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ANIMAL PRODUCTS |
Animal Science Department, Iowa State University, Ames 50011-3150
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Key Words: Beef • Calpain • Irradiation • Tenderization • Vitamin E
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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). The development of meat tenderness is due, in part, to degradation of key muscle proteins (Huff-Lonergan et al., 1995; Taylor et al., 1995). The calpain system is implicated in this process and is composed of several isoforms of calcium-dependent cysteine proteases (calpains) and their specific competitive inhibitor, calpastatin (for review, see Koohmaraie, 1992; Goll et al., 2003).
During the conversion of muscle to meat, dramatic changes occur within the microenvironment of the muscle cell (Winger and Pope, 1980) that can affect calpain activity (Huff-Lonergan et al., 1996; Veiseth et al., 2004). In addition to a decrease in pH and an increase in ionic strength, postmortem changes in muscle are accompanied by weakening of the antioxidant defense system and a marked increase in indices of oxidation (Martinaud et al., 1997; Harris et al., 2001). Because µ- and m-calpain have an oxidizable cysteine residue at their active site, they require reducing conditions to be active. Thus, oxidizing conditions influence the activity of µ-calpain (Guttmann et al., 1997). It is highly likely, therefore, that oxidative processes in postmortem muscle affect the rate of tenderization by negatively influencing calpain activity. Irradiation (used for preservation and food safety) applied early in the postmortem period creates highly oxidizing conditions and significantly decreases the tenderness of whole muscle products (DeFremery and Pool, 1959; Yoon, 2003; Rowe et al., 2004). Therefore, the hypothesis of this study was that exposure of postmortem bovine muscle tissue to oxidation (via irradiation at 24 h postmortem) would result in inactivation of µ- and m-calpain and decreased proteolysis of myofibrillar proteins and higher shear force values in aged beef steaks.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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. Steers were slaughtered at the Iowa State University Meat Laboratory at an average BW of 634 kg using approved humane procedures. Four steers, two fed the CON diet and two fed the VITE diet, were slaughtered on each slaughter date.
At approximately 22 h postmortem, 2.54-cm-thick, boneless strip loin steaks were collected from the LM from each carcass and vacuum packaged. All LM steaks (10 steaks per strip loin) from one loin of each carcass were assigned as nonirradiated controls, whereas all LM steaks from the opposite side strip loin of each carcass were assigned to be irradiated. Assignment of the side to be irradiated was random. Two adjacent steaks from each strip loin were assigned to an aging period of 0, 1, 3, 7, or 14 d postirradiation (1, 2, 4, 8, or 15 d postmortem) at 4°C. One steak from each aging period was designated for biochemical analysis, and one steak was designated for Warner-Bratzler shear force (WBSF) determination. To determine whether dietary treatment increased vitamin E content of the LM steaks before irradiation, an additional steak was taken from each carcass at the posterior end of the strip loin, vacuum packaged, frozen at –20°C, and then sent to the University of Wisconsin, Soil and Plant Analysis Laboratory (Madison), for analysis of 
-tocopherol content according to the procedures of Liu et al. (1996).
Irradiation
Irradiation was conducted at the Linear Accelerator Facility at the Iowa State University Meat Laboratory. At 22 to 24 h postmortem, vacuum-packaged steaks from one side of each carcass were irradiated (average dose = 6.4 kGy). Steaks from the opposite side strip loins were not irradiated but were held at the same temperature (approximately 20°C) for the same length of time (approximately 10 min) as the irradiated steaks. Samples were irradiated with a CIRCE IIIR electron beam irradiator (Thomson-CSF Linac, St. Aubin, France) at an energy level of 10 MeV, a power level of 10 kW, and a conveyor speed of 0.223 m/min. After irradiation, all steaks (irradiated and nonirradiated) were held at 4°C for 0, 1, 3, 7, or 14 d postirradiation (1, 2, 4, 8, and 15 d postmortem). At the completion of each aging period, steaks designated for WBSF were frozen until subsequent analysis. Steaks designated for biochemical analysis were used immediately.
