial fraction from the dark muscle of tuna

Abstract

Desirable bright cherry-red meat color of tuna depends on the redox state of myoglobin (Mb). Mitochondria involved in energy production in live muscle, also influence the redox state. However, no research has determined metmyoglobin (metMb) reduction in tuna muscle via mitochondria. This study aimed to assess the metMb reductase activity in the mitochondrial fraction from the dark muscle of tuna with a high concentration of Mb. As a result, elevated assay temperature resulted in increasing mitochondrial metMb reductase activity in the range of 10–30 °C. There was no significant change in the activity between pH 5.7–6.8, but a significant decrease was observed at pH 7.0. Furthermore, NADH, as the cofactor, was found to initiate the metMb reduction, while exhaustion of NADH resulted in metMb formation. Therefore, maintenance of NADH concentration was considered to be essential to maintain the color of tuna meat during chilled storage.

Introduction

Meat color, an extremely important factor, can influence consumers' acceptability and purchase decisions (Faustman and Cassens, 1990). Color depends on the content and state of muscle pigment, most of which is myoglobin (Mb). Mb is an oxygen-binding heme protein, found in the heart and skeletal muscles of vertebrates (Livingston et al., 1983). Mb is responsible for intracellular oxygen storage and oxygen transport from the plasma membrane to the mitochondria. Mb forms various derivatives depending on its redox state, and accumulation of metmyoglobin (metMb) produced by oxidation of heme iron from divalent to trivalent states results in the discoloration (browning) of meat. Many migratory fish species, such as tuna, have considerable Mb content in their dark muscle, and consequently, undesirable fast discoloration is encountered during storage.

Generally, in living muscles, metMb does not accumulate, as metMb-reducing enzyme systems maintain the physiological role of Mb and could be relevant to the maintenance of fresh meat color (Giddings and Hultin, 1974; Hagler et al., 1979; Echevarne et al., 1990). The presence of NADH-cytochrome b₅ reductase and an electron carrier (cytochrome b₅ located in the outer membrane of mitochondria) in the mitochondrial fraction of bovine muscle was reported by Arihara et al. (1995). Furthermore, mitochondria showed an ability to affect color stability of beef via mitochondria-mediated metMb reducing activity (Ledward, 1985; Ramanathan et al., 2011; English et al., 2016). Although mitochondrial reducing ability related to beef color and its stability have been frequently discussed in research (Tang et al., 2005a,b; Ramanathan et al., 2010; Ramanathan and Mancini, 2010; Mancini and Ramanathan, 2014; Belskie et al., 2015), no previous study has examined the role of mitochondrial function in fish muscle in relation to metMb reduction and color stability.

According to Giddings and Hultin (1974), the loss of metMb reducing activity in post-rigor meat was attributed to factors such as decrease in pH, depletion of substrates such as NADH, oxidative deteriorative changes, and decreasing enzymatic activities including decomposition of mitochondrial particles. Stewart et al. (1965) found that the effect of temperature on metMb reducing activity depended on meat form (intact or minced). Similarly, optimum pH for metMb reducing activity seems to be dependent on the reducing activity source (purified preparation or crude extract) and assay conditions (Bekhit and Faustman, 2005). In the case of tuna meat, color stability is much lower compared with that of livestock meat, and thus discoloration proceeds much faster. The activation of the metMb reduction system, if any, would greatly help maintain meat color during subsequent chilled storage. Therefore, the objective of this study was to explore the relationship of mitochondrial fraction and metMb reduction in the dark muscle of tuna. Factors, such as assay temperature, pH, and cofactors like NADH, that could influence the mitochondrial functions related to the appearance of tuna dark muscle were also investigated.

Materials and Methods

Materials    Fresh specimens of the dark muscle of bigeye tuna (Thunnus obesus) were purchased at Shiogama Seafood Market, Miyagi Prefecture, Japan, and stored at -80 °C until use.

