- Email: [email protected]
Heat-induced formation of myosin oligomer-soluble filament complex in high-salt solution
International Journal of Biological Macromolecules 73 (2015) 17–22
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Heat-induced formation of myosin oligomer-soluble filament complex in high-salt solution Masato Shimada a,1 , Eisuke Takai a,1 , Daisuke Ejima b , Tsutomu Arakawa c , Kentaro Shiraki a,∗ a
Faculty of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8573, Japan AminoScience Laboratories, Ajinomoto Inc., 1-1 Kawasaki, Japan c Alliance Protein Laboratories, San Diego, CA 92121, USA b
a r t i c l e
i n f o
Article history: Received 29 August 2014 Received in revised form 17 October 2014 Accepted 7 November 2014 Available online 15 November 2014 Keywords: Myosin Dynamic light scattering Filament formation Thermal aggregation Heat-induced gelation
a b s t r a c t Heat-induced aggregation of myosin into an elastic gel plays an important role in the water-holding capacity and texture of meat products. Here, we investigated thermal aggregation of porcine myosin in high-salt solution over a wide temperature range by dynamic light scattering experiments. The myosin samples were readily dissolved in 1.0 M NaCl at 25 ◦ C followed by dilution into various salt concentrations. The diluted solutions consistently contained both myosin monomers and soluble filaments. The filament size decreased with increasing salt concentration and temperature. High temperatures above Tm led to at least partial dissociation of soluble filaments and thermal unfolding, resulting in the formation of soluble oligomers and binding to the persistently present soluble filaments. Such a complex formation between the oligomers and filaments has never been observed. Our results provide new insight into the heat-induced myosin gelation in high-salt solution. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Myosin is a major muscle protein. The myosin molecule has an asymmetric structure with two globular heads and a rod-like tail. It associates into insoluble filaments through electrostatic tail-totail interaction under physiological conditions [1,2]. Accordingly, disruption of electrostatic interactions by high salt concentration, e.g., above 0.3 M, leads to reversible dissociation into monomers, which are in equilibrium with varying sizes of associated species, e.g., soluble oligomers, as determined by differential velocity sedimentation [3,4] or flow birefringence and electron microscopy [5,6]. Temperature also affects self-association of dissociated myosin monomers or oligomers. When heated in salt solution, myosin monomers and oligomers are denatured and aggregate into an elastic gel, which plays an important role in the water-holding capacity and texture of myosin-containing food products [7–9]. The heat-induced myosin gels are classified into two types: the aggregate-type gel in high-salt solution [10] and the strand-type gel in low-salt solution [11]. The dependence of heat-induced myosin
∗ Corresponding author. Tel.: +81 29 8535306; fax: +81 29 8535215. E-mail address: [email protected] (K. Shiraki). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ijbiomac.2014.11.005 0141-8130/© 2014 Elsevier B.V. All rights reserved.
gel formation on salt concentration is attributed to the association state of myosin. Thus, it is likely that gel formation of myosin by heating is closely linked to association states of myosin in solution as a function of temperature and salt concentration. This study was performed to investigate the size and structure of myosin under different solution conditions.
2. Materials and methods 2.1. Purification of myosin Porcine myosin extract was prepared as follows [12]. The myosin used in this study was extracted from the lean portion of porcine inner thigh meat. A sample of 250 g of the starting meat was suspended in 450 ml of buffer containing 0.10 M pyrophosphate and 5.0 mM MgCl2 (pH 7.0) and homogenized at 5 ◦ C for 60 min. The resultant myosin extract suspension was stored frozen at −25 ◦ C. After thawing, the myosin extract was dialyzed against 1.0 mM KCl and 2.0 mM sodium phosphate buffer (pH 7.0), resulting in myosin precipitation. The precipitates were washed with 1.0 mM KCl and 2.0 mM sodium phosphate buffer (pH 7.0) and stored frozen. The purity of the myosin extract was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Aliquots of the sample were dissolved in reducing sample buffer
18
M. Shimada et al. / International Journal of Biological Macromolecules 73 (2015) 17–22
containing 2% (w/v) SDS, 10% glycerol, 0.04 M DTT, 0.01% (w/v) bromophenol blue, and 62.5 mM Tris–HCl (pH 6.8). The samples were boiled for 5 min and loaded onto 10% polyacrylamide gels. 2.2. Preparation of myosin solution The frozen precipitate prepared above was used as a starting material of purified myosin, which was dissolved with 1.0 M NaCl and 20 mM sodium phosphate buffer (pH 7.5), i.e., at high salt concentration. After incubation for 1 h at 25 ◦ C, the 1.0 M NaCl solution containing soluble myosin was centrifuged at 18,800 × g for 20 min. The supernatants were diluted with 20 mM sodium phosphate buffer (pH 7.5) containing 0.20–1.0 M NaCl to generate a solution containing an appropriate concentration of myosin and different concentrations of NaCl for various solution measurements. The diluted myosin solutions were incubated at room temperature for 1 h and then centrifuged. The myosin concentrations in the supernatant were determined by the bicinchoninate method [13].
Fig. 1. Solubility of myosin in the presence of 0.20–1.0 M NaCl in 20 mM sodium phosphate buffer (pH 7.5) at 25 ◦ C. (Inset) Time course of changes in myosin concentration in 0.20–1.0 M NaCl solution.