Warner-Bratzler Shear Force
All procedures were done in accordance to AMSA (1995) guidelines. Frozen 2.54-cm-thick steaks were thawed at 2°C and used for WBSF determination. Steaks were broiled in an electric broiler (model 6850, General Electric, Chicago Heights, IL.) 15 cm from the heat source. Steaks were broiled to an internal temperature of 30°C, turned, and then broiled to a final temperature of 70°C. Temperature was monitored using an Electrotherm digital probe (model TM99A, Cooper Instrument Corp., Middlefield, CT). Steaks were covered with plastic wrap and allowed to chill overnight at 4°C. Steaks were equilibrated to room temperature (23°C), and six 1-cm-diameter cores were removed parallel to the muscle fibers. Each core was sheared perpendicular to the fiber direction using a TA.XT2 texture analyzer with a 5-kg load cell (Texture Technologies Corp., Scarsdale, NY). All tests were performed using the Warner-Bratzler probe and guillotine set (TA-7B USDA; Texture Technologies Corp.). The probe was lowered 30 mm from the point of resistance, and the penetration speed was 3.3 mm/s. All data were collected using Texture Expert software (Version 1.22; Texture Technologies, Corp.).
Extraction Procedure
Sarcoplasmic proteins were extracted according to Shackelford et al. (1994) with modifications. At 0, 1, 3, 7, and 14 d postirradiation, 10 g of finely diced fresh meat were homogenized in three volumes of ice-cold extraction buffer (10 mM EDTA, 2 µM E-64, 100 mg/L of trypsin inhibitor, and 2 mM phenylmethylsulfonylfluoride, 100 mM Tris-HCl, pH 8.3) using a polytron PT 3100 (Kinmetaica AG, Littau, Switzerland) set at 22,000 rpm. Samples were centrifuged (27,000 x g) for 30 min at 4°C. Supernatant fractions were filtered through cheesecloth, and sample volume was recorded. Protein concentration of each sample was determined using a Bradford assay (BioRad protein assay kit, BioRad Laboratories, Hercules, CA; Bradford, 1976). The supernatant fraction was used for casein zymography, calpastatin activity determination, SDS-PAGE, and immunoblotting. The pellet fractions were collected and used for myofibrillar protein purification.
Myofibrillar Protein Purification
Four grams of pellet from each sarcoplasmic protein extraction was weighed and homogenized in 10 volumes of standard salt solution (100 mM KCl, 2 mM MgCl2, 1 mM ethylenebis(oxyethylenenitrilo) tetraacetic acid, 1 mM NaN3, 20 mM K2HPO4, pH 7.0). Myofibrils were further purified by differential centrifugation (Huff-Lonergan et al., 1995), and protein concentration was determined using the Biuret method as modified by Robson et al. (1968). Purified myofibrils were used for SDS-PAGE and immunoblotting.
SDS-PAGE Gel System and Western Blotting
Gel Composition.
Myofibril and supernatant samples in sample buffer/tracking dye were run on acrylamide gels (acrylamide: N,N'-bis-methylene acrylamide = 100:1 [wt/wt], 0.1% SDS [wt/vol], 0.05% N,N,N'N-tetramethylethylenediamine (TEMED), 0.05% ammonium persulfate [wt/vol], and 0.375 M Tris-HCl, pH 8.8). A 15% acrylamide gel was used for troponin-T analysis, a 10% acrylamide gel was used for desmin analysis, and 9% acrylamide gels were used for µ-calpain and myofibril-bound µ-calpain analysis. A 5% acrylamide stacking gel (acrylamide: N,N'-bis-methylene acrylamide = 100:1 [wt/wt], 0.1% SDS [wt/vol], 0.125% TEMED, 0.075% ammonium persulfate [wt/vol], and 0.125 M Tris-HCl, pH 6.8) was used on all of the acrylamide gels. For titin and nebulin analysis, 5% continuous gels composed of acrylamide (N,N'-bis-methylene acrylamide = 100:1 [wt/wt]), 0.1% SDS (wt/vol), 0.067% TEMED, 0.1% ammonium persulfate (wt/vol), 2 mM EDTA, and 200 mM Tris-HCl, pH 8.0 were used.
