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Activities of calpastatin, μ-calpain and m-calpain are stable during frozen storage of meat
MEAT SCIENCE Meat Science 72 (2006) 116–120 www.elsevier.com/locate/meatsci
Activities of calpastatin, l-calpain and m-calpain are stable during frozen storage of meat L. Kristensen *, M. Christensen, P. Ertbjerg Department of Food Science, The Royal Veterinary and Agricultural University, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark Received 12 December 2004; received in revised form 10 May 2005; accepted 21 June 2005
Abstract The stability of l-calpain, m-calpain and calpastatin activity during frozen storage of pork was studied in two experiments. In experiment 1, pork longissimus muscle was stored at either 20 or 80 C, and the samples were assayed at 2–3 weeks interval for calpain activity and calpastatin activity using a m-calpain stock solution stored at 4 C. No effects on calpain activity at either temperature were observed for up to 123 days of storage. Calpastatin activity was stable the first few weeks of storage, where after it decreased up to 143 days of storage independently of meat storage temperature. At day 143, calpastatin activity was also assayed using a newly purified stock solution of m-calpain giving a calpastatin activity equal to the activity measured day 0 using the original m-calpain stock solution. The m-calpain stock solution was unstable during storage at 4 C and the activity decreased in a linear manner and was highly related to the observed decrease in calpastatin activity during storage. In experiment 2, meat was stored as in experiment 1 and was assayed at 2–3 week intervals for calpastatin activity using a m-calpain stock solution stored at either 4 or 80 C. As in experiment 1, the measured activity of calpastatin decreased during storage using m-calpain stock solution stored at 4 C and this decrease was highly correlated to the decrease in the activity of the m-calpain stock solution. The activity of the mcalpain stock solution stored at 80 C was constant during storage period of 153 days and likewise was the calpastatin activity measured using this stock solution. The relation between measured calpastatin activity and storage time of m-calpain stock solution was tested by adding, to a calpastatin assay, up to 10 lL of a partly inactivated m-calpain solution. A negative relationship was observed between added inactivated m-calpain and measured calpastatin activity which suggests that the inactive m-calpain molecules mask some of the binding sites on calpastatin and thereby prevent some of the active m-calpain molecules from binding to calpastatin. This would underestimate the measured calpastatin activity. In conclusion, the calpains as well as calpastatin are stable during frozen storage of meat, and the observed decreased in calpastatin activity is due to instability of the m-calpain stock solution used in the calpastatin assay. 2005 Elsevier Ltd. All rights reserved. Keywords: Calpastatin; Calpain; Freezing; Pork
1. Introduction Meat samples used in research are often stored frozen for various times before. e.g., determination of enzyme activity or sensory/instrumental determination of eating quality. Larger samples for Warner–Bratzler shear force determination are routinely stored at 20 C and smal*
Corresponding author. Fax: +45 35283341. E-mail address: [email protected] (L. Kristensen).
0309-1740/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2005.06.010
ler samples for activity determination of the calpain system at 80 C. Koohmaraie (1990) reports that the activities of l- and m-calpain in beef are stable for 6 weeks when stored at 70 C while the activity of calpastatin decreases during frozen storage of muscle samples. According to Koohmaraie (1990) only 45% of the original activity of calpastatin could be measured after 6 weeks of frozen storage. The instability of calpastatin activity in beef during frozen storage has also been observed by Whipple and Koohmaraie (1992) in muscle
L. Kristensen et al. / Meat Science 72 (2006) 116–120
samples stored for 4 weeks at 30 C and in lamb by Duckett et al. (1998) in samples stored for 6 weeks at 20 C. Ingolfsson and Dransfield (1991) observed a 50% reduction in calpastatin activity in lamb after 1 week of storage at 20 C. The objectives of the present work were to study the stability of l-calpain, m-calpain and calpastatin activity during frozen storage of pork for 22 weeks at 20 and 80 C, and to clarify the underlying mechanism for the reported instability of calpastatin during the frozen storage of meat.