Chemicals    Mb from equine skeletal muscle (95–100%) and NADH (≥ 97%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Potassium hexacyanoferrate (II) trihydrate was obtained from Kanto Chemical Co., Inc. (Tokyo, Japan). EDTA was from Dojindo Laboratories (Kumamoto, Japan). Tris (hydroxymethyl) aminomethane, hydrochloric acid, sucrose, dithiothreitol (DTT) and 0.1 mol/L phosphate buffer solution (pH 6.0, 6.4 and 7.0) were obtained from Fuji Film Wako Pure Chemical (Osaka, Japan). All the chemicals used were of analytical grade. DAPI (4, 6-diamidino-2-phenylindole) solution (10 mg/mL) was from PromoCell (Heidelberg, Germany). Pierce BCA protein assay kit was from Thermo Fisher Scientific (Fair Lawn, New Jersey, USA).

Preparation of mitochondrial fraction    The dark muscle was homogenized with 5 volumes of iced-cold grinding solution (0.5 M sucrose; 10 mM EDTA; 1 mM DTT; 50 mM Tris-HCl buffer, pH 7.5) using a grinder (Coyote Tissue Grinder G50) at 3 000 rpm for 1 min, and centrifuged at 1 000 × g for 10 min. The supernatant was further centrifuged at 10 000 × g for 20 min to precipitate the mitochondria. The sediment was dissolved with 5 volumes of wash solution (0.5 M sucrose; 1 mM DTT; 20 mM Tris-HCl buffer, pH 7.5) and precipitated again at 10 000 × g. The crude mitochondria fraction thus obtained was dissolved in 40% sucrose, and used for the following experiments.

Aliquots of 3 mL of 40% and 60% sucrose were placed into 6.5 mL centrifuge seal tubes and stratified depending on the difference in specific gravity of the solutions. The mitochondria were precipitated at the boundary of the solutions by centrifugation at 120 000 × g for 1 h, and subsequently subtracted with a 1-mL syringe equipped with a needle (0.60 φ× 25 mm). The mitochondria fraction was suspended in 1 mL of 5% sucrose and centrifuged at 10 000 × g for 30 min. The sediment was re-dissolved in 200 µL of the wash solution and stored at 4 °C until use (for up to 1 week). All the steps were performed at 0–4 °C.

DAPI staining    Mitochondrial fraction was incubated in 1 µg/mL DAPI solution at 37 °C for 20 min. The mitochondria were viewed using a fluorescence microscope (Olympus BX53) with 360-nm excitation and 460-nm emission filters.

Mitochondria-mediated metMb reduction    The reduction activity of metMb was measured according to Hagler et al. (1979) in a 1-mL-cuvette which contained the mixture (final pH 4.9) of 0.50 µmol EDTA, 5.0 µmol citrate buffer (pH 4.7), 0.30 µmol K₄Fe(CN)₆, 0.15 µmol metMb in 20 mM sodium phosphate buffer (pH 6.0), 50 µL mitochondrial fraction, 10 µmol NADH, and a total volume was adjusted to 1.0 mL with distilled water. The absorption spectra were measured at 25 °C, and the ratios of deoxyMb, oxyMb, and metMb were calculated by the following formulae (Tang et al., 2004):

• [deoxyMb] = -0.543R₁ + 1.594R₂ + 0.552R₃-1.329
• [oxyMb] = 0.722R₁-1.432R₂-1.659R₃ + 2.599
• [metMb] = -0.159R₁-0.085R₂ + 1.262R₃-0.520
• where R₁= A₅₈₂/A₅₂₅, R₂= A₅₅₇/A₅₂₅, R₃= A₅₀₃/A₅₂₅.

The control absorption spectrum was measured in the absence of NADH, and metMb reduction was initiated by the addition of NADH. One unit of metMb reductase activity was defined as 1 micromole of reduced metMb per min per g of mitochondria protein.

The assay mixture contained citrate-phosphate buffer combinations, because equine Mb was dissolved in phosphate buffer. For the other experimental groups, three types of buffers (citrate, phosphate and Tris-HCl buffers) were used to substitute for the citrate-phosphate buffer combination.