Western Blotting.
After electrophoresis, samples were transferred onto Poly Screen polyvinylidene difluoride transfer membrane (NEN Life Science Products, Inc., Boston, MA) according to the procedures of Lonergan et al. (2001). After transfer, polyvinylidene difluoride membranes were placed in blocking solution composed of 80 mM disodium hydrogen orthophosphate, 20 mM sodium dihydrogen orthophosphate, 100 mM sodium chloride, 0.1% polyoxyethylene sorbitan monolaurate (Tween-20; vol/vol; PBS-Tween), and 5% nonfat dry milk (wt/vol) for 1 h at room temperature (23°C). After blocking, membranes were placed in their respective primary antibody diluted in PBS-Tween. Troponin-T blots were incubated for 1 h at room temperature (23°C) with the primary antibody (monoclonal antitroponin-T antibody, JLT-12; Sigma Chemical Co., St. Louis, MO) diluted 1:5,000. For µ-calpain and myofibril bound µ-calpain blots, the primary antibody (monoclonal anti-µ-calpain antibody, MA3-940; Affinity Bioreagents, Inc., Golden, CO) was diluted 1:10,000 and incubated overnight at 4°C. The desmin primary antibody (polycolonal rabbit antidesmin antibody, V2022; Biomedia, Foster City, CA) was used at a dilution of 1:10,000 for 2 h at room temperature (23°C). After primary antibody incubations were complete, membranes were washed three times (10 min/wash,) using PBS-Tween at room temperature (23°C) before incubation with the secondary antibody. Troponin-T, sarcoplasmic µ-calpain, and myofibril-bound µ-calpain blots were incubated 1 h at room temperature with the secondary antibody (goat anti-mouse conjugated with horseradish peroxidase, catalog No. A2554; Sigma Chemical Co.) diluted 1:5,000. Desmin blots were incubated 1 h at room temperature (23°C), with the secondary antibody (goat anti-rabbit conjugated with horseradish peroxidase, catalog No. A9169, Sigma Chemical Co.) diluted 1:5,000. After completion of the secondary antibody incubation, all membranes were washed three times (10 min/wash) using PBS-Tween at room temperature (23°C). Detection was initiated using premixed reagents (ECL Plus kit; Amersham Pharmacia Biotech, Piscataway, NJ). Chemiluminescence was detected using a 16-bit mega-pixel CCD camera FluorChem 8800 (Alpha Innotech Corp., San Leandro, CA) and FluorChem IS-800 software (Alpha Innotech Corp.). Densitometric measurements were completed using the AlphaEaseFC software (Alpha Innotech Corp.).
Casein Zymography
Casein zymography (Raser et al., 1995) was conducted to measure µ- and m- calpain activity at 0, 1, 3, 7, and 14 d postirradiation. Immediately after extraction, nondenaturing gel samples were made using the supernatant collected from the initial extraction. Gel samples were made by diluting the supernatant at a ratio of 60:40 (supernatant:ice-cold electrophoresis sample buffer [20% {vol/vol} glycerol, 0.1% {wt/vol} bromophenol blue, 0.75% {vol/vol} 2-mercaptoethanol {MCE}, 150 mM Tris-HCl, pH 6.8]). Samples were immediately loaded onto nondenaturing (12.5%) acrylamide gels containing casein. Gels were run on the Hoefer SE260 Mighty Small II (10 cm wide x 10 cm tall x 1.5 mm thick) gel system (Hoefer Scientific Instruments, San Francisco, CA) for analysis. Gels were run at a constant voltage of 20 V for approximately 22 h. Gels were incubated in CaCl2 and stained according to Melody et al. (2004). Images were taken using a 16-bit megapixel CCD camera FluorChem 8800 (Alpha Innotech Corp.) and FluroChem IS-800 software (Alpha Innotech Corp.).