2. Materials and methods 2.1. Experimental Pork longissimus muscle from one pig was obtained from a local commercial slaughter house 45 min postmortem (PM) and transported to the laboratory (experiment 1). Approximately 2 h PM, the muscle was cut into samples of 2 cm3. Using two randomly selected samples the activities of l-calpain, m-calpain and calpastatin were determined (day 0). The remaining samples were immediately frozen in liquid nitrogen. The frozen samples were mixed thoroughly and divided into two batches which were stored at either 20 or 80 C for 22 weeks. At 2–3 week intervals during storage, a sample was randomly selected from each batch for determination of extractable activity of l-calpain, m-calpain and calpastatin. Sampling was repeated on a second animal (experiment 2). One week before each animal was slaughtered; a stock solution of partially purified m-calpain was prepared and stored in 30% glycerol at either 4 or 80 C. 2.2. Calpain determination The activities of l- and m-calpain were determined by modification of the method described by Ertbjerg, Henckel, Karlsson, Larsen, and Møller (1999). Samples were finely chopped using a handheld knife and 10 g of meat was homogenized (Ultra Turrax Mixer T25, 2 · 30 s at 9500 rpm and 2 · 30 s at 13,500 rpm, 30 s cooling between bursts) in 60 mL of buffer (50 mM Tris–HCl, 5 mM EDTA (Fluka Chemie AG, CH-9471, Buchs, Switzerland), 10 mM monothioglycerol, 1 lM leupeptin, pH 8.00). The homogenate was centrifuged for 20 min at 25,000g at 4 C and the supernatant was filtered through a 0.2 lm pore size filter (Sartorius, Go¨ttingen, Germany). One milliliter of filtrate was heated at 100 C for 3 min for determination of total calpastatin activity. The two calpain isoforms were separated using an ionexchange column: 20 mL of the filtrate were loaded on a Resource-Q 6 mL column (Amersham Phamacia Biotech, Uppsala, Sweden) and eluted with an NaCl gradient from 0 to 0.6 M NaCl in 20 mM Tris–HCl, 1 mM
117
EDTA, 10 mM monothioglycerol, pH 7.5, using a run time of 20 min. Fractions eluting between 0.08 and 0.23 M NaCl were pooled and analyzed for l-calpain activity using the method described by Geesink and Koohmaraie (1999). Explained briefly, the calpastatin activity in the pooled fractions was measured before and after heat inactivation of l-calpain using partially purified l-calpain in the assay (see below). The difference in calpastatin activity between heated and not heated elute equals the activity of l-calpain. Fractions eluting between 0.29 and 0.35 M NaCl were pooled and analyzed for m-calpain activity using the assay described by Ertbjerg et al. (1999). Determination of total calpastatin activity was achieved using partially purified m-calpain (Ertbjerg et al., 1999). 2.3. Partial purification of l- and m-calpain Calpastatin assays require the use of partially purified calpain. A porcine longissimus muscle was obtained 2 h PM from a local slaughter house. The muscle was trimmed of fat and connective tissue and 150 g was homogenized (Ultra Turrax Mixer T25, 2 · 30 s at 9500 rpm and 2 · 30 s at 13,500 rpm, 30 s cooling between burst) in 900 mL buffer (50 mM Tris–HCl, 5 mM EDTA, 10 mM monothioglycerol, 1 lM leupeptin, pH 8.00). The homogenate was centrifuged for 20 min at 25,000g, 4 C and the supernatant was filtered through nylon net. (NH4)2SO4 was added slowly to the supernatant to a final saturation of 50% and was hereafter kept at 0 C while slowly stirring for 30 min. The solution was centrifuged at 20,000g for 20 min, the supernatant was discharged and the pellet was dissolved in 60 mL buffer-A (20 mM Tris–HCl, 1 mM EDTA, 10 mM monothioglycerol, pH 7.5) and afterwards dialyzed (cut-off: 12,000–14,000 kDa) overnight in 3 L buffer-A. The dialysate was centrifuged for 10 min at 20,000g and the supernatant was adjusted to 0.80 M (NH4)2SO4 which was loaded on a butyl sepharose four fast flow column (26 · 40 cm, Amersham Pharmacia Biotech) previously equilibrated with buffer-B (0.80 M (NH4)2SO4, 20 mM Tris–HCl, 1 mM EDTA, 10 mM monothioglycerol, pH 7.5). Calpastatin and the calpain isoforms were separated using a linear gradient from 100% to 0% buffer-B in buffer-A in 90 min and 4 mL fractions were collected and screened for calpain activity using the method described by Ertbjerg et al. (1999). Fractions containing calpain activity were pooled and the ionic strength was adjusted to 5 mS by dilution with buffer-A. To separate l- and m-calpain the adjusted solution was loaded on a Resource-Q 6 mL column (Amersham Phamacia Biotech, Uppsala, Sweden) and eluted with an NaCl gradient from 0 to 0.6 M NaCl in 20 mM Tris–HCl, 1 mM EDTA, 10 mM monothioglycerol, pH 7.5, using a run time of 20 min. Fractions containing l- or m-calpain activity were pooled and glycerol
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was added to a final concentration of 30% and stored at either 4 or 80 C until use.