Protein concentration assay    Protein concentration of the mitochondrial fraction was determined according to the bicinchoninic acid (BCA) protein assay using bovine serum albumin (Thermo Fisher Scientific) as a standard. The protein concentration thus determined was used for calculation of metMb reductase activity.

Statistical analysis    Statistical analysis was performed using Microsoft Excel 2010 and Statcel 4 software, OMS Publishing Inc. (Saitama, Japan). Significant differences (p < 0.05) were determined by analysis of variance.

Results

When the prepared mitochondrial fraction was subjected to DNA staining with DAPI solution, the shapes of mitochondria were clearly visualized without any sign of rupture during the preparation procedure (Fig. 1). The yield of the prepared mitochondrial fraction was about 0.36 ± 0.16 mg/g muscle. The fraction was used for all the following experiments.

Fig. 1.

DAPI staining of the mitochondrial fraction from the dark muscle. Scale bar, 10 µm.

Temperature-dependence of metMb reductase activity    MetMb reduction was accelerated with increased temperature (10–30 °C) and showed the highest activity at 30°C (Fig. 2(a)). However, during the 10-min incubation, there was no significant difference in relative metMb ratio (%). Elevated temperature could promote the metMb reducing activity of mitochondrial fraction, but did not influence the metMb reducing capacity (Fig. 2(b)).

Fig. 2.

Effect of temperature on reduction of the metMb. (a) relative activity (%): metMb reductase activity relative to the control (25 °C); metMb (µmol) was reduced by 1 g of mitochondrial protein per min. (b) changes in relative metMb (%): changes in metMb ratio against total Mb after 10 min reduction relative to the control (25 °C). All the data are shown as means ± S.D. (n = 3). Different superscripts in the same graph indicate significant differences (p < 0.05, Tukey-Kramer).

pH-dependence of metMb reductase activity    MetMb reducing activity and changes in metMb ratio (%) decreased with increased pH values (Fig. 3(a)(b)), but metMb reducing activities were shown to vary in the different buffers, even at the same pH. The highest pH values of citrate buffer and phosphate buffer were 5.0 and 6.0, respectively (Fig. 3(a)(b)).

Fig. 3.

Effect of pH on metMb reduction. (a) relative activity (%): metMb reductase activity relative to the control; metMb (µmol) was reduced by 1 g of mitochondrial protein per min. (b) relative metMb changes (%): changes in metMb ratio against total Mb after 10 min reduction relative to the control. All the data are shown as means ± S.D. (n = 3). Open triangles: citrate buffer; open circles: phosphate buffer; closed circles: Tris-HCl buffer.

When metMb reduction was measured in the pH range of 5.7–7.0 using phosphate buffers, there was no significant difference in the activity from pH 5.7 to 6.8, but a significant decrease was found at pH 7.0 (Fig. 4(a)). The maximal metMb reducing capacity was found at around pH 6.6–6.8 in the phosphate buffer after the 10-min incubation (Fig. 4(b)).

Fig. 4.

Effect of pH on metMb reduction in phosphate buffer. (a) relative activity (%): metMb reductase activity relative to the control; metMb (µmol) was reduced by 1 g of mitochondrial protein per min. (b) relative metMb changes (%): changes in metMb ratio against total Mb after 10 min reduction relative to the control. The control was measured using citrate buffer (pH 4.9). All the data are shown as means ± S.D. (n = 3). Different superscripts in the same graph indicate significant differences (p < 0.05, Tukey-Kramer).

NADH concentration-dependence of metMb reductase activity    When metMb reduction under different NADH concentrations was measured, metMb ratio (%) decreased rapidly after NADH addition (Fig. 5(a)), and during the first minute of incubation, similar patterns in the changes of metMb ratio were observed in the range of 0.05–5 mM NADH, except at 0.01 mM NADH, suggesting very low metMb reductase activity at the low NADH concentrations. However, the metMb ratio (%) maintained the level after 2 min, probably due to the exhaustion of NADH in the reduction system. Furthermore, the metMb ratio (%) was recovered after approximately 2 min and 5 min of incubation in the presence of 0.05 mM and 0.1 mM NADH, respectively.