2.3. DLS analysis of myosin Dynamic light scattering (DLS) experiments were performed using a light scattering photometer (Zetasizer Nano ZS; Malvern Instruments, Worcestershire, UK) equipped with a 4 mW He–Ne ion laser ( = 633 nm). Data analysis was performed as reported previously [14]. The size of myosin in aqueous salt solutions was determined as follows. All buffer and salt solutions used to make myosin samples were filtered through 0.4-m filter. The myosin solutions at 0.50 mg/ml in 20 mM sodium phosphate (pH 7.5) containing 0.30, 0.50, 0.80, and 1.0 M NaCl were placed, immediately after sample preparation, in a 1-cm path-length quartz cuvette and subjected to DLS measurement at different temperatures (25 ◦ C, 35 ◦ C, 45 ◦ C, 50 ◦ C, 55 ◦ C, 65 ◦ C, and 75 ◦ C) at a detection angle of 173◦ . No apparent turbidity or precipitation was observed before and after the DLS experiments. 2.3.1. CD for thermal unfolding of myosin Circular dichroism (CD) spectra were measured using a spectropolarimeter J-720W (Japan Spectroscopic Co., Ltd., Tokyo, Japan). A 0.30 mg/ml myosin solution in 0.30 M NaCl and 20 mM sodium phosphate buffer (pH 7.5) was transferred to a quartz cell with a 1-mm path length and subjected to CD measurements at a scan rate of 100 nm/min. Thermal unfolding was measured by following the CD intensity at 222 nm with an increasing temperature rate of 1.0 ◦ C/min. The thermal unfolding transition curve was analyzed using a van’t Hoff plot assuming a two-state transition and linear baselines for native and unfolded states, from which the apparent mid-transition temperature (Tm ) was determined [15], though the thermal unfolding of myosin may be more complex than two-state transition. The composition of ␣-helix and -sheet was estimated by using K2D3 method [16]. 2.4. TEM observation of myosin Transmission electron microscopy (TEM) of myosin in aqueous salt solution was performed with an acceleration voltage of 80 kV (JEM-1400; JEOL, Tokyo, Japan). Samples of 0.50 mg/ml myosin in 0.30 M NaCl, 20 mM sodium phosphate buffer (pH 7.5) were incubated at 25 ◦ C or 55 ◦ C for 90 min. Aliquots of 2 l of the above sample solution were placed on a 150-mesh copper grid covered with a carbon-coated hydrophilic film, to which 10 volumes of pure water were added. The samples were then negatively stained with 2 l of 2% (w/v) silicotungstic acid solution. The solution of the grid was dried for several minutes.
Fig. 2. Size distributions of soluble myosin in 0.30 M (solid lines) or 1.0 M (dot lines) NaCl, 20 mM sodium phosphate buffer (pH 7.5) monitored by DLS at 25 ◦ C.
3. Results Insoluble myosin filaments prepared from porcine inner thigh meat are expected to dissociate into monomers at high salt concentration, i.e., above 0.3 M, resulting in soluble myosin solutions. We determined the solubility of myosin at 25 ◦ C as a function of salt concentration. Fig. 1 shows the solubility of myosin in 0.20–1.0 M NaCl after incubation for 1 h at 25 ◦ C. The myosin concentration in the supernatant increased sharply with increasing NaCl concentration from 0.20 M to 0.25 M, consistent with the previous report [17] and reached a plateau at 0.25 M, above which the solubility increased only slightly. The myosin concentrations of the supernatant in the presence of NaCl at or above 0.20 M changed little over incubation for 1–48 h at 25 ◦ C (see Fig. 1 inset): the myosin concentrations remained constant around 0.25–0.275 mg/ml. These results suggest that the myosin solution was stable over the course of the experiments. The associated state of myosin molecules at high salt concentrations was investigated by DLS. Fig. 2 shows the size distribution of ∼0.30 mg/ml myosin in 0.30 M NaCl (solid line) and 1.0 M NaCl (dotted line) at 25 ◦ C. The distribution was expressed as volume percentage calculated from the hydrodynamic radius based on the assumption of the spherical shape of myosin in these solutions. These samples were prepared from the supernatant of myosin solubilized by 1.0 M NaCl (pH 7.5) followed by dilution in buffer solution containing the indicated NaCl concentration. Two peaks at 18.9 and
M. Shimada et al. / International Journal of Biological Macromolecules 73 (2015) 17–22
19
Fig. 3. Dependence of myosin monomer and filament sizes on salt concentration and temperature. (a) NaCl dependence of soluble myosin filament (circles) and monomer (triangles) at 25 ◦ C (closed symbols) and 55 ◦ C (open symbols). (b) Temperature dependence of soluble myosin filament (circles) and monomer (triangles) in 0.30 M (closed symbols) and 1.0 M (open symbols) NaCl.