Fig. 1 shows the changes in activities of the calpain system extracted during frozen storage of meat at 20 and 80 C in experiment 1. The stability was similar at the two storage temperatures for all three components of the calpain system. The extractable activity of l-calpain and m-calpain did not change during the storage period indicating that the enzymes were stable at both storage temperatures. The activity of calpastatin was constant in the first few weeks of the storage period, thereafter the activity decreased until day 123. The decreased activity of calpastatin is in agreement with Koohmaraie (1990), Whipple and Koohmaraie (1992), Duckett et al. (1998) and Ingolfsson and Dransfield (1991). The previous studies, however, observed a more rapid decrease in calpastatin activity during frozen storage than reported in Fig. 1. Meat samples are stored at 80 C because chemical and biological events are expected to be negligible at this temperature. Thus, it is surprising that calpastatin seems to be unstable at this temperature and that no difference is observed between storage at 20 and 80 C. The rates of chemical reactions are normally highly dependent on temperature. Fig. 2 shows that the activity of the m-calpain stock
22 Experiment 1 20 18
Activity of m-calpain stock (U/mL)
3. Results and discussion
140 120 100 80 60 40 20 0 0
25
50 75 100 Days of storage at 4˚C
125
150
Fig. 2. Activity of m-calpain stock solution during storage at 4 C: (closed symbols) experiment 1; (open symbols) experiment 2. A linear regression line is added (R2 = 0.97 and 0.98 for experiments 1 and 2, respectively).
solution decreased during storage in 30% glycerol at 4 C due to inactivation of the m-calpain molecules. The observed decrease in calpastatin activity (Fig. 1) was related to the change in activity of the m-calpain stock solution, i.e., the lower the activity of m-calpain stock solution, the lower the measured activity of calpastatin (Fig. 3). To test whether this relation was causal, the activity of calpastatin in meat stored at 80 C for 143 days was assayed using both the original stock solution of m-calpain and a newly purified stock solution. The resulting calpastatin activity using the new stock solution was similar with the initial levels observed at the start of the storage period (Table 1). The new
y = 17,0(1-e
19 14
-0,053x
)
2
R = 0,70 17
12
Calpastatin activity (U/g)
Activity (U/g)
16
10 8 6 4
15 13
-0,053x
y = 15,1(1-e
)
2
11
R = 0,77
9 7
2 0
20
40
60 80 Days of storage
100
120
140
5 0
Fig. 1. Measured activity of l-calpain (circles), m-calpain (squares) and calpastatin (triangles) in meat stored at 20 C (closed marks) and 80 C (open marks). A polynomial regression line is added the calpastatin data (R2 = 0.75) and a linear regression the calpain data (R2 = 0.01 and 0.10 for m- and l-calpain, respectively).
25 50 75 100 m-calpain stock solution activity (U/mL)
Fig. 3. Measured calpastatin activity as a function of the activity of mcalpain stock solution: (closed symbols) experiment 1; (open symbols) experiment 2.
L. Kristensen et al. / Meat Science 72 (2006) 116–120 Table 1 Effects of time of storage of the m-calpain used in the calpastatin activity assay on the measured activity of calpastatin in pork longissimus muscle stored at 80 C for 143 days m-Calpain stock solution
A
Meat storage time (d) Storage time of m-calpain (d)A Activity (U/g) SD (U/g)
0 5 16.8a 1.5
B 143 148 11.5b 0.8
143 5 16.6a 0.8
Different superscripts indicate significant different (P < 0.05). A Time that the m-calpain used in the calpastatin assay was stored at 4 C.
stock solution gave a 44% higher calpastatin activity at a meat storage time of 143 days than the original stock solution and there was no difference between the calpastatin activity originally measured at day 0 and calpastatin activity measured at day 143 using the new stock solution. Thus, the observed decrease in calpastatin activity during frozen storage of meat (Fig. 1) appears related to the instability of the m-calpain stock solution. The second experiment tested whether the measured calpastatin activity of meat stored at either 20 or 80 C was influenced by the storage temperature of the m-calpain stock solution (Fig. 4). As found in experiment 1 (Fig. 1), no effect of storage temperature of meat was observed. The samples measured using m-calpain stored at 4 C had a constant level of calpastatin in the first half of the storage period followed by a decreased calpastatin activity in the second half of the per-
20 Experiment 2
Calpastatin activity (U/g)
18
16
14 12
10
8
6 0
20
40
60 80 100 Days of storage
120
140
160
Fig. 4. Measured activity of calpastatin in meat stored at 20 C (triangles) or 80 C (squares). The regression lines are fitted to the pooled data from determinations made using m-calpain stock solution stored at 4 C (open marks, R2 = 0.74) or 80 C (closed marks, R2 = 0.05), respectively.