Fig. 5.

Changes in the metMb ratio under different NADH concentrations. (a) the assay was carried out in the presence of 5 mM (closed circles), 1 mM (open circles), 0.5 mM (closed triangles), 0.1 mM (open triangles), 0.05 mM (closed squares), 0.01 mM (open squares) NADH at 25 °C for 10 min. (b) 0.05 mM (open triangles): metMb reduction under 0.05 mM NADH at 25 °C for 20 min but supplemented with the same concentration of NADH as the beginning of reduction at 11 min of incubation; 0.01 mM (open squares): metMb reduction under 0.01 mM NADH at 25 °C for 20 min but supplemented with the same concentration of NADH as the beginning of reduction at 11 min of incubation. The arrow: NADH supplementation.

When NADH was added into the assay mixture after 11 min of incubation (Fig. 5(b)), metMb ratio (%) showed the similar pattern to that at the beginning of metMb reduction indicating that NADH depletion resulted in a decrease in metMb reduction.

Discussion

The mitochondrial fraction after DNA staining with DAPI (Fig. 1) showed very similar appearance to that reported by Zoladek et al. (1995). Equine metMb was clearly reduced by the mitochondrial fraction from the dark muscle of bigeye tuna as detected by the spectrophotometric assay. The rate of metMb reduction was calculated by Tang's formulae (Tang et al., 2004). The results supported the presence of a metMb reductase system in the mitochondrial fraction from the dark muscle of tuna. NADH was essential for metMb reduction as metMb was not reduced at all in its absence (data not shown). This was in good agreement with the finding that equine and porcine metMbs were enzymatically reduced by the porcine longissimus dorsi muscle extract (Mikkelsen et al., 1999).

Effect of temperature    Optimum temperature for metMb reducing activity appears to be species- and reductase source (purified reductase vs. crude one)-dependent (Bekhit and Faustman, 2005). In this study, increased temperature resulted in acceleration of metMb reduction. Similarly, Reddy and Carpenter (1991) found that elevation of temperature from 4 to 30 °C tripled metMb reducing activity in the bovine l. dorsi muscle extract at pH 6.4 and 7.0. Mikkelsen et al. (1999) reported that the rates of enzymatic and non-enzymatic reduction of metMb in the porcine l. dorsi muscle increased in a temperature-dependent manner in the range of 15–30 °C. Regarding the activity from fish sources, purified yellowfin tuna (T. albacares) metMb reductase was most active in the temperature range of 33–35 °C (Levy et al., 1985), while the purified metMb reductase from bluefin tuna (T. thynnus) was found to show the optimal temperature of 25 °C, but was mostly inactivated above 30 °C. Al-Shaibani et al. (1977) also showed the optimum temperature of the purified metMb reductases from bluefin tuna and jack mackerel (T. symmetricus) was 25 °C. Consistent with the finding of Stewart et al. (1965), the effect of temperature on metMb formation might not be the same for all the sources, since elevated temperature not only accelerates metMb reductase activity but also Mb autoxidation. In the present study, there was no significant difference in metMb reductase activity between 25 °C and 30 °C. Therefore, the assays in the following experiments were performed at 25 °C.

