Denaturation of Myofibrillar Proteins from Chicken as Affected by pH,
Temperature, and Adenosine Triphosphate Concentration
R.L.J.M. Van Laack1 and J. L. Lane
Department of Food Science and Technology, University of Tennessee, Knoxville, Tennessee 37901-1071
ABSTRACT The susceptibility to denaturation of myofibrillar protein from chicken muscles was investigated
and compared with denaturation of myofibrillar protein
from pork. Immediately postmortem, the Pectoralis profundus (white muscle) and the Pubo-ishio femorale (red
muscle) of six Arbor Acres chickens were collected. The
Semimembranosus (white muscle) and Psoas major (red
muscle) of three Yorkshire × Landrace and three Yorkshire × Landrace × Duroc pigs were collected at 45 min
postmortem. Protein denaturation was prevented by
keeping the muscles at 0 to 2 C in a buffer (pH 7.2)
containing ethylene glycol-bis (β-aminoethyl ether)
N,N,N′,N′-tetraacetic acid (EGTA) (to sequester Ca ions).
After purification, myofibrils were incubated at 25 or 40
C, pH 5.4 or 6.5, with 0, 0.68, or 3.4 mM adenosine triphosphate (ATP). Protein solubility, an indicator of denatur-
ation, was assessed after 0, 10, 20, and 60 min incubation.
Protein solubility of chicken pectoralis myofibrils was not
affected by any of the conditions. In the other myofibrils,
pH 5.4 caused significantly (P < 0.05) more protein denaturation than pH 6.5, and incubation at 40 C resulted in
significantly more protein denaturation than incubation
at 25 C. The presence of ATP (tested at pH 6.5) affected
denaturation; higher ATP concentrations resulted in increased loss of solubility. We concluded that chicken red
myofibrillar proteins are equally susceptible to denaturation as are pork red and white myofibrils. Chicken pectoralis (white) muscle fibers are least susceptible to denaturation. The results of this study indicate that factors other
than protein denaturation are responsible for the low
water-holding capacity of pale, soft, exudative chicken
breast muscle.
(Key words: pale, soft, exudative, protein denaturation, poultry)
2000 Poultry Science 79:105–109
stimulation on drip loss from beef and pork can be explained (Offer, 1991).
The postmortem processes in poultry are similar to
those in mammalian species. However, as the rate of these
processes is faster in poultry than in pork, ultimate pH
values can be achieved within as little as 5 min postmortem (Khan and Nakamura, 1970; Ma et al., 1971; Ma and
Addis, 1973; Kijowski et al., 1982; Addis, 1986). Despite
the fact that poultry muscle has a pH below 6.0 while the
temperature of the carcass is still above 37 C, not all
poultry is pale and exudative. According to Offer (1991),
it is not only the severity of conditions that influences
myosin denaturation but also the time that myosin is
exposed to these conditions that needs to be taken into
consideration. Rigor, i.e., the binding of actin and myosin
to form actomyosin, protects myosin against further denaturation. Thus, it is suggested that the rapid rate of postmortem glycolysis in poultry reduces the sensitivity of
myosin to denaturation because the rapid glycolysis results in a rapid rigor. Although this may explain why
INTRODUCTION
The condition of pale, soft, and exudative (PSE) pork
has been studied extensively (Bendall and Swatland,
1988). The main cause of PSE pork is rapid postmortem
glycolysis, resulting in a low pH (5.4 to 5.6) at a time that
temperature is still high (>35 C). Such conditions cause
protein denaturation, specifically denaturation of myosin.
When myosin denatures, the length of the heads of the
myosin molecule is reduced (Offer and Knight, 1988). As
myosin is attached to actin, shrinkage of the myosin heads
results in a reduction of filament spacing, inducing water
to be expelled from the muscle cells into the extracellular
space (Offer and Knight, 1988).
Offer (1991) developed a model to predict the drip
(water) loss from meat. This model was based on the
assumption that myosin denaturation (causing a reduction in filament spacing) is the primary cause of drip
loss. By using this model, effects of chilling and electrical
Received for publication October 13, 1998.
Accepted for publication September 1, 1999.
1
To whom correspondence should be addressed: Riëtte van Laack,
Department of Food Science and Technology, PO Box 1071, Knoxville,
TN 37901-1071; e-mail: [email protected].
Abbreviation Key: ATP = adenosine triphosphate; EGTA = ethylene
glycol-bis(β-aminoethyl ether)N,N,N′,N′-tetraacetic acid; PSE = pale,
soft, and exudative; PP = Pectoralis profundus; PF = Pubo-ishio femorale;
SM = semimembranosus; PM = Psoas major.
105
106
VAN LAACK AND LANE
most poultry meat does not display PSE, it does not explain why, under certain conditions, poultry does become
PSE. Similar to pork, high temperatures and stress of
poultry result in occurrence of PSE characteristics (Vimini, 1996; Barbut, 1998; Sosnicki et al., 1998).