198 nm were observed for myosin in 0.30 M NaCl solution and may be assigned as myosin monomers and soluble filaments, respectively. Although it is not possible to assign the myosin structure based on DLS experiments, the term “soluble filament” is used based on TEM observations described below. Two peaks at 14.1 and 127 nm were observed for myosin in 1.0 M NaCl solution, also likely corresponding to myosin monomers and soluble filaments, respectively. These results indicated that soluble myosin solution is not homogeneous with monomeric species and contains significant amounts of soluble filaments at high salt concentrations even at 25 ◦ C. The hydrodynamic radius of myosin was investigated at various NaCl concentrations and temperatures. Fig. 3a shows the hydrodynamic radius of myosin as a function of NaCl concentration at 25 ◦ C and 55 ◦ C. Myosin monomer aggregates into soluble oligomers through myosin head–head interactions above 55 ◦ C in the presence of salt. The size of the myosin monomer depended little on salt concentration at both 25 ◦ C (closed triangles) and 55 ◦ C (open triangles). The size of the soluble myosin filaments also depended little on salt concentration at 55 ◦ C (open circles). In contrast, the size of soluble myosin filaments at 25 ◦ C (closed circles) decreased gradually with increasing salt concentration, e.g., from 180 nm at 0.30 M to 130 nm at 1.0 M, suggesting disassembly of self-associated myosin filaments. Fig. 3b shows the temperature dependence of myosin monomers and soluble filaments at 0.3 and 1.0 M NaCl. The size of the myosin monomers at both 0.30 M NaCl (closed triangles) and 1.0 M NaCl (open triangles) decreased slightly with increasing temperature, i.e., from 40 nm at 25 ◦ C to 25 nm at 75 ◦ C. Such a decrease in size of the monomer peak may simply have been due to temperature-dependent viscosity change. In contrast, the size of soluble myosin filaments decreased sharply with increasing temperature between 25 ◦ C and 45 ◦ C following a slight increase at lower temperature at both 0.30 M NaCl (closed circles) and 1.0 M NaCl (open circles). This sharp drop in the hydrodynamic size of the filaments was followed by a gradual decrease above 45 ◦ C. It should be noted that the size of the filaments approached the level of the monomer at 75 ◦ C. These data indicated that the size of myosin filaments depends on both temperature and NaCl concentration, and higher temperature causes dissociation of selfassociated soluble myosin filaments. DLS experiments were carried out at four different salt concentrations and seven different temperatures. Under each set of conditions, the hydrodynamic radius range of the soluble filaments was determined and plotted as a phase diagram, i.e., the size range of soluble myosin filaments as a function of salt concentration and temperature; data related to the monomer peak were ignored
(Fig. 4). It is clear that hydrodynamic radius of soluble filaments decreased monotonically with increasing temperature and NaCl concentration within time frame of DLS experiments, i.e., 1 min. It appears that heat-induced dissociation of myosin filaments play a major role in reduction of hydrodynamic radius under such a short incubation time and a low protein concentration, 0.50 mg/ml. The size of the soluble filaments showed a maximum at about 0.30 M NaCl and 35 ◦ C and a minimum at about 0.80 M NaCl and 75 ◦ C. As shown in Fig. 3a, the hydrodynamic size of the soluble filaments was relatively constant at 55 ◦ C over the salt concentration range from 0.30 to 1.0 M. The phase diagram showed that the hydrodynamic size was fairly constant on salt concentration between 40 ◦ C and 60 ◦ C. Below and above these temperatures, the filament size became dependent on the salt concentration. In contrast, the filament size was dependent on temperature at all salt concentrations examined, more so at lower and higher salt concentrations. The stability of soluble filaments was investigated at different salt concentrations and temperatures. Fig. 5 shows the time course of changes in hydrodynamic radius of soluble filaments. Fig. 5a shows the time course of changes in filament size at 25 ◦ C in the presence of 0.30 M (black) and 1.0 M NaCl (red). At this temperature, the myosin filaments were stable over 90-min incubation at both salt concentrations. Fig. 5b shows the time course at 45 ◦ C in the presence of 0.50 M NaCl. A gradual decrease in filament size was observed with time. In contrast, the hydrodynamic size increased sharply with time at 55 ◦ C in the presence of 0.30 M NaCl (Fig. 5c). The hydrodynamic size changed linearly with time at least under certain conditions. The rate of change was determined at different temperatures and salt concentrations, from which a phase diagram
Fig. 4. Phase diagram of soluble myosin filament size in 0.30–1.0 M NaCl at 25–75 ◦ C.
20
M. Shimada et al. / International Journal of Biological Macromolecules 73 (2015) 17–22
Fig. 5. Representative data for hydrodynamic radius of myosin filament. (a) 0.30 M NaCl (black) or 1.0 M NaCl (red) at 25 ◦ C. (b) 0.50 M NaCl at 45 ◦ C. (c) 0.30 M NaCl at 55 ◦ C. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
was constructed (Fig. 6). At 25 ◦ C, the filaments were stable for over 90 min of incubation at all salt concentrations examined, as shown in Fig. 5a. At 35–45 ◦ C, the slope of the time course curve was negative at all salt concentrations examined, as shown Fig. 5b. Above 50 ◦ C, the slope of the time course curve was positive, especially at 0.30 M NaCl and 60 ◦ C, as shown in Fig. 5c. The temperature dependence of the filamentous myosin hydrodynamic radius was observed at all salt concentrations examined (Fig. 4). Whether such changes in size are associated with structural changes was examined by far-UV CD spectroscopy. Fig. 7
Fig. 6. Phase diagram of rate of change in hydrodynamic radius of soluble myosin filament during 90-min incubation.