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iod. This is in accordance with the results presented in Fig. 1 except the calpastatin levels was constant for a longer period in Fig. 4 before the activity decreased. This might be due to a higher initial activity of the mcalpain stock solution used in experiment 2 (Fig. 2). As also observed in experiment 1, there was a relationship between the activity of the m-calpain stock solution and the measured calpastatin activity (Fig. 3); however, only below an activity of approximately 60 U/mL, above this no relation to calpastatin activity was observed. If the partially purified m-calpain stock solution was stored at 80 C, then the activity of the stock solution did not change during the storage period (results not shown). The calpastatin activities measured using m-calpain stored at 80 C were constant during the whole storage period of the meat stored at both 20 and 80 C (Fig. 4). The results obtained in the present study support the conclusion that the decrease in calpastatin activity during frozen storage of meat is due to instability of the m-calpain stock solution and not due to instability of calpastatin in the meat during frozen storage. At least two mechanisms might explain the observed decrease in calpastatin activity: an effect of glycerol and/ or an effect of inactivated m-calpain in the stock solution. The m-calpain used in the calpastatin assay was adjusted to 0.2 U/mL by diluting the stock solution with a Tris buffer. However, the dilution factor decreased during the storage period because of the decreased activity of the stock solution (Fig. 2). The stock solution contained 30% glycerol, so the final glycerol concentration in the calpastatin assay increased during the storage period. The effect of glycerol in the assay was therefore tested by including from 0% to 1.2% glycerol in a calpastatin assay, which is within the range of glycerol used in the assays (Figs. 1 and 4). No effect of up to 1.2% glycerol on calpastatin activity was observed (results not shown). The second mechanism is due to inactivated m-calpain in the stock solution. Since the dilution factor of the m-calpain stock solution decreases during storage, the concentration of inactivated m-calpain molecules in the adjusted solution increases during storage. Calpastatin binds to m-calpain in a ratio of 1:4 (Goll, Thompson, Li, Wei, & Cong, 2003). If calpastatin binds to inactivated m-calpain in the assay and the inactive mcalpain molecules thereby mask the binding sites on calpastatin for active m-calpain molecules, then the calpastatin activity would be underestimated. This suggestion was tested by including increasing amounts of partly inactivated m-calpain to a calpastatin assay (Fig. 5). The inactivation was achieved by storing a mcalpain solution at 4 C for 10 month in which the activity decreased to 20% of the initial value. The measured calpastatin activity was highly dependent on the amount of added partly inactivated m-calpain. Thus, the observed decrease in calpastatin activity during frozen
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frozen and that it is safe to freeze store meat samples, intended for calpastatin and calpain activity measurements, for several month at 20 C. Since differences exists between the purity, activity and content of inactivated m-calpain in stock solutions produced in different labs and from different sources, caution should be taken when comparing results. When planning experiments only one batch of m-calpain stock solution should be used for the entire experiment and this solution should be stored frozen.
10 9
Calpastatin activity (U/g)
8 7 6 5 4 3
Acknowledgment
2
Ahmad Abdal-Kader Kabel and Julie C. Simonsgaard are gratefully acknowledged for their technical assistance in this study.
1 0 0
6 10 2 4 8 Added µL of inactivated m-calpain stock to the assay
Fig. 5. Measured activity of calpastatin as a function of added partly inactivated m-calpain stock solution to the assay. The partly inactivated m-calpain was produced by storing a m-calpain solution at 4 C for 10 month in which the activity decreased to 20% of the initial value. Calpastatin assay were as described in material and method section except 0–10 lL of inactivated m-calpain solution and from 50 to 40 lL of a 30% glycerol solution were added giving a total addition of 50 lL to all assays. The final assay volume was 450 lL.
storage of meat most probably is an artifact due to a masking effect of inactivated m-calpain molecules in the calpastatin assay.