Calpastatin Activity
Calpastatin activity was determined on samples aged 0 (n = 20), 3 (n = 20), and 14 (n = 12) d postirradiation. The sarcoplasmic protein extract was dialyzed against 40 volumes of 40 mM Tris, 1 mM EDTA (pH 7.4) overnight at 4°C. The dialysate was heated to 95°C for 20 min and immediately chilled on ice. Following centrifugation at 27,000 x g, calpastatin activity was determined as described by Koohmaraie et al. (1995). One unit of calpastatin activity was defined as the ability to inhibit one unit of bovine m-calpain activity (Koohmaraie, 1990). Calpastatin activity (total units/g of tissue) and calpastatin specific activity (total units/g of protein extracted in the sarcoplasmic protein extraction) were reported.
Statistical Analyses
Data were analyzed as a split-plot design. The whole plot was vitamin E treatment (supplemented with 0 or 1,000 IU vitamin E daily). The split plot was irradiation treatment (0 or 6.4 kGy). Specific comparisons were made within aging period. Each slaughter date served as a replication. Replication x vitamin E was the whole-plot error term, whereas replication x vitamin E x irradiation was the split-plot error term. Treatment effects were considered significant at P < 0.05.
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Results |
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). Supplementing the diets of finishing steers with vitamin E to increase vitamin E content in muscle was used as a treatment to slow overall oxidation in the tissue (Liu et al., 1995). Conversely, irradiation of the meat was used as a treatment to induce protein oxidation at an early time postmortem (Rowe et al., 2004). Both strategies influenced protein oxidation in these samples. Vitamin E supplementation increased the -tocopherol content of the muscle by almost 3.5-fold (4.19 and 1.22 µg/g muscle for VITE and control diets, respectively; Rowe et al., 2004). In the samples that were not irradiated, the increased concentration of -tocopherol in the meat limited the number of sarcoplasmic proteins that were oxidized during postmortem aging. In addition, oxidized sarcoplasmic proteins were oxidized to a lesser extent in the steaks from animals fed supranutritional levels of vitamin E (Rowe et al., 2004).
The irradiation treatment was successful in inducing protein oxidation (as measured by carbonyl content) in both the sarcoplasmic and myofibrillar proteins from samples taken through 7 d of aging (P < 0.05; Rowe et al., 2004). Most importantly, however, Rowe et al. (2004) showed that increasing protein oxidation (carbonyl content) of both the myofibrillar and sarcoplasmic proteins was significantly correlated with increased WBSF values.
In the current study, all comparisons were made within an individual aging period. This specific approach was used to investigate the effect that protein oxidation had on shear force, protein degradation, and the activity of the calpain system at discrete time points rather than over time. Vitamin E supplementation had no effect (P = 0.4) on shear force within any of the time points; however, at 15 d postmortem (14 d postirradiation) in the nonirradiated samples, the steaks from the VITE-fed steers tended (P = 0.09) to have a lower shear force (0.8 kg lower) than steaks from steers fed the CON diet (2.51 ± 0.158 vs. 3.31 ± 0.412 kg, respectively). Early postmortem irradiation of steaks had a more pronounced effect than did the vitamin E treatment. At 1, 3, 7, and 14 d postirradiation, irradiated steaks had higher (P < 0.05) shear force values than steaks (from the same steers) that were not irradiated (Figure 1). The difference between the two irradiation treatments by 14 d postirradiation was 0.8 kg.
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). This indicates that degradation of troponin-T occurred earlier in nonirradiated meat from the steers fed high levels of vitamin E than in nonirradiated meat from steers not supplemented with vitamin E. The differences were not detected (P > 0.05) at other time points and there was no interaction (P > 0.05) between vitamin E and irradiation treatments.
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). There were no irradiation treatment differences (P > 0.05) in the intensity of the 30-kDa degradation product in samples analyzed immediately after irradiation or 1 d later (0 and 1 d postirradiation; results not shown).
Desmin.
Diet (VITE vs. CON) did not influence (P > 0.05) degradation of desmin. However, irradiation did influence (P < 0.05) the degradation of desmin in LM steaks analyzed at 3, 7 and 14 d after irradiation, but not (P > 0.05) on 0 or 1 d after irradiation. Less (P < 0.05) intact desmin immunoreactive bands and more intense (P < 0.05) desmin degradation bands were detected in the nonirradiated samples at these time points (3, 7 and 14 d postirradiation; Figure 3). This indicated that there was less degradation of desmin at later aging times in the irradiated samples.