shows the CD signal at 222 nm, which began to increase around 35 ◦ C followed by a sharp increase up to 55 ◦ C and a plateau above 60 ◦ C. The midpoint temperature was estimated to be ∼46 ◦ C. Fig. 7 (inset) shows the far UV CD spectrum at 25 ◦ C characterized by double minima for ␣-helix and the spectrum at 98 ◦ C with a 217 nm minimum for -sheet structure. Thus, the thermal unfolding was characterized as a structure change from ␣ to  transition. As temperature increased from 25 ◦ C to 98 ◦ C, apparent ␣-helix content decreased from 77% to 13% and apparent -sheet content increased from 0% to 24%. Comparison of Fig. 4 and the CD melting curve suggested that the observed decrease in hydrodynamic size was due to thermal unfolding of myosin molecules accompanied by dissociation of soluble ␣-helical myosin filaments. Above 55 ◦ C, the filament size was less dependent on temperature at all salt concentrations examined, possibly due to complete unfolding at this temperature. However, a strong dependence of size on incubation temperature was observed above 50 ◦ C (Fig. 6), most likely reflecting the tendency of the unfolded -structure myosin to aggregate upon incubation. To examine the protein structure, myosin solution containing 0.30 M NaCl was incubated at 25 ◦ C or 55 ◦ C for 90 min and examined by TEM. When incubated at 25 ◦ C in 0.3 M NaCl, myosin was observed mostly as monomers (Fig. 8a), along with soluble filaments (Fig. 8b). The soluble filaments were 600 nm in length and about 10 nm in thickness (Fig. 8b). When incubated at 55 ◦ C, typical protein aggregates composed of unfolded myosin were observed as soluble oligomers (Fig. 8c). In addition, soluble filaments 500 nm in length were observed (Fig. 8d). Interestingly, the soluble oligomers formed complexes with the soluble filaments (Fig. 8d), consistent with the sharp increase in hydrodynamic radius corresponding to the soluble filaments upon incubation at 55 ◦ C (Fig. 5d). These results indicated that heat-denatured myosin molecules form soluble oligomers at 0.30 M NaCl and bind to soluble filaments most likely via the myosin heads.
4. Discussion
Fig. 7. Thermal unfolding of myosin monitored by CD 222 nm intensity change. The continuous curve indicates a theoretical line calculated on the basis of the twostate thermal unfolding equation. (Inset) Far-UV CD spectra of myosin in 0.30 M NaCl, 20 mM sodium phosphate buffer (pH 7.5) were measured at 25 ◦ C or 98 ◦ C.
The overall changes in myosin solution are depicted schematically in Fig. 9. Soluble myosin samples were prepared by 1.0 M NaCl at 25 ◦ C followed by dilution to different salt concentrations. The diluted solutions consistently contained both myosin monomers and soluble filaments. The filament size decreased with increasing salt concentration and temperature (Fig. 4). High temperatures above Tm led to dissociation of soluble filaments and unfolding followed by formation of soluble oligomers and a complex between the oligomers and soluble filaments.
M. Shimada et al. / International Journal of Biological Macromolecules 73 (2015) 17–22
21
Fig. 8. TEM observations and graphical representations of myosin in 0.30 M NaCl incubated at 25 ◦ C or 55 ◦ C for 90 min. (a, b) 25 ◦ C. (c, d) 55 ◦ C. The scale bars indicate 200 nm.
Fig. 9. Schematic illustration of 1.0 M salt solubilization, dilution, and heating of porcine myosin.
4.1. Structural properties of soluble myosin filaments in high-salt solution The findings of this study indicated the coexistence of soluble myosin filaments and myosin monomers in the presence of NaCl above 0.30 M (Fig. 2, also schematic illustration in Fig. 9). It has been shown that myosin forms (1) insoluble filaments in physiological saline solution [1,2], (2) monomers at high salt concentrations (typically >0.3 M) [10], and (3) soluble short filaments induced by dilution or dialysis of high-salt-dissociated monomeric myosin in low-salt solution (typically <0.2 M) [5,18–21]. However, this study showed that high salt concentration stabilized soluble myosin filaments below the melting temperature (46 ◦ C). The presence of soluble myosin filaments was confirmed by systematic DLS measurements discussed here as well as by electrophoresis (data not shown). In addition, TEM experiments also confirmed the presence of both monomeric and filamentous myosin molecules above 0.30 M NaCl. We observed filamentous myosin in the presence of salt, which may have implications for heat-induced myosin aggregation. In
the previous aggregation model, it was assumed that the soluble fraction contains only monomeric myosin below the melting temperature. Above the melting temperature, the myosin monomers unfold and form aggregates by head–head interaction [10], followed by tail–tail cross-linking [22] with a ball-like structure. In agreement with this model, we also observed a similar balllike structure (Fig. 8). Unlike the previous model, however, we observed stable soluble myosin filaments as well as monomers below the melting temperature in the presence of NaCl above 0.30 M. At around the melting temperature, soluble myosin filaments dissociated into smaller filaments or monomers due to thermal unfolding. Above 55 ◦ C, myosin molecules unfolded and formed soluble oligomers, which bound to the persistent soluble filaments. 4.2. Implications of myosin gelation We propose a new potential mechanism of heat-induced gelation of soluble myosin in the presence of NaCl at high concentrations. High temperature plays a major role in unfolding,
22
M. Shimada et al. / International Journal of Biological Macromolecules 73 (2015) 17–22
while high salt concentration dissociates myosin filaments within a short period during DLS experiments (1 min). Upon prolonged incubation, both parameters, i.e., temperature and salt concentration, affect aggregation of unfolded and dissociated unfolded myosin filaments. Heat-induced gelation of myosin has classically been defined as either aggregate-type gel derived from myosin monomers in high-salt solution above 0.3 M [10] or strand-type gel derived from myosin filaments in low-salt solution below 0.3 M [11]. However, the results of the present study suggest that heatinduced myosin gel may be composed of both myosin oligomers and myosin filaments. The composition of oligomers and filaments depends on the solution conditions. TEM showed spherical aggregates bound by the filaments, consistent with the myosin network in low-salt solution [11,23]. We have recently demonstrated that insoluble myosin can be solubilized in physiological salt solution using arginine as an additive [12]. In addition, arginine causes protein aggregates to adopt a more compact morphology [14]. Thus, it is possible that arginine improves the elasticity of the heat-induced myosin gel. Our study provided useful information for investigating heat-induced myosin aggregation with gel formation in physiological salt solution containing arginine. In conclusion, myosin in high salt solution above 0.3 M consistently contained both myosin monomers and soluble filaments. Above melting temperature, the soluble filament dissociated and unfolded, resulting in formation of soluble oligomers and binding to the persistently present soluble filament. It would be a new potential mechanism of heat-induced gelation of myosin in high salt solution.