4. Conclusion In conclusion, the calpains as well as calpastatin are stable during frozen storage of pork. The results presented strongly suggest that m-calpain stock solutions used to determine calpastatin activity should be stored
References Duckett, S. K., Klein, T. A., Leckie, R. K., Thorngate, J. H., Busboom, J. R., & Snowder, G. D. (1998). Effect of freezing on calpastatin activity and tenderness of callipyge lamb. Journal of Animal Science, 7, 1869–1874. Ertbjerg, P., Henckel, P., Karlsson, A., Larsen, L. M., & Møller, A. J. (1999). Combined effect of epinephrine and exercise on calpain/ calpastatin and cathepsin B and L activity in porcine longissimus muscle. Journal of Animal Science, 77, 2428–2436. Geesink, G. H., & Koohmaraie, M. (1999). Technical note: a rapid method for quantification of calpain and calpastatin activities in muscle. Journal of Animal Science, 77, 3225–3229. Goll, D. E., Thompson, V. F., Li, H. Q., Wei, W., & Cong, J. Y. (2003). The calpain system. Physiological Reviews, 83, 731–801. Ingolfsson, R., & Dransfield, E. (1991). The effects of low-voltage electrical stimulation and freezing on tenderisation, enzyme activities, drip losses and cooking losses of lamb. Icelandic Journal of Agricultural Science, 5, 63–80. Koohmaraie, M. (1990). Quantification of Ca2+-dependent protease activities by hydrophobic and ion-exchange chromatography. Journal of Animal Science, 68, 659–665. Whipple, G., & Koohmaraie, M. (1992). Effects of lamb age, muscle type, and 24-hour activity of endogenous proteinases on postmortem proteolysis. Journal of Animal Science, 70, 798–804.
Activities of calpastatin, l-calpain and m-calpain are stable during frozen storage of meat L. Kristensen *, M. Christensen, P. Ertbjerg Department of Food Science, The Royal Veterinary and Agricultural University, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark Received 12 December 2004; received in revised form 10 May 2005; accepted 21 June 2005
Abstract The stability of l-calpain, m-calpain and calpastatin activity during frozen storage of pork was studied in two experiments. In experiment 1, pork longissimus muscle was stored at either 20 or 80 C, and the samples were assayed at 2–3 weeks interval for calpain activity and calpastatin activity using a m-calpain stock solution stored at 4 C. No effects on calpain activity at either temperature were observed for up to 123 days of storage. Calpastatin activity was stable the first few weeks of storage, where after it decreased up to 143 days of storage independently of meat storage temperature. At day 143, calpastatin activity was also assayed using a newly purified stock solution of m-calpain giving a calpastatin activity equal to the activity measured day 0 using the original m-calpain stock solution. The m-calpain stock solution was unstable during storage at 4 C and the activity decreased in a linear manner and was highly related to the observed decrease in calpastatin activity during storage. In experiment 2, meat was stored as in experiment 1 and was assayed at 2–3 week intervals for calpastatin activity using a m-calpain stock solution stored at either 4 or 80 C. As in experiment 1, the measured activity of calpastatin decreased during storage using m-calpain stock solution stored at 4 C and this decrease was highly correlated to the decrease in the activity of the m-calpain stock solution. The activity of the mcalpain stock solution stored at 80 C was constant during storage period of 153 days and likewise was the calpastatin activity measured using this stock solution. The relation between measured calpastatin activity and storage time of m-calpain stock solution was tested by adding, to a calpastatin assay, up to 10 lL of a partly inactivated m-calpain solution. A negative relationship was observed between added inactivated m-calpain and measured calpastatin activity which suggests that the inactive m-calpain molecules mask some of the binding sites on calpastatin and thereby prevent some of the active m-calpain molecules from binding to calpastatin. This would underestimate the measured calpastatin activity. In conclusion, the calpains as well as calpastatin are stable during frozen storage of meat, and the observed decreased in calpastatin activity is due to instability of the m-calpain stock solution used in the calpastatin assay. 2005 Elsevier Ltd. All rights reserved. Keywords: Calpastatin; Calpain; Freezing; Pork
1. Introduction Meat samples used in research are often stored frozen for various times before. e.g., determination of enzyme activity or sensory/instrumental determination of eating quality. Larger samples for Warner–Bratzler shear force determination are routinely stored at 20 C and smal*
Corresponding author. Fax: +45 35283341. E-mail address: [email protected] (L. Kristensen).
0309-1740/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2005.06.010
ler samples for activity determination of the calpain system at 80 C. Koohmaraie (1990) reports that the activities of l- and m-calpain in beef are stable for 6 weeks when stored at 70 C while the activity of calpastatin decreases during frozen storage of muscle samples. According to Koohmaraie (1990) only 45% of the original activity of calpastatin could be measured after 6 weeks of frozen storage. The instability of calpastatin activity in beef during frozen storage has also been observed by Whipple and Koohmaraie (1992) in muscle
L. Kristensen et al. / Meat Science 72 (2006) 116–120
samples stored for 4 weeks at 30 C and in lamb by Duckett et al. (1998) in samples stored for 6 weeks at 20 C. Ingolfsson and Dransfield (1991) observed a 50% reduction in calpastatin activity in lamb after 1 week of storage at 20 C. The objectives of the present work were to study the stability of l-calpain, m-calpain and calpastatin activity during frozen storage of pork for 22 weeks at 20 and 80 C, and to clarify the underlying mechanism for the reported instability of calpastatin during the frozen storage of meat.