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). Evidence of nebulin degradation was noted in some samples as early as 1 d postirradiation (2 d postmortem). Within 3 d postirradiation (4 d postmortem), none of the samples exhibited detectable intact nebulin (Figure 4a). Within the irradiated LM steaks, no intact titin or intact nebulin was detected on the SDS-PAGE gels at any time sampled (0, 1, 3, 7, or 14 d postirradiation) indicating that irradiation affected the structure of these two giant myofibrillar proteins.
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; Melody et al., 2004). This technique allows simultaneous detection of both µ- and m-calpain activity. In this system, sarcoplasmic protein extracts were run under reducing (presence of MCE) conditions on nondenaturing PAGE gels that contained casein. Gels were then incubated in calcium chloride plus a reducing agent (MCE), and were subsequently stained. Active calpain degraded the casein in the polyacrylamide gel during the calcium chloride/reducing agent incubation, leaving zones of low casein concentration within the gel that appeared as clear bands after the gels were stained and subsequently destained. Because reducing conditions are maintained throughout the procedure, this technique allows detection of calpain that may have been reversibly oxidized and potentially inactive in the tissue, but is still capable of becoming active once it was reduced (Guttmann et al., 1997).
µ-Calpain Activity.
In all samples, regardless of diet or irradiation treatment, substantial µ-calpain activity was observed in samples taken on the day of irradiation (d 0, not shown) and on 1 d postirradiation (Figure 5); however, by 3 d after irradiation, the irradiated samples (regardless of diet) showed larger clear zones in the region of the gel to which µ-calpain migrated (Figure 5). At 7 and 14 d after irradiation, the differences between irradiated and nonirradiated samples were even more evident (Figure 5). Irradiated samples had obviously larger, brighter clear zones, indicating more µ-calpain activity in those sarcoplasmic extracts. In fact, by 14 d postirradiation in the nonirradiated samples, there was no detectable µ-calpain activity, as was expected. Because µ-calpain autolyzes in postmortem tissue and the activity of µ-calpain typically decreases markedly within 1 to 3 d after slaughter (Veiseth et al., 2001), the fact that substantial activity remained as late as 15 d postmortem (14 d postirradiation) in the irradiated samples is remarkable. This likely indicates that the calpain in the irradiated LM steaks was not active, or at least minimally active, in the tissue and that activity was restored when reducing conditions were introduced during analysis.
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); however, irradiation influenced the extent of autolysis noted in the samples at 3, 7, and 14 d after irradiation (Figure 6). Just as there was no observable difference in µ-calpain activity in the sarcoplasmic extracts in the casein zymograms at 0 and 1 d after irradiation, there was no difference in the amount of µ-calpain autolysis in the sarcoplasmic extracts from irradiated and nonirradiated meat at those same time points. However, by 3 d postirradiation, all irradiated samples had less extensive autolysis that did the nonirradiated samples (Figure 6). Similar results were seen at 7 and 14 d after irradiation. In fact, by 7 and 14 d postirradiation, in the nonirradiated samples, the only µ-calpain large subunit immunoreactive band that was detected was a band that migrates at 76 kDa, a sign of extensive autolysis. In sarcoplasmic extracts of irradiated samples from the opposite side strip loins, the presence of the intermediate autolysis product that migrates at 78 kDa (in addition to the 76-kDa band) was noted; this was indicative of less extensive autolysis.
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; Melody et al., 2004). Diet did not (P > 0.05) seem to influence this in the current study. However, irradiation resulted in more (P < 0.05) detectable µ-calpain associated with myofibrils isolated at on the same day than the samples that were irradiated (0 d postirradiation or 1 d postmortem; Figure 7). At later times postirradiation (1, 3, 7, and 14 d) there was no treatment difference (P > 0.05) in the amount of immunoreactive µ-calpain large subunit detected in the purified myofibrils (Figure 7). The degree of autolysis of the µ-calpain large subunit associated with the myofibrils mirrored the degree of autolysis noted in the µ-calpain large subunit in the sarcoplasmic fraction on a given day of aging (Figures 6 and 7).