Acknowledgments The authors are grateful to Dr. Katsuhiro Yamamoto and Dr. Yasuhiro Funatsu for helpful discussions. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]
[14] [15] [16] [17] [18] [19] [20] [21] [22] [23]
A.D. McLachlan, J. Karn, Nature 299 (1982) 226–231. Y. Tsunashima, T. Akutagawa, Biopolymers 75 (2004) 264–277. J.E. Godfreyt, W.F. Harrington, Biochemistry 580 (1969) 886–893. J.E. Godfreyt, W.F. Harrington, Biochemistry 581 (1969) 894–908. I. Katsura, H. Noda, J. Biochem. 69 (1971) 219–229. B.L. Eaton, F.A. Pepe, J. Mol. Biol. 82 (1974) 421–423. T. Fukazawa, Y. Hashimoto, T. Yasu, J. Food Sci. 26 (1961) 541–549. K. Samejima, Y. Hashimoto, T. Yasui, T. Fukazawa, J. Food Sci. 34 (1969) 242–245. E. Puolanne, M. Halonen, Meat Sci. 86 (2010) 151–160. K. Yamamoto, J. Biochem. 108 (1990) 896–898. K. Yamamoto, K. Samejima, Agric. Biol. Chem. 52 (1988) 1803–1811. E. Takai, S. Yoshizawa, D. Ejima, T. Arakawa, K. Shiraki, Int. J. Biol. Macromol. 62 (2013) 647–651. P.K. Smith, R.I. Krohn, G.T. Hermanson, A.K. Mallia, F.H. Gartner, M.D. Provenzano, E.K. Fujimoto, N.M. Goeke, B.J. Olson, D.C. Klenk, Anal. Biochem. 150 (1985) 76–85. S. Tomita, H. Yoshikawa, K. Shiraki, Biopolymers 95 (2011) 695–701. J.K. Amisha Kamal, D.V. Behere, Biochemistry 41 (2002) 9034–9042. C. Louis-Jeune, M.A. Andrade-Navarro, C. Perez-Iratxeta, Proteins 80 (2012) 374–381. M. Ishioroshi, K. Samejima, T. Yaw, J. Food Sci. 44 (1979) 1280–1284. E. Reisler, C. Smith, G. Seegax, J. Mol. Biol. 143 (1980) 129–145. E. Reisler, P. Cheung, C. Oriol-Audit, J.A. Lake, Biochemistry 21 (1982) 701–707. K.M. Trybus, S. Lowey, Cell Biol. 105 (1987) 3007–3019. J.H. Sinard, W.F. Stafford, T.D. Pollard, Cell Biol. 109 (1989) 1537–1547. M. Ishioroshi, K. Samejima, T. Yasui, J. Food Sci. 46 (1981) 1412–1418. C. Boyer, S. Joandel, A. Ouali, J. Culioli, J. Food Sci. 61 (1996) 1138–1143.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Heat-induced formation of myosin oligomer-soluble filament complex in high-salt solution Masato Shimada a,1 , Eisuke Takai a,1 , Daisuke Ejima b , Tsutomu Arakawa c , Kentaro Shiraki a,∗ a
Faculty of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8573, Japan AminoScience Laboratories, Ajinomoto Inc., 1-1 Kawasaki, Japan c Alliance Protein Laboratories, San Diego, CA 92121, USA b
a r t i c l e
i n f o
Article history: Received 29 August 2014 Received in revised form 17 October 2014 Accepted 7 November 2014 Available online 15 November 2014 Keywords: Myosin Dynamic light scattering Filament formation Thermal aggregation Heat-induced gelation
a b s t r a c t Heat-induced aggregation of myosin into an elastic gel plays an important role in the water-holding capacity and texture of meat products. Here, we investigated thermal aggregation of porcine myosin in high-salt solution over a wide temperature range by dynamic light scattering experiments. The myosin samples were readily dissolved in 1.0 M NaCl at 25 ◦ C followed by dilution into various salt concentrations. The diluted solutions consistently contained both myosin monomers and soluble filaments. The filament size decreased with increasing salt concentration and temperature. High temperatures above Tm led to at least partial dissociation of soluble filaments and thermal unfolding, resulting in the formation of soluble oligomers and binding to the persistently present soluble filaments. Such a complex formation between the oligomers and filaments has never been observed. Our results provide new insight into the heat-induced myosin gelation in high-salt solution. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Myosin is a major muscle protein. The myosin molecule has an asymmetric structure with two globular heads and a rod-like tail. It associates into insoluble filaments through electrostatic tail-totail interaction under physiological conditions [1,2]. Accordingly, disruption of electrostatic interactions by high salt concentration, e.g., above 0.3 M, leads to reversible dissociation into monomers, which are in equilibrium with varying sizes of associated species, e.g., soluble oligomers, as determined by differential velocity sedimentation [3,4] or flow birefringence and electron microscopy [5,6]. Temperature also affects self-association of dissociated myosin monomers or oligomers. When heated in salt solution, myosin monomers and oligomers are denatured and aggregate into an elastic gel, which plays an important role in the water-holding capacity and texture of myosin-containing food products [7–9]. The heat-induced myosin gels are classified into two types: the aggregate-type gel in high-salt solution [10] and the strand-type gel in low-salt solution [11]. The dependence of heat-induced myosin
∗ Corresponding author. Tel.: +81 29 8535306; fax: +81 29 8535215. E-mail address: [email protected] (K. Shiraki). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ijbiomac.2014.11.005 0141-8130/© 2014 Elsevier B.V. All rights reserved.
gel formation on salt concentration is attributed to the association state of myosin. Thus, it is likely that gel formation of myosin by heating is closely linked to association states of myosin in solution as a function of temperature and salt concentration. This study was performed to investigate the size and structure of myosin under different solution conditions.
2. Materials and methods 2.1. Purification of myosin Porcine myosin extract was prepared as follows [12]. The myosin used in this study was extracted from the lean portion of porcine inner thigh meat. A sample of 250 g of the starting meat was suspended in 450 ml of buffer containing 0.10 M pyrophosphate and 5.0 mM MgCl2 (pH 7.0) and homogenized at 5 ◦ C for 60 min. The resultant myosin extract suspension was stored frozen at −25 ◦ C. After thawing, the myosin extract was dialyzed against 1.0 mM KCl and 2.0 mM sodium phosphate buffer (pH 7.0), resulting in myosin precipitation. The precipitates were washed with 1.0 mM KCl and 2.0 mM sodium phosphate buffer (pH 7.0) and stored frozen. The purity of the myosin extract was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Aliquots of the sample were dissolved in reducing sample buffer
18
M. Shimada et al. / International Journal of Biological Macromolecules 73 (2015) 17–22
containing 2% (w/v) SDS, 10% glycerol, 0.04 M DTT, 0.01% (w/v) bromophenol blue, and 62.5 mM Tris–HCl (pH 6.8). The samples were boiled for 5 min and loaded onto 10% polyacrylamide gels. 2.2. Preparation of myosin solution The frozen precipitate prepared above was used as a starting material of purified myosin, which was dissolved with 1.0 M NaCl and 20 mM sodium phosphate buffer (pH 7.5), i.e., at high salt concentration. After incubation for 1 h at 25 ◦ C, the 1.0 M NaCl solution containing soluble myosin was centrifuged at 18,800 × g for 20 min. The supernatants were diluted with 20 mM sodium phosphate buffer (pH 7.5) containing 0.20–1.0 M NaCl to generate a solution containing an appropriate concentration of myosin and different concentrations of NaCl for various solution measurements. The diluted myosin solutions were incubated at room temperature for 1 h and then centrifuged. The myosin concentrations in the supernatant were determined by the bicinchoninate method [13].
Fig. 1. Solubility of myosin in the presence of 0.20–1.0 M NaCl in 20 mM sodium phosphate buffer (pH 7.5) at 25 ◦ C. (Inset) Time course of changes in myosin concentration in 0.20–1.0 M NaCl solution.
2.3. DLS analysis of myosin Dynamic light scattering (DLS) experiments were performed using a light scattering photometer (Zetasizer Nano ZS; Malvern Instruments, Worcestershire, UK) equipped with a 4 mW He–Ne ion laser ( = 633 nm). Data analysis was performed as reported previously [14]. The size of myosin in aqueous salt solutions was determined as follows. All buffer and salt solutions used to make myosin samples were filtered through 0.4-m filter. The myosin solutions at 0.50 mg/ml in 20 mM sodium phosphate (pH 7.5) containing 0.30, 0.50, 0.80, and 1.0 M NaCl were placed, immediately after sample preparation, in a 1-cm path-length quartz cuvette and subjected to DLS measurement at different temperatures (25 ◦ C, 35 ◦ C, 45 ◦ C, 50 ◦ C, 55 ◦ C, 65 ◦ C, and 75 ◦ C) at a detection angle of 173◦ . No apparent turbidity or precipitation was observed before and after the DLS experiments. 2.3.1. CD for thermal unfolding of myosin Circular dichroism (CD) spectra were measured using a spectropolarimeter J-720W (Japan Spectroscopic Co., Ltd., Tokyo, Japan). A 0.30 mg/ml myosin solution in 0.30 M NaCl and 20 mM sodium phosphate buffer (pH 7.5) was transferred to a quartz cell with a 1-mm path length and subjected to CD measurements at a scan rate of 100 nm/min. Thermal unfolding was measured by following the CD intensity at 222 nm with an increasing temperature rate of 1.0 ◦ C/min. The thermal unfolding transition curve was analyzed using a van’t Hoff plot assuming a two-state transition and linear baselines for native and unfolded states, from which the apparent mid-transition temperature (Tm ) was determined [15], though the thermal unfolding of myosin may be more complex than two-state transition. The composition of ␣-helix and -sheet was estimated by using K2D3 method [16]. 2.4. TEM observation of myosin Transmission electron microscopy (TEM) of myosin in aqueous salt solution was performed with an acceleration voltage of 80 kV (JEM-1400; JEOL, Tokyo, Japan). Samples of 0.50 mg/ml myosin in 0.30 M NaCl, 20 mM sodium phosphate buffer (pH 7.5) were incubated at 25 ◦ C or 55 ◦ C for 90 min. Aliquots of 2 l of the above sample solution were placed on a 150-mesh copper grid covered with a carbon-coated hydrophilic film, to which 10 volumes of pure water were added. The samples were then negatively stained with 2 l of 2% (w/v) silicotungstic acid solution. The solution of the grid was dried for several minutes.
Fig. 2. Size distributions of soluble myosin in 0.30 M (solid lines) or 1.0 M (dot lines) NaCl, 20 mM sodium phosphate buffer (pH 7.5) monitored by DLS at 25 ◦ C.
3. Results Insoluble myosin filaments prepared from porcine inner thigh meat are expected to dissociate into monomers at high salt concentration, i.e., above 0.3 M, resulting in soluble myosin solutions. We determined the solubility of myosin at 25 ◦ C as a function of salt concentration. Fig. 1 shows the solubility of myosin in 0.20–1.0 M NaCl after incubation for 1 h at 25 ◦ C. The myosin concentration in the supernatant increased sharply with increasing NaCl concentration from 0.20 M to 0.25 M, consistent with the previous report [17] and reached a plateau at 0.25 M, above which the solubility increased only slightly. The myosin concentrations of the supernatant in the presence of NaCl at or above 0.20 M changed little over incubation for 1–48 h at 25 ◦ C (see Fig. 1 inset): the myosin concentrations remained constant around 0.25–0.275 mg/ml. These results suggest that the myosin solution was stable over the course of the experiments. The associated state of myosin molecules at high salt concentrations was investigated by DLS. Fig. 2 shows the size distribution of ∼0.30 mg/ml myosin in 0.30 M NaCl (solid line) and 1.0 M NaCl (dotted line) at 25 ◦ C. The distribution was expressed as volume percentage calculated from the hydrodynamic radius based on the assumption of the spherical shape of myosin in these solutions. These samples were prepared from the supernatant of myosin solubilized by 1.0 M NaCl (pH 7.5) followed by dilution in buffer solution containing the indicated NaCl concentration. Two peaks at 18.9 and
M. Shimada et al. / International Journal of Biological Macromolecules 73 (2015) 17–22
19
Fig. 3. Dependence of myosin monomer and filament sizes on salt concentration and temperature. (a) NaCl dependence of soluble myosin filament (circles) and monomer (triangles) at 25 ◦ C (closed symbols) and 55 ◦ C (open symbols). (b) Temperature dependence of soluble myosin filament (circles) and monomer (triangles) in 0.30 M (closed symbols) and 1.0 M (open symbols) NaCl.
198 nm were observed for myosin in 0.30 M NaCl solution and may be assigned as myosin monomers and soluble filaments, respectively. Although it is not possible to assign the myosin structure based on DLS experiments, the term “soluble filament” is used based on TEM observations described below. Two peaks at 14.1 and 127 nm were observed for myosin in 1.0 M NaCl solution, also likely corresponding to myosin monomers and soluble filaments, respectively. These results indicated that soluble myosin solution is not homogeneous with monomeric species and contains significant amounts of soluble filaments at high salt concentrations even at 25 ◦ C. The hydrodynamic radius of myosin was investigated at various NaCl concentrations and temperatures. Fig. 3a shows the hydrodynamic radius of myosin as a function of NaCl concentration at 25 ◦ C and 55 ◦ C. Myosin monomer aggregates into soluble oligomers through myosin head–head interactions above 55 ◦ C in the presence of salt. The size of the myosin monomer depended little on salt concentration at both 25 ◦ C (closed triangles) and 55 ◦ C (open triangles). The size of the soluble myosin filaments also depended little on salt concentration at 55 ◦ C (open circles). In contrast, the size of soluble myosin filaments at 25 ◦ C (closed circles) decreased gradually with increasing salt concentration, e.g., from 180 nm at 0.30 M to 130 nm at 1.0 M, suggesting disassembly of self-associated myosin filaments. Fig. 3b shows the temperature dependence of myosin monomers and soluble filaments at 0.3 and 1.0 M NaCl. The size of the myosin monomers at both 0.30 M NaCl (closed triangles) and 1.0 M NaCl (open triangles) decreased slightly with increasing temperature, i.e., from 40 nm at 25 ◦ C to 25 nm at 75 ◦ C. Such a decrease in size of the monomer peak may simply have been due to temperature-dependent viscosity change. In contrast, the size of soluble myosin filaments decreased sharply with increasing temperature between 25 ◦ C and 45 ◦ C following a slight increase at lower temperature at both 0.30 M NaCl (closed circles) and 1.0 M NaCl (open circles). This sharp drop in the hydrodynamic size of the filaments was followed by a gradual decrease above 45 ◦ C. It should be noted that the size of the filaments approached the level of the monomer at 75 ◦ C. These data indicated that the size of myosin filaments depends on both temperature and NaCl concentration, and higher temperature causes dissociation of selfassociated soluble myosin filaments. DLS experiments were carried out at four different salt concentrations and seven different temperatures. Under each set of conditions, the hydrodynamic radius range of the soluble filaments was determined and plotted as a phase diagram, i.e., the size range of soluble myosin filaments as a function of salt concentration and temperature; data related to the monomer peak were ignored
(Fig. 4). It is clear that hydrodynamic radius of soluble filaments decreased monotonically with increasing temperature and NaCl concentration within time frame of DLS experiments, i.e., 1 min. It appears that heat-induced dissociation of myosin filaments play a major role in reduction of hydrodynamic radius under such a short incubation time and a low protein concentration, 0.50 mg/ml. The size of the soluble filaments showed a maximum at about 0.30 M NaCl and 35 ◦ C and a minimum at about 0.80 M NaCl and 75 ◦ C. As shown in Fig. 3a, the hydrodynamic size of the soluble filaments was relatively constant at 55 ◦ C over the salt concentration range from 0.30 to 1.0 M. The phase diagram showed that the hydrodynamic size was fairly constant on salt concentration between 40 ◦ C and 60 ◦ C. Below and above these temperatures, the filament size became dependent on the salt concentration. In contrast, the filament size was dependent on temperature at all salt concentrations examined, more so at lower and higher salt concentrations. The stability of soluble filaments was investigated at different salt concentrations and temperatures. Fig. 5 shows the time course of changes in hydrodynamic radius of soluble filaments. Fig. 5a shows the time course of changes in filament size at 25 ◦ C in the presence of 0.30 M (black) and 1.0 M NaCl (red). At this temperature, the myosin filaments were stable over 90-min incubation at both salt concentrations. Fig. 5b shows the time course at 45 ◦ C in the presence of 0.50 M NaCl. A gradual decrease in filament size was observed with time. In contrast, the hydrodynamic size increased sharply with time at 55 ◦ C in the presence of 0.30 M NaCl (Fig. 5c). The hydrodynamic size changed linearly with time at least under certain conditions. The rate of change was determined at different temperatures and salt concentrations, from which a phase diagram
Fig. 4. Phase diagram of soluble myosin filament size in 0.30–1.0 M NaCl at 25–75 ◦ C.
20
M. Shimada et al. / International Journal of Biological Macromolecules 73 (2015) 17–22
Fig. 5. Representative data for hydrodynamic radius of myosin filament. (a) 0.30 M NaCl (black) or 1.0 M NaCl (red) at 25 ◦ C. (b) 0.50 M NaCl at 45 ◦ C. (c) 0.30 M NaCl at 55 ◦ C. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
was constructed (Fig. 6). At 25 ◦ C, the filaments were stable for over 90 min of incubation at all salt concentrations examined, as shown in Fig. 5a. At 35–45 ◦ C, the slope of the time course curve was negative at all salt concentrations examined, as shown Fig. 5b. Above 50 ◦ C, the slope of the time course curve was positive, especially at 0.30 M NaCl and 60 ◦ C, as shown in Fig. 5c. The temperature dependence of the filamentous myosin hydrodynamic radius was observed at all salt concentrations examined (Fig. 4). Whether such changes in size are associated with structural changes was examined by far-UV CD spectroscopy. Fig. 7
Fig. 6. Phase diagram of rate of change in hydrodynamic radius of soluble myosin filament during 90-min incubation.
shows the CD signal at 222 nm, which began to increase around 35 ◦ C followed by a sharp increase up to 55 ◦ C and a plateau above 60 ◦ C. The midpoint temperature was estimated to be ∼46 ◦ C. Fig. 7 (inset) shows the far UV CD spectrum at 25 ◦ C characterized by double minima for ␣-helix and the spectrum at 98 ◦ C with a 217 nm minimum for -sheet structure. Thus, the thermal unfolding was characterized as a structure change from ␣ to  transition. As temperature increased from 25 ◦ C to 98 ◦ C, apparent ␣-helix content decreased from 77% to 13% and apparent -sheet content increased from 0% to 24%. Comparison of Fig. 4 and the CD melting curve suggested that the observed decrease in hydrodynamic size was due to thermal unfolding of myosin molecules accompanied by dissociation of soluble ␣-helical myosin filaments. Above 55 ◦ C, the filament size was less dependent on temperature at all salt concentrations examined, possibly due to complete unfolding at this temperature. However, a strong dependence of size on incubation temperature was observed above 50 ◦ C (Fig. 6), most likely reflecting the tendency of the unfolded -structure myosin to aggregate upon incubation. To examine the protein structure, myosin solution containing 0.30 M NaCl was incubated at 25 ◦ C or 55 ◦ C for 90 min and examined by TEM. When incubated at 25 ◦ C in 0.3 M NaCl, myosin was observed mostly as monomers (Fig. 8a), along with soluble filaments (Fig. 8b). The soluble filaments were 600 nm in length and about 10 nm in thickness (Fig. 8b). When incubated at 55 ◦ C, typical protein aggregates composed of unfolded myosin were observed as soluble oligomers (Fig. 8c). In addition, soluble filaments 500 nm in length were observed (Fig. 8d). Interestingly, the soluble oligomers formed complexes with the soluble filaments (Fig. 8d), consistent with the sharp increase in hydrodynamic radius corresponding to the soluble filaments upon incubation at 55 ◦ C (Fig. 5d). These results indicated that heat-denatured myosin