2. Materials and methods 2.1. Experimental Pork longissimus muscle from one pig was obtained from a local commercial slaughter house 45 min postmortem (PM) and transported to the laboratory (experiment 1). Approximately 2 h PM, the muscle was cut into samples of 2 cm3. Using two randomly selected samples the activities of l-calpain, m-calpain and calpastatin were determined (day 0). The remaining samples were immediately frozen in liquid nitrogen. The frozen samples were mixed thoroughly and divided into two batches which were stored at either 20 or 80 C for 22 weeks. At 2–3 week intervals during storage, a sample was randomly selected from each batch for determination of extractable activity of l-calpain, m-calpain and calpastatin. Sampling was repeated on a second animal (experiment 2). One week before each animal was slaughtered; a stock solution of partially purified m-calpain was prepared and stored in 30% glycerol at either 4 or 80 C. 2.2. Calpain determination The activities of l- and m-calpain were determined by modification of the method described by Ertbjerg, Henckel, Karlsson, Larsen, and Møller (1999). Samples were finely chopped using a handheld knife and 10 g of meat was homogenized (Ultra Turrax Mixer T25, 2 · 30 s at 9500 rpm and 2 · 30 s at 13,500 rpm, 30 s cooling between bursts) in 60 mL of buffer (50 mM Tris–HCl, 5 mM EDTA (Fluka Chemie AG, CH-9471, Buchs, Switzerland), 10 mM monothioglycerol, 1 lM leupeptin, pH 8.00). The homogenate was centrifuged for 20 min at 25,000g at 4 C and the supernatant was filtered through a 0.2 lm pore size filter (Sartorius, Go¨ttingen, Germany). One milliliter of filtrate was heated at 100 C for 3 min for determination of total calpastatin activity. The two calpain isoforms were separated using an ionexchange column: 20 mL of the filtrate were loaded on a Resource-Q 6 mL column (Amersham Phamacia Biotech, Uppsala, Sweden) and eluted with an NaCl gradient from 0 to 0.6 M NaCl in 20 mM Tris–HCl, 1 mM
117
EDTA, 10 mM monothioglycerol, pH 7.5, using a run time of 20 min. Fractions eluting between 0.08 and 0.23 M NaCl were pooled and analyzed for l-calpain activity using the method described by Geesink and Koohmaraie (1999). Explained briefly, the calpastatin activity in the pooled fractions was measured before and after heat inactivation of l-calpain using partially purified l-calpain in the assay (see below). The difference in calpastatin activity between heated and not heated elute equals the activity of l-calpain. Fractions eluting between 0.29 and 0.35 M NaCl were pooled and analyzed for m-calpain activity using the assay described by Ertbjerg et al. (1999). Determination of total calpastatin activity was achieved using partially purified m-calpain (Ertbjerg et al., 1999). 2.3. Partial purification of l- and m-calpain Calpastatin assays require the use of partially purified calpain. A porcine longissimus muscle was obtained 2 h PM from a local slaughter house. The muscle was trimmed of fat and connective tissue and 150 g was homogenized (Ultra Turrax Mixer T25, 2 · 30 s at 9500 rpm and 2 · 30 s at 13,500 rpm, 30 s cooling between burst) in 900 mL buffer (50 mM Tris–HCl, 5 mM EDTA, 10 mM monothioglycerol, 1 lM leupeptin, pH 8.00). The homogenate was centrifuged for 20 min at 25,000g, 4 C and the supernatant was filtered through nylon net. (NH4)2SO4 was added slowly to the supernatant to a final saturation of 50% and was hereafter kept at 0 C while slowly stirring for 30 min. The solution was centrifuged at 20,000g for 20 min, the supernatant was discharged and the pellet was dissolved in 60 mL buffer-A (20 mM Tris–HCl, 1 mM EDTA, 10 mM monothioglycerol, pH 7.5) and afterwards dialyzed (cut-off: 12,000–14,000 kDa) overnight in 3 L buffer-A. The dialysate was centrifuged for 10 min at 20,000g and the supernatant was adjusted to 0.80 M (NH4)2SO4 which was loaded on a butyl sepharose four fast flow column (26 · 40 cm, Amersham Pharmacia Biotech) previously equilibrated with buffer-B (0.80 M (NH4)2SO4, 20 mM Tris–HCl, 1 mM EDTA, 10 mM monothioglycerol, pH 7.5). Calpastatin and the calpain isoforms were separated using a linear gradient from 100% to 0% buffer-B in buffer-A in 90 min and 4 mL fractions were collected and screened for calpain activity using the method described by Ertbjerg et al. (1999). Fractions containing calpain activity were pooled and the ionic strength was adjusted to 5 mS by dilution with buffer-A. To separate l- and m-calpain the adjusted solution was loaded on a Resource-Q 6 mL column (Amersham Phamacia Biotech, Uppsala, Sweden) and eluted with an NaCl gradient from 0 to 0.6 M NaCl in 20 mM Tris–HCl, 1 mM EDTA, 10 mM monothioglycerol, pH 7.5, using a run time of 20 min. Fractions containing l- or m-calpain activity were pooled and glycerol
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was added to a final concentration of 30% and stored at either 4 or 80 C until use.