The fact that, despite a rapid glycolysis and a high
temperature, most poultry does not become PSE, and the
fact that further acceleration of the already rapid postmortem glycolysis does result in PSE, suggests that the model
developed by Offer (1991) is not applicable to poultry. The
myosin isoenzyme of mammalian species is genetically
different from that of avian species (Gauthier and Lowey,
1977, 1979). It may be suggested that different isoforms
of myosin respond differently to denaturing conditions.
The purpose of this study was to determine whether
myofibrillar protein from chicken denatures under similar conditions as myofibrillar protein from pork. In addition, we compared the response of myofibrillar protein
from a mostly red and a mostly white muscle to pH
and temperature.
MATERIALS AND METHODS
Six 42-d-old Arbor Acre chickens were stunned with
an electrical knife and were bled for 2 min. Immediately
after the bleeding, the Pectoralis profundus (PP) and the
Pubo-ishio femorale (PF) were excised.
Six market pigs, three Landrace × Yorkshire and three
Yorkshire × Landrace × Duroc, were slaughtered at the
University Meat Laboratory. After scalding, dehairing,
and evisceration, at about 45 min postmortem, the Semimembranosus (SM) and Psoas major (PM) were excised.
The muscle samples were cut parallel to the muscle
fiber into strips of 2 to 3 in long and 0.5 to 1 in wide.
These strips were tied to a bamboo stick, wrapped in
aluminum foil, and frozen in liquid nitrogen. The frozen
samples were stored at −60 C. When needed, the samples
were thawed overnight in ‘rigor’ buffer of 0 to 2 C, containing 75 mM KCl, 5 mM K-phosphate, 2 mM ethylene
glycol-bis(β-aminoethyl ether)N,N,N′,N′-tetraacetic acid
(EGTA), and 2 mM MgCl2, pH 7.2. This buffer prevents
muscle contraction (EGTA chelates Ca2+) and denaturation (pH 7.2).
After overnight thawing, myofibrils were purified using procedures described by Warner (1994). The protein
concentration of the myofibrillar suspension was determined using the biuret procedure (Gornall et al., 1949)
with bovine serum albumin as standard. Myofibrils (10
mg/mL) were then suspended in one of four incubation
buffers: 1) 0.05 M K-phosphate and 2 mM MgCl2, pH 6.5;
2) same as buffer 1 but pH 5.4; 3) same as buffer 1 but
containing 0.68 mM adenosine triphosphate (ATP); and
4) same as buffer 1 but containing 3.4 mM ATP. The
buffers without ATP (buffers 1 and 2) represented rigor
conditions. The buffers containing ATP represented early
2
Denver Instrumental Company, Denver, CO 80004.
TABLE 1. Myofibrillar protein solubility (mg/mL, least square
means) of myofibrils from pork and chicken muscle after
0 min of incubation
ATP1 concentration (mM)
02
0.683
3.44
Chicken muscle
Pectoralis profundus
Pubo-ishio femorale
2.78a
1.70b
2.79a
1.58b
2.88a
1.63b
Pork muscle
Semimembranosus
Psoas Major
2.36a
2.31a
2.36a
2.27a
2.42a
2.18a
a,b
Means within each column with no common letter differ significantly (P < 0.05).
1
Adenosine triphosphate.
2
SE = 0.19.
3
SE = 0.18.
4
SE = 0.17.
postmortem (0.68 mM ATP) and in vivo (3.4 mM ATP)
conditions.
Myofibrillar suspensions (500-µL portions) were transferred into microfuge tubes. To start incubations, the
tubes were placed in a water bath set at either 25 or 40
C. After 0, 10, 20, or 60 min at these temperatures, two
tubes for each of the four buffers were removed from
the water bath and were placed into an ice-water bath.
Subsequently, 500 µL of ice-cold myosin solubilization
buffer (1.0 M KCl, 0.2 M K phosphate, 2 mM MgCl2, and
20 mM tetrapyrophosphate, pH 6.8) was added. To allow
protein solubilization, the tubes were stored at 0 to 2 C.
The next morning, tubes were centrifuged at 16,000 ×
g for 10 min in a Micro 16 micro centrifuge.2 Protein
concentration of the supernatant was assessed using the
biuret procedure (Gornall et al., 1949) with bovine serum
albumin as standard.
Data were analyzed using PROC MIXED of SAS威 Version 6.11 (SAS, 1996). Split-split plot with pork and
chicken in the whole plot, muscle type in the subplot,
and ATP concentration, pH, and time in the sub-subplot
were analyzed. Each animal was used as a separate meat
source, resulting in six replications. Because of the complexity of the higher order interactions, the subanalyses
were run by levels of various factors. Analysis options
in PROC MIXED were used to preserve the estimated
values of the error terms from the complete analysis.