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). This band was most consistently seen in the sarcoplasmic extracts from the nonirradiated samples and was rarely seen in sarcoplasmic extracts from the irradiated samples (Figure 5). It has been hypothesized that these two bands are seen on casein zymographs when m-calpain is partially autolyzed (Veiseth et al., 2001).
Calpastatin Activity.
Calpastatin activity was measured at 0, 3, and 14 d after irradiation. Diet did influence the activity of calpastatin in the sarcoplasmic extracts from samples taken the day of irradiation (d 0). Calpastatin activity (expressed both as units/g tissue and as specific activity = units/g extracted protein) was lower (P < 0.05) in the d-0 extracts of the LM steaks from steers fed the VITE diet (Table 1) compared with d-0 extracts of the LM steaks from steers fed the CON diet.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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; Melody et al., 2004).
It is commonly accepted that activity of the calpain enzyme system is involved in governing the rate and extent of proteolysis of the key proteins that regulate meat tenderness. Although the calpain system has been extensively studied over the past few decades, the mechanisms that control its activity in postmortem muscle have not been fully elucidated. The two most studied isoforms of the calpain enzymes, µ- and m-calpain, are cysteine proteases. As such, calpains require the reactive sulfhydryl groups in the active site to be reduced to be functional. Indeed, assays for the activity of these enzymes include the presence of reducing agents (Koohmaraie et al., 1990).
One factor that has not been examined to date is the influence that protein oxidation has on the tenderization process of beef. Oxidation of proteins changes their functional properties and thereby influences how those proteins perform in the tissue or during further processing (Xiong, 2000). Oxidation of myofibrillar proteins has been shown to occur naturally in meat during aging (Martinaud et al., 1997). Because cysteine residues contain sulfhydryl groups, they are one target in proteins for oxidation. The calpains are cysteine proteases and, thus, their activity might be compromised under oxidative conditions. These proteases are involved in postmortem tenderization. Therefore, examination of the influence of protein oxidation on the proteolysis of key myofibrillar proteins and on the calpain system itself can lend valuable information regarding postmortem control of meat tenderization.
Influence of Higher Levels of Endogenous Antioxidant (Vitamin E) in the Muscle
Slowing the oxidation that occurs in muscle tissue by incorporating high levels of vitamin E in the muscle has been shown to result in earlier production of protein degradation products, the appearance of which are often correlated with tenderness (Harris et al., 2001). In addition, it has been shown that high levels of vitamin E in muscle tissue can result in product that has lower shear forces at early times postmortem (Harris et al., 2001). In the current study, it was again shown that nonirradiated meat with high levels of endogenous vitamin E had earlier degradation of the protein troponin-T, an indicator of proteolysis that correlates with tenderization (Lonergan et al., 2001). This infers that very low levels of oxidation can influence proteolysis. In addition, although it was not significant, there was a trend (P = 0.09) for the meat from vitamin E fed cattle to be more tender after 15 d of aging (difference of 0.8 kg). These results, in conjunction with the results from Harris et al. (2001), support the concept that oxidative processes in early postmortem meat may hinder tenderization by interfering with proteolytic processes. These results allow for the possibility that increasing the level of antioxidants in meat may improve tenderness. Indeed, compared with the nonirradiated meat from conventionally fed steers, the nonirradiated meat with higher levels of vitamin E had less oxidation of sarcoplasmic proteins (Rowe et al., 2004). This finding is intriguing because this is the fraction in which µ- and m-calpain are found, leaving open the possibility of calpain oxidation being involved in governing meat tenderness.
Influence of Irradiation/Higher Levels of Oxidation
To more closely examine the effects of oxidation, irradiation was used in this study as a means to uniformly create oxidizing conditions within early postmortem meat. The results from this portion of the study strengthen the hypothesis that oxidative processes can interfere with the tenderization process by suppressing calpain activity and slowing the rate of proteolysis occurring in meat during aging. This is supported by the fact that the more oxidized samples (from irradiated meat) had significantly higher shear force values and dramatically less degradation of troponin-T and desmin at 3, 7, and 14 d after irradiation.