Fig. 1 shows the changes in activities of the calpain system extracted during frozen storage of meat at 20 and 80 C in experiment 1. The stability was similar at the two storage temperatures for all three components of the calpain system. The extractable activity of l-calpain and m-calpain did not change during the storage period indicating that the enzymes were stable at both storage temperatures. The activity of calpastatin was constant in the first few weeks of the storage period, thereafter the activity decreased until day 123. The decreased activity of calpastatin is in agreement with Koohmaraie (1990), Whipple and Koohmaraie (1992), Duckett et al. (1998) and Ingolfsson and Dransfield (1991). The previous studies, however, observed a more rapid decrease in calpastatin activity during frozen storage than reported in Fig. 1. Meat samples are stored at 80 C because chemical and biological events are expected to be negligible at this temperature. Thus, it is surprising that calpastatin seems to be unstable at this temperature and that no difference is observed between storage at 20 and 80 C. The rates of chemical reactions are normally highly dependent on temperature. Fig. 2 shows that the activity of the m-calpain stock
22 Experiment 1 20 18
Activity of m-calpain stock (U/mL)
3. Results and discussion
140 120 100 80 60 40 20 0 0
25
50 75 100 Days of storage at 4˚C
125
150
Fig. 2. Activity of m-calpain stock solution during storage at 4 C: (closed symbols) experiment 1; (open symbols) experiment 2. A linear regression line is added (R2 = 0.97 and 0.98 for experiments 1 and 2, respectively).
solution decreased during storage in 30% glycerol at 4 C due to inactivation of the m-calpain molecules. The observed decrease in calpastatin activity (Fig. 1) was related to the change in activity of the m-calpain stock solution, i.e., the lower the activity of m-calpain stock solution, the lower the measured activity of calpastatin (Fig. 3). To test whether this relation was causal, the activity of calpastatin in meat stored at 80 C for 143 days was assayed using both the original stock solution of m-calpain and a newly purified stock solution. The resulting calpastatin activity using the new stock solution was similar with the initial levels observed at the start of the storage period (Table 1). The new
y = 17,0(1-e
19 14
-0,053x
)
2
R = 0,70 17
12
Calpastatin activity (U/g)
Activity (U/g)
16
10 8 6 4
15 13
-0,053x
y = 15,1(1-e
)
2
11
R = 0,77
9 7
2 0
20
40
60 80 Days of storage
100
120
140
5 0
Fig. 1. Measured activity of l-calpain (circles), m-calpain (squares) and calpastatin (triangles) in meat stored at 20 C (closed marks) and 80 C (open marks). A polynomial regression line is added the calpastatin data (R2 = 0.75) and a linear regression the calpain data (R2 = 0.01 and 0.10 for m- and l-calpain, respectively).
25 50 75 100 m-calpain stock solution activity (U/mL)
Fig. 3. Measured calpastatin activity as a function of the activity of mcalpain stock solution: (closed symbols) experiment 1; (open symbols) experiment 2.
L. Kristensen et al. / Meat Science 72 (2006) 116–120 Table 1 Effects of time of storage of the m-calpain used in the calpastatin activity assay on the measured activity of calpastatin in pork longissimus muscle stored at 80 C for 143 days m-Calpain stock solution
A
Meat storage time (d) Storage time of m-calpain (d)A Activity (U/g) SD (U/g)
0 5 16.8a 1.5
B 143 148 11.5b 0.8
143 5 16.6a 0.8
Different superscripts indicate significant different (P < 0.05). A Time that the m-calpain used in the calpastatin assay was stored at 4 C.
stock solution gave a 44% higher calpastatin activity at a meat storage time of 143 days than the original stock solution and there was no difference between the calpastatin activity originally measured at day 0 and calpastatin activity measured at day 143 using the new stock solution. Thus, the observed decrease in calpastatin activity during frozen storage of meat (Fig. 1) appears related to the instability of the m-calpain stock solution. The second experiment tested whether the measured calpastatin activity of meat stored at either 20 or 80 C was influenced by the storage temperature of the m-calpain stock solution (Fig. 4). As found in experiment 1 (Fig. 1), no effect of storage temperature of meat was observed. The samples measured using m-calpain stored at 4 C had a constant level of calpastatin in the first half of the storage period followed by a decreased calpastatin activity in the second half of the per-
20 Experiment 2
Calpastatin activity (U/g)
18
16
14 12
10
8
6 0
20
40
60 80 100 Days of storage
120
140
160
Fig. 4. Measured activity of calpastatin in meat stored at 20 C (triangles) or 80 C (squares). The regression lines are fitted to the pooled data from determinations made using m-calpain stock solution stored at 4 C (open marks, R2 = 0.74) or 80 C (closed marks, R2 = 0.05), respectively.