Effect of pH    In bovine cardiac muscle, a similar curve of metMb reductase activity related to assay pH was observed, when citrate buffer or acetate buffer was added to the assay mixture, and the optimal pH values in these two buffers were approximately 6.5 in the pH range of 5.7–7.3 (Hagler et al., 1979). In contrast, different optimal pH was observed in the citrate and phosphate buffers. Based on the postmortem pH change in muscle, the assay of pH effect on metMb reduction was investigated in the pH range of 5.7–7.0 using phosphate buffers. The results were consistent with those of porcine metMb reduction (Mikkelsen et al., 1999), showing that the metMb reductase activity decreased at higher pH. Nevertheless, maximal metMb reducing capacity (the amount of reduced metMb) was found at around pH 6.6–6.8 as suggested by the changes in the metMb ratio in the pH range of 5.7–7.0. Although metMb reductase activity of the lamb muscle was not affected in the pH range of 5.5–7.5, the numerically highest activity was observed when the pH was 7.4 (Bekhit et al., 2001). Similarly, Echevarne et al. (1990) reported metMb reducing activity of bovine muscle homogenates was maximal at around pH 7.3, when phosphate buffers of various pH were used. Al-Shaibani et al. (1977) showed that metMb reductase activity from bluefin tuna (T. thynnus) muscle was highest at around pH 7.0–7.3. It has been shown that optimum pH for metMb reductase activity is dependent on the enzyme source and assay conditions (Bekhit and Faustman, 2005). It was thus considered that the enzyme in the mitochondrial fraction was stable against pH changes, as there was no obvious effect on its pH-dependency of metMb reduction.

Effect of NADH concentration    NADH, as a coenzyme and an electron carrier, was essential for the conversion of ferric Mb to its ferrous form (Bekhit and Faustman, 2005). The initial NADH in bluefin tuna muscle was reported to be around 130 nmol/g (Pong et al., 2000a), which was higher than that found in beef longissimus steaks aged for 3 days (29 nmol/g tissue) (Mitacek et al., 2019). NADH content decreased in postmortem dark muscle, and it seems to be one of the main limiting factors decreasing the metMb reducing activity (Echevarne et al. 1990). Bekhit et al. (2001) reported that the highest metMb reduction was observed at 0.05 mM NADH in the presence of lamb muscle extract. The apparent K_m of metMb reductase for NADH was 2.5 × 10⁻⁶ M for the enzyme from the dark muscle of yellowfin tuna (Levy et al., 1985). However, the higher value (24.4 × 10⁻⁵ M) was estimated for bluefin tuna reductase (Pong et al., 2000b) as this reductase was probably isolated from the white muscle. In our study, there was less reducing activity in the presence of 0.01 mM NADH, whereas no difference in the activity was found in the NADH concentration range of 0.05–1 mM. Hence, 0.05 mM NADH can be considered as a saturation level in the metMb reduction assay. Along with the increase in NADH concentration, the activity of porcine metMb reductase increased to a saturation level at 0.1 mM NADH (Mikkelsen et al., 1999).

The supplementation of NADH during the assay resulted in the resumption of metMb reduction (Fig. 5(b)). It could be hypothesized that regeneration of NADH in the dark muscle during storage might retain the metMb reduction system. Upon slaughter, most of muscle glycogen is to be anaerobically converted to lactate which is accumulated in the muscle. Watts et al. (1966) hypothesized that in live muscles, lactate may be oxidized to pyruvate by the lactate dehydrogenase (LDH) and subsequently, hydrogen from lactate would be used for NADH production. Incidentally, Kim et al. (2006) tested this hypothesis and concluded that non-enzymatic reduction of metMb occurred in an equine lactate-LDH-NAD system, but was inhibited by the exclusion of NAD⁺, L-lactic acid or LDH.

Conclusions

The presence of a metMb reductase system was demonstrated in the mitochondrial fraction from the dark muscle of bigeye tuna, and NADH played a pivotal role in metMb reduction. This metMb reductase showed higher stability against temperature and pH variations owing to its localization in the mitochondria membrane. Future research should focus on revealing the effect of NADH on the maintenance of metMb reductase activity in postmortem muscle and determining the importance of the lactate-LDHNAD system in the enzymatic metMb reduction.

Acknowledgements    The authors would like to thank Dr. Kazue Nagasawa, Tohoku University, for his technical assistance with microscopy. This work was partly supported by the Japan Society for Promotion of Sciences (KAKENHI # 22380015 to Y.O.).

Conflict of Interest    The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Abbreviations

Mb

myoglobin

metMb

metmyoglobin

DTT

dithiothreitol

DAPI

4, 6-diamidino-2-phenylindole

BCA

bicinchoninic acid

LDH

lactate dehydrogenase

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