Ultrasonication of Chicken Natural Actomyosin: Effect on ATPase Activity, Turbidity and SDS-PAGE Profiles at Different Protein Concentrations
With the increasing application of ultrasonics in meat tenderization and processing,
physicochemical events initiated by sonic radiation at myofibrillar level and
propagated in complex tissue such as meat require a clear understanding. The
enormous amount of basic information collected by studying myofibrils, actomyosin
or their individual constituents has already clarified intricacies of muscular
contraction and, part of such basic information has find application in meat
sciences. In this investigation, chicken Natural actomyosin (NAM) has been taken
as a simple model to work out some effects of ultrasonication in a concentration
range of 0.5 to 1.8 mg protein mL-1. At each concentration, NAM solution
in 0.6 M NaCl (2.0 mL) was individually exposed to 20 kHz sonic waves for a
total of 10 min. Cooling was maintained by keeping NAM containers in crushed
ice and a lag of 5 sec after each 10 sec long sonic burst. Aliquots from each
sonicated NAM were subjected to biochemical analyses. Most striking differences
were observed in Ca2+-ATPase activity, which displayed a steady decline
that corresponded with the decreasing protein concentration. Ultrasonication
of NAM for 10 min caused a loss of ~47% of Ca2+-ATPase activity at
the highest dilution (0.5 mg mL-1). In the same order of protein
concentration, turbidity of ultrasonicated NAM also decreased which denotes
increasing transparency. Thus, ATPase and turbidity data demonstrate that due
to sonic radiation, interactions among constituents of chicken actomyosin complex
alter and these structural changes are devoid of any fragmentation. Under present
experimental conditions, SDS-PAGE profiles did not reveal any novel band which
could be attributed to ultrasonic fragmentation or proteolytic contamination.
The findings also suggest that unlike myofibrils, actomyosin is a model where
interactions and substructural changes of constituent polypeptides can be investigated
without interference of endogenous muscle proteases.
Received: August 11, 2012;
Accepted: October 10, 2012;
Published: April 18, 2013
High intensity ultrasonication is getting increasing application in various
food technologies and several reviews are available on this subject (Dolatowski
et al., 2007; Chemat et al., 2011).
In meat industry, sonic radiation is being used for curing (Siro
et al., 2009) and tenderizing carcass meat (Lyng
et al., 1997; Got et al., 1999; Jayasooriya
et al., 2004). The impact of ultrasonication on meat involves generation
of heat by pressure waves, changes in pH or color and water holding capacity
as well as substructural disorganization (Latoch, 2010).
Even fragmentation or solubilization of some of the structural proteins might
take place in majority of meat types (Ito et al.,
2003; Stadnik et al., 2008). At high intensity
sonic radiation, microstructure of collagen fibers might get severely altered
and products be spilled over to occupy intracellular spaces (Chang
et al., 2012). Aging and endogenous proteases, released during lysosomal
disruption, are inherent participants in tenderization process attributed to
fragmentation of various structural proteins (Jayasooriya
et al., 2007).
A problem associated with meat of higher vertebrates is the substantial amount
of collagen network. Chicken meat has quite low collagen contents and chicken
myofibrillar proteins have been successfully solubilized by ultrasonication
at 20 kHz for 10 min (Ito et al., 2004). Natural
actomyosin (NAM) is the contractile complex that constitutes bulk of muscle
mass extractable in high salt solutions, either directly from muscle mince or
myofibrillar preparations. SDS-PAGE profiles of chicken NAM have so far revealed
no contamination originating from collagen. NAM thus represents the principal
contractile mass of myofibrillar proteins in vitro. Because of the above
mentioned advantages, chicken Natural actomyosin (NAM) has been chosen here
as the model to initially investigate some of the biochemical events after exposure
to 20 kHz sonic radiation for 10 min. Virtually no information has been published
on isolated NAM till now. The investigation takes in to account protein concentration
dependence as the variable of the sonication-induced changes in Ca2+-ATPase,
turbidity and SDS-PAGE profiles. The selected parameters reflect changes in
actin-myosin interaction, dissociability or aggregation of the complex and substructural
MATERIALS AND METHODS
Location of studies and design of research plan: The experiments were carried
out at Zoology Department of Aligarh Muslim University, Aligarh (Uttar Pradesh,
India) during the last one year. The proposal originated from a need to elucidate
biochemical events at the basic level of organization of contractile complex
(natural actomyosin, NAM) without interference from lysosomal proteases and
other myofibrillar proteins of chicken skeletal muscle. It was envisaged that
Ca2+ activated ATPase, turbidity measurements and polypeptide composition
on SDS-PAGE profiles will provide selective information on sonic radiation-induced
changes in chicken NAM, in terms of actin-myosin interaction and structural
changes or fragmentation.
Source of chemicals: All the chemicals and reagents used in this study
were of analytical grade. Acrylamide, bis-acrylamide, Phenyl methane sulfonylfluoride
(PMSF), Adenosine 5-triphosphate
(ATP) disodium salt, ammonium per sulphate and TEMED were procured from authorized
dealers of Sigma-Aldrich Chemicals Pvt. Ltd. (USA) in India. Sodium chloride,
1-amino-2-naphthol-4-sulphonic acid, bovine serum albumin, Tris buffer and all
other reagents were purchased from SRL Chemicals, India.
Source of animal and muscle type: Pectoralis major from the breast
muscle mass of the broilers of 3 month age were dissected out post-sacrifice
and immersed in ice bath. Muscle was chopped finely and washed 3 times with
several volumes of 5 mM phosphate buffer (pH 7.0) containing 2 mM PMSF before
proceeding for actomyosin extraction.
Extraction and purification of actomyosin: Natural actomyosin (NAM)
from the washed pellet obtained as above was extracted as described previously
(Ahmad and Hasnain, 2006; Hasnain
and Ahmad, 2006) with additional step of eliminating contamination of free
myosin. Briefly, the pellet of muscle mince was suspended in extraction buffer
(0.45 M KCl containing 25 mM phosphate buffer, pH 7.0). After gentle mixing
and overnight storage under crushed ice, viscous NAM was centrifuged at 10 K
rpm (4°C) and precipitated by 10-fold dilution with chilled distilled water.
Pellet was saved and dissolved in solvent buffer (0.6 M NaCl with 20 mM Tris-maleate,
pH 7.0). Traces of free myosin were removed by washing two times with 0.2 M
NaCl. It was followed by two cycles of dissolving NAM pellet in solvent buffer
and precipitation with distilled water to 0.06 M NaCl, to eliminate trace low
ionic strength salt soluble impurities. Finally, NAM dissolved in solvent buffer
was dialyzed overnight against the solvent buffer, cleared by centrifugation
at 10 K rpm (4°C) and stored under crushed ice during investigations which
were completed within 24 h.
Protein estimation: Protein concentration was determined by Biuret method
of Gornall et al. (1949) using bovine serum albumin
as standard. Three dilutions (0.5, 1.3 and 1.8 mg mL-1) of NAM were
subjected to ultrasonication and subsequent biochemical analyses. All dilutions
were made in NAM solvent buffer (0.6 M NaCl with 20 mM Tris-maleate, pH 7.0).
Ultrasonic treatment: Ultrasonication was performed using Ralco immersible-probe
ultrasonicator for a total of 10 min at 20 kHz. During ultrasonic treatment,
the NAM containing glass beaker was kept in ice bath intervened by cooling lags
of 5 sec after each 10 sec of ultrasonic burst. The duration of 10 min is the
sum of only the ultrasonication bursts of 10 sec and does not include cooling
intervals of 5 sec. Samples were subjected to biochemical analyses immediately.
Ca2+-ATPase assay and electrophoretic profiling: Ca2+-ATPase
was assayed at 20°C in final concentrations of 50 mM NaCl, 20 mM Tris-maleate
of pH 7.0, 5 mM CaCl2 and 1 mM ATP and, the liberated Pi measured
as reported by Hasnain et al. (1979). Turbidity
was monitored at 340 nm using UV-VIS 118 Spectrophotometer (Systronics). SDS-PAGE
in 12% gels was performed essentially as described previously (Ahmad
et al., 2012). Following overnight fixing and washing in 10% acetic
acid-5% methanol, protein bands were visualized by staining with Coomassie Brilliant
Blue R-250. Background was cleared by washing in 7% acetic acid.
Documentation and densitometry: Stained SDS PA-gels were documented
at different contrasts using digital camera (SONYCYBERSHOT: Zoom-4X, 12 Megapixels)
and by scanning on an all-in-one HP Deskjet (F370) computer setup. Densitometry
of the gel-lanes was carried out using Scion Imaging (Scion Corporation; Beta
release, 4.0) and GelPro (Media Cybernetics, USA) software programs.
Figure 1 demonstrates the effect of ultrasonication (20 kHz)
on chicken NAM at the three concentrations. In comparison with the control (unsonicated
NAM), sonication for 10 min resulted in a loss of ~23%, ~19% and ~14% Ca2+-ATPase
activity at protein concentrations of 1.8, 1.3 and 0.5, respectively. Turbidity
measurements at these NAM concentrations showed a decline of 10, 25, 50%, respectively,
indicating increasing transparency subsequent to sonication (Fig.
SDS-PAGE profiles of ultrasonicated chicken NAM along with the control are
shown in Fig. 3a. Each lane displays the same number of polypeptides
with characteristic molecular weights of 200, 46 and 39 kDa for myosin heavy
chain, actin and tropomyosin, respectively.
||Protein concentration dependence of ultrasonication on Ca2+-ATPase
activity of chicken natural actomyosin (NAM), Unsonicated NAM has been taken
as the control
||Sonication induced decrease in turbidity of chicken natural
actomyosin (NAM) as a function of decreasing protein concentration
|| (a) SDS-PAGE profiles of chicken natural actomyosin (NAM)
control and sonicated dilutions, Chicken control polypeptides were taken
as the markers, Established molecular weights (Mr) of myosin
heavy chain, actin, troponin-I, tropomyosin and light chains are labelled
on the left of the profiles, (b) Densitometric tracings of the four lanes
of Fig. 3a in the same order, The scans display only those
polypeptides which stack below actin
Polypeptides below tropomyosin are troponin-I (23 kDa) and troponin-C (19 kDa)
and the rest (25, 20 and 16 kDa) myosin light chains. No novel band was visualized.
The relative intensities of these bands correspond to the increasing concentrations
of NAM from 0.5 to 1.8 mg mL-1. Densitometry of the polypeptides
stacking below the actin (46 kDa band) also does not reveal the presence of
any new band and accords with the protein dilutions (Fig. 3b).
Scan of the portion of gels above the actin band was kinky due to background
and has, therefore, been excluded. However, as the Fig. 3
shows there is no apparent cleavage in the main band of myosin heavy chain.
Reports which described fragmentation of isolated myosin and heavy meromyosin
(HMM) by ultrasonication are rather old (Barany et al.,
1963a, b). Isolated actin, the other integral protein
component of actomyosin complex is a thin filament protein of myofibrils and
its G-monomer does not fragment under sonication (Asakura,
1961; Asakura et al., 1963). Its fibrous
polymer (F-actin) fragments under ultrasonic influence; but, the fragments polymerize
into larger than the usual polymers (Asakura et al.,
1963; Carlier et al., 1985). In an earlier
study, ATP hydrolysis by actin and the polymerization was shown to display veritable
responses to millimolar vs. micromolar concentrations of calcium (Dancker
and Low, 1977). Anyhow, the above literature deals with isolated actin and
not as a constituent of natural actomyosin complex. Whereas even F-actin polymer
will stack as a single band in SDS-PAGE due to dissociation by SDS, fragmentation
of myosin can be reliably monitored by this technique, though the number and
intensity of novel bands depend on a multitude of factors. Another problem with
conventionally prepared tryptic subfragments of myosin (e.g., HMM) is that they
are already heterogenous in gel profiles (Samejima et
al., 1976). More stable and homogenous myosin subfragments are generated
by other enzymes and chemical cleavages (Samejima et
Therefore, with the objective to understand the basics of the mechanism of
ultrasonic effects, natural actomyosin was chosen as a model that emulates conditions
within the myofibrils. Actomyosin is the contractile proteins complex that constitutes
the bulk of myofibrillar proteins and muscle mass. It was perceived that Natural
actomyosin (NAM) is a model wherein the impact of ultrasonication can be directly
observed in terms of ATPase activities, interactions of actin with myosin along
with regulatory proteins (troponins and tropomyosin) or overall aggregation,
fragmentation or dissolution behavior. As huge amount of literature suggests,
SDS-PAGE provides a direct look into fragmentation and several of the events
related with the above aspects. Use of actomyosin also eliminates fragmentation
of myosin and associated proteins by intrinsic proteases which may be released
or activated post mortem (Koohmaraie and Geesink, 2006;
Goll et al., 2008). The use of fresh actomyosin
is also recommended, since myosin heavy chain may get fragmented even in highly
purified myosin preparation when aged and kept at warm temperature (Hasnain,
The results of this study (Fig. 1) demonstrate that after
10 min of ultrasonication and irrespective of the nature of other changes in
NAM, Ca2+-ATPase declines. The data also suggests that the magnitude
of ultrasonication effect would be protein concentration dependent and, therefore,
protein concentration variables should be carefully considered for a meaningful
comparative study. An initial activation of Ca2+-ATPase of avian
myofibrils was reported by Talesara and Narang (1977),
however, under present conditions no such activation was observed for chicken
NAM. No fragmentation was visualized in SDS-PAGE profiles also, that is in agreement
with those obtained even on meat slices (Lyng et al.,
1997, 1998). However, a clearing effect, as apparent
by reduction in turbidity, suggests change in interactions of NAM constituents,
in particular actin-myosin interaction as initially suggested by decline in
Since conformation of actin as well as myosin undergoes changes under certain
conditions (Asakura et al., 1963; Hayakawa
et al., 2010), the state of either of these proteins can potentially
alter the actin-myosin interaction. The later authors have shown that myosin
can be made soluble at low ionic strength solutions instead of normal 0.2 M
salts (Hayakawa et al., 2009). More importantly,
the conformation dependent size variation within a portion of myosin rod can
depolymerize myosin filaments (Hayakawa et al., 2010).
The important role played by myosin tail domain has been emphasized in non-muscle
myosin, as well (Guthrie, 2012). It is well established
that subsequent to mixing myosin with actin, an abrupt increase in turbidity
occurs due to actomyosin complex formation and the salt concentration to keep
actomyosin in solution is 0.6 M KCl. In contrast, individual preparations of
actin as well as myosin are transparent and have lower salt solubility characteristics.
Therefore, a conformation dependent change in the solubility profiles of actin
and myosin will eventually affect the nature of actomyosin complex and, in turn,
ATPase and other properties of such a complex. It is likely that some of the
changes in interaction of actomyosin (cell-free system) and myofibrillar proteins
(myofibril-trapped actomyosin) due to ultrasonic effect may be common in some
of details; but they may differ in several other respects, such as the action
and consequences of intracellular proteases.
The results of this study suggest that sonication of chicken Natural actomyosin
(NAM) at 20 kHz for 10 min causes no fragmentation of constituent polypeptides.
Rather a reduction in actin-myosin interaction (dissociation) occurs that is
evident from reduced turbidity which is a measure of increasing transparency.
The remarkable decline in Ca2+-ATPase appears to be the consequence
of actin-myosin dissociation. The findings support use of actomyosin as a simple
cell-free model to follow ultrasonication-induced changes which initiate in
the contractile apparatus and subsequently affect state of muscle. The change
in investigated properties is protein concentration dependent, suggesting caution
in comparative studies.
Corresponding author is grateful to the University Grants Commission (UGC),
New Delhi for funding this work. Authors are obliged to the University and the
Chairman, Department of Zoology for laboratory facilities and to Prof. Abbas
Abidi for his help. We also acknowledge with thanks the technical support by
Miss Areeba Ahmad.
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