119
iod. This is in accordance with the results presented in Fig. 1 except the calpastatin levels was constant for a longer period in Fig. 4 before the activity decreased. This might be due to a higher initial activity of the mcalpain stock solution used in experiment 2 (Fig. 2). As also observed in experiment 1, there was a relationship between the activity of the m-calpain stock solution and the measured calpastatin activity (Fig. 3); however, only below an activity of approximately 60 U/mL, above this no relation to calpastatin activity was observed. If the partially purified m-calpain stock solution was stored at 80 C, then the activity of the stock solution did not change during the storage period (results not shown). The calpastatin activities measured using m-calpain stored at 80 C were constant during the whole storage period of the meat stored at both 20 and 80 C (Fig. 4). The results obtained in the present study support the conclusion that the decrease in calpastatin activity during frozen storage of meat is due to instability of the m-calpain stock solution and not due to instability of calpastatin in the meat during frozen storage. At least two mechanisms might explain the observed decrease in calpastatin activity: an effect of glycerol and/ or an effect of inactivated m-calpain in the stock solution. The m-calpain used in the calpastatin assay was adjusted to 0.2 U/mL by diluting the stock solution with a Tris buffer. However, the dilution factor decreased during the storage period because of the decreased activity of the stock solution (Fig. 2). The stock solution contained 30% glycerol, so the final glycerol concentration in the calpastatin assay increased during the storage period. The effect of glycerol in the assay was therefore tested by including from 0% to 1.2% glycerol in a calpastatin assay, which is within the range of glycerol used in the assays (Figs. 1 and 4). No effect of up to 1.2% glycerol on calpastatin activity was observed (results not shown). The second mechanism is due to inactivated m-calpain in the stock solution. Since the dilution factor of the m-calpain stock solution decreases during storage, the concentration of inactivated m-calpain molecules in the adjusted solution increases during storage. Calpastatin binds to m-calpain in a ratio of 1:4 (Goll, Thompson, Li, Wei, & Cong, 2003). If calpastatin binds to inactivated m-calpain in the assay and the inactive mcalpain molecules thereby mask the binding sites on calpastatin for active m-calpain molecules, then the calpastatin activity would be underestimated. This suggestion was tested by including increasing amounts of partly inactivated m-calpain to a calpastatin assay (Fig. 5). The inactivation was achieved by storing a mcalpain solution at 4 C for 10 month in which the activity decreased to 20% of the initial value. The measured calpastatin activity was highly dependent on the amount of added partly inactivated m-calpain. Thus, the observed decrease in calpastatin activity during frozen
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frozen and that it is safe to freeze store meat samples, intended for calpastatin and calpain activity measurements, for several month at 20 C. Since differences exists between the purity, activity and content of inactivated m-calpain in stock solutions produced in different labs and from different sources, caution should be taken when comparing results. When planning experiments only one batch of m-calpain stock solution should be used for the entire experiment and this solution should be stored frozen.
10 9
Calpastatin activity (U/g)
8 7 6 5 4 3
Acknowledgment
2
Ahmad Abdal-Kader Kabel and Julie C. Simonsgaard are gratefully acknowledged for their technical assistance in this study.
1 0 0
6 10 2 4 8 Added µL of inactivated m-calpain stock to the assay
Fig. 5. Measured activity of calpastatin as a function of added partly inactivated m-calpain stock solution to the assay. The partly inactivated m-calpain was produced by storing a m-calpain solution at 4 C for 10 month in which the activity decreased to 20% of the initial value. Calpastatin assay were as described in material and method section except 0–10 lL of inactivated m-calpain solution and from 50 to 40 lL of a 30% glycerol solution were added giving a total addition of 50 lL to all assays. The final assay volume was 450 lL.
storage of meat most probably is an artifact due to a masking effect of inactivated m-calpain molecules in the calpastatin assay.
4. Conclusion In conclusion, the calpains as well as calpastatin are stable during frozen storage of pork. The results presented strongly suggest that m-calpain stock solutions used to determine calpastatin activity should be stored
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