Fisher’s least significant differences were used to assess
significance of differences between means in pairwise
comparisons. In all cases P < 0.05 was used.
RESULTS AND DISCUSSION
Initial solubilities (0 min of incubation) are presented
in Table 1. Initial solubility of chicken myofibrillar protein
from the white (PP) muscle was greater than of myofibrillar protein from the red (PF) muscle. These results agreed
with those of Cassens and Cooper (1971) and Xiong and
Brekke (1989), who indicated that muscles containing
mostly red myofibrils contain lower amounts of extractable myosin than muscles containing mostly white myo-
107
DENATURATION OF MYOFIBRILLAR PROTEINS FROM CHICKEN
1
TABLE 2. Protein solubility (% of solubility after 0 min
incubation) of myofibrils from pork and chicken muscles
incubated at 40 C and at pH 5.4 or 6.5; effect of
duration of incubation (min)
Incubation time
Muscle
Chicken
Pectoralis profundus
Pubo-ishio femorale
Pork
Semimembranosus
Psoas major
pH
10
20
60
6.5
5.4
6.5
5.4
100
96
97a
79a,2
103
104
93a
73a
101
89
80b
57b
6.5
5.4
6.5
5.4
952
108a
98
94a
87
85b
95
90a
84
70c
89
70b
a,b
Means within each row with no common letter differ significantly
(P < 0.05).
1
Least square means (n = 6), SE = 5.
2
Significantly different from 100 (the initial value after 0 min incubation).
fibrils. In pork, there was no difference in solubility of
myofibrillar protein from white (SM) vs red (PM) muscle
(Table 1). Possibly, there was a smaller difference in fiber
type between these two pork muscles than between the
two chicken muscles; in general, pork muscles are a mixed
type, whereas the chicken PP is almost homogeneously
white (Pearson and Young, 1989).
To determine if there is a difference in rate (or extent)
of denaturation (decrease in solubility), solubility values
were expressed relative to solubility at the start of the
incubation, i.e., the ratio of protein solubility after 0, 10,
20, or 60 min of incubation to protein solubility after 0
min of incubation × 100%. Thus, initial differences in
absolute solubility would not influence the results.
Incubation at 25 C and pH 6.5 did not induce denaturation of the myofibrillar proteins (results not shown). This
treatment was included as a control. In previous studies,
temperatures below 30 C did not cause protein denaturation (Penny, 1967a,b; Offer, 1991).
In myofibrils incubated at 40 C, a progressive decrease
of protein solubility occurred (Tables 2 and 3). Only in
chicken PP, was protein solubility not affected by incuba-
tion at 40 C. Protein denaturation at 40 C was significantly
greater than at 25 C for all treatments, except for chicken
PP (Table 3).
The effects of ATP and pH on denaturation are presented only for 60 min incubation. As can be seen in Table
4, pH 5.4 resulted in more protein denaturation than pH
6.5 in all myofibrils except in the PP myofibrils. Irrespective of pH, solubility of chicken PP myofibrillar protein
did not change significantly.
We tested the denaturation of myofibrillar protein and
the effect of different levels of ATP (Table 5). Early postmortem conditions were simulated by 0.68 mM ATP, and
3.4 mM ATP was used to simulate in vivo conditions.
The ATP can influence protein (myosin) denaturation in
various ways. The presence of ATP will result in muscle
relaxation; cross-bridges between actin and myosin will
be dissociated. Consequently, the presence of ATP may be
expected to result in more protein denaturation (Penny,
1967b; Offer, 1991). Enzymes are more resistant to denaturation in the presence of their substrate (Stryer, 1988).
The ATP is the substrate of myosin and, thus, may be
expected to protect this protein against denaturation. As
can be seen in Table 5, protein solubilities were lower in
the presence of ATP than in the absence of ATP. This
result indicates that the effect of increased exposure of
myosin to denaturation overrides the protective effect of
ATP. Again, protein solubility of chicken PP was not
affected by any of the conditions.
Jacobson and Henderson (1973) reported that myosin
incubated in the presence of 1 mM ATP and 10 mM Ca2+,
pH 7.4, at 45 C did not denature. They determined that
under those conditions (1 mM ATP), the actomyosin complex was not completely dissociated and, consequently,
myosin was protected against denaturation. The discrepancy between Jackson and Henderson (1973) and our
results may be due to a difference in incubation buffer;
in the present study we did not have Ca2+ in the buffer.
The absence of Ca2+ probably resulted in more dissociation and, thus, increased exposure of myosin to denaturing conditions.
The results of the present study indicate that chicken
PF and pork SM and PM myofibrillar proteins are equally
sensitive to denaturation. The function of the red muscle
TABLE 3. Protein solubility1 (% of initial solubility) of myofibrils from pork and chicken muscles
after 60 min incubation; the effect of temperature 25 vs 40 C
Condition 1
Chicken muscle
Pectoralis profundus
Pubo-ishio femorale
Pork muscle
Semimembranosus
Psoas Major
Condition 2
Condition 3
Condition 4
25 C
40 C
25 C
40 d
25 C
40 C
25 C
40 C
103a
91a
89b
57b
97
98a
101
80b
102a
99a
98b
72b
99a
85a
90b
64b
100a
89a
70b
70b
96
98a
84
89b
90a
93
70b
82
60
61
56
66
Means within each treatment with no common letter differ significantly (P < 0.05).
Least square means (n = 6), SE = 5.
2
Condition 1: 0 mM adenosine triphosphate (ATP), pH 5.4; Condition 2: 0 mM ATP, pH 6.5; Condition 3:
0.68 mM ATP, pH 6.5; and Condition 4: 3.4 mM ATP pH 6.5.
a,b
1
108
VAN LAACK AND LANE
1
TABLE 4. Effect of pH on protein solubility (% of initial
solubility) of myofibrils from chicken and pork
muscles incubated for 60 min at 40 C
pH
Chicken muscle
Pectoralus profundus
Pubo-ishio femorale
Pork muscle
Semimembranosus
Psoas major
6.5
5.4
101
80a
89
57b
84a
89a
70b
70b
a,b
Means within each treatment with no common letter differ significantly (P < 0.05).
1
Least square means (n = 6), SE = 5.
fibers (such as PM and PF) is to perform repetitive actions
for long periods of time. White muscles (such as SM and
PP) produce fast, high-energy repetitions over a short
period of time. Because of their oxidative metabolism,
rigor onset in red fibers is rapid, and the ultimate pH
will be higher than in white fibers. In white fibers, with
a glycolytic metabolism, postmortem glycolysis continues
longer, and the ultimate pH will be lower than in red
fibers. Although proteins are equally sensitive to certain
denaturing conditions, denaturation of proteins in red
fibers is limited; red muscle fibers enter rigor rapidly
and have a higher ultimate pH. Consequently, protein
denaturation will be limited.
Proteins in chicken white myofibrils (PP) seem more
resistant to denaturation than the proteins from the
chicken red myofibrils (PF) and those from pork myofibrils. It may be suggested that during collection of the
samples from the chicken pectoralis muscle, proteins denatured completely. In that situation, any further denaturation during incubation would not be noticeable or would
not occur; however, the high initial solubility of proteins
from the PP muscle (Table 1) indicates that this was not
the case. Thus, it can be concluded from the present results that proteins from chicken PP are more resistant to
denaturation than proteins from pork muscles or those
from red chicken muscle. Possibly, this resistance is re-
TABLE 5. Influence of adenosine triphosphate (ATP) concentration
on protein solubility1 (% of initial solubility) of myofibrils from
pork and chicken, incubated for 60 min at 40 C at pH 6.5
ATP concentration (mM)
Chicken muscle
Pectoralis profundus
Pubo-ishio femorale
Pork muscle
Semimembranosus
Psoas major
0
0.68
3.4
101w
80a,x
98w
72ab,x
90w
64b,x
84a,x
89a,x
70b,x
82a,x
56c,x
66b,x
a,b
Means within each row with no common letter differ significantly
(P < 0.05).
w,x
Means within each column with no common letter differ significantly (P < 0.05).
1
Least square means (n = 6), SE = 5.
lated to the genetic makeup of the avian myofibrillar
protein. According to Moore et al. (1993), genetic make up
of avian muscle is sufficiently different from mammalian
muscle to cause differences in biochemical properties.
Results of the present study are similar and in
agreement with those of Xiong et al. (1987). Using differential scanning calorimetry, they determined that denaturation of myofibrillar protein from chicken pectoralis muscle occurred at 60.8 C, whereas proteins from pork longissimus muscle occurred at 58.5 C.
CONCLUSIONS
The model proposed by Offer (1991) predicts the formation of PSE by using kinetics of denaturation of myosin
as a guide. The results of the present study indicate that
myosin from chicken pectoralis muscle is resistant to denaturation, suggesting that the PSE quality in chicken is
different from that in pork (in which it is associated with
protein denaturation).
Based upon the results of the present study, it seems
unlikely that myosin denaturation is the cause of PSE in
chicken. Further research on the cause of the low waterholding capacity and the pale color of PSE chicken is
needed.
ACKNOWLEDGMENTS
The chickens in this study were generously donated
by Burnett Poultry, Morristown, TN. The advise and
guidance by Clark J. Brekke and H. Dwight Loveday
and the technical assistance of Emayet Spencer, George
Wilson, and Salena Stevens is greatly appreciated.
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