This study also showed that oxidation slowed µ-calpain autolysis as oxidized samples had more µ-calpain activity on casein zymographs as late as 15 d postmortem. These results also indicate that the nonirradiated samples lost µ-calpain activity during aging at a relatively normal rate (similar to what has been noted in numerous other studies: Veiseth et al., 2001; Boehm et al., 1998), whereas the irradiated (oxidized) samples did not. This is an important observation because loss of µ-calpain activity and extensive autolysis by 14 to 15 d postmortem is expected in postmortem muscle (Koohmariaie 1992b; Bohem et al., 1998). Because calpain loses activity after extensive autolysis, loss of calpain activity during postmortem aging of meat indicates prior activation. Calpain that is prevented from being active will not fully autolyze in the same time-frame and will, thus, be able to be activated once the conditions for activity are satisfied (for example, provision of ample free calcium and reducing conditions). The fact that a substantial amount of activity was recovered in the oxidized samples at such late times postmortem indicates two important things. First, it indicates that µ-calpain was likely not fully active in oxidized meat; thus, it was unable to autolyze and subsequently become inactivated at the same rate as in the nonoxidized meat. In vitro studies have shown that oxidative conditions do slow autolysis and arrest activity of µ-calpain (Guttmann et al., 1997; Guttmann and Johnson, 1998), similar to what was seen in the tissue samples in the current study. Second, these results indicate that µ-calpain may have been reversibly oxidized because the reducing conditions used in the assay (casein zymogram) did restore activity. Taken together, these results provide new, novel evidence that oxidative conditions inhibit calpain activity and that this inhibition may be reversible.
The results of the current study indicate that m-calpain may not be affected by oxidation in the same manner as µ-calpain in postmortem muscle. Whether this is due to an innate difference in susceptibility to oxidation or simply due to the fact that m-calpain may not be fully activated in postmortem muscle cannot be definitely proven in this study. However, there was evidence that m-calpain may have autolyzed to some extent in the nonirradiated samples. In the casein zymographs a lower migrating clear zone below the main band of m-calpain activity was noted at later aging times in the nonirradiated samples (Figure 5; d 7, 14). The apparent lack of this lower zone of activity in the irradiated samples could indicate that there was impedance of m-calpain activity. The origin of this lower zone of activity cannot be proven, but it has been noted in other studies and has been attributed to autolysis of m-calpain (Veiseth et al., 2001; Kent et al., 2004).
The fact that calpastatin activity was higher in the irradiated samples is also significant. Calpastatin has been shown to be a substrate for calpain in postmortem muscle (Doumit and Koohmaraie, 1999). Digestion of calpastatin with either µ- or m-calpain does result in a reduction of calpastatin activity (Doumit and Koohmaraie, 1999). In fact, it has been suggested that, in postmortem muscle, degradation of calpastatin by µ-calpain limits the calpain inhibitory activity of calpastatin and may be an additional factor that influences tenderization (Doumit and Koohmaraie, 1999). If oxidation slowed calpain activity in the irradiated samples in the current study, then it is possible that a lack of calpain induced degradation, and, thus, less inactivation of calpastatin may be a side result of protein oxidation. Alternatively, oxidation itself may have altered calpastatin in some manner to render it less susceptible to inactivation during aging.
It was interesting to note that the giant proteins, titin and nebulin, responded differently to irradiation than did the relatively smaller proteins, troponin-T and desmin. Irradiation seemed to severely disrupt the structure of these proteins to the point that they were not detectable on the high porosity gels normally used for monitoring intact titin and nebulin. Others have also noted that titin and nebulin from irradiated muscle were apparently fragmented and were not detectable on SDS-PAGE gels, even though the mobility of other smaller proteins (including myosin) did not seem to be affected (Horowits et al., 1986). The role that irradiation-induced disruption of titin and nebulin has on influencing meat tenderness has yet to be ascertained.
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2 Correspondence: 2278 Kildee Hall (phone: 515-294-9125; fax: 515-294-9143; e-mail: elonerga{at}iastate.edu).
Received for publication June 12, 2003. Accepted for publication July 8, 2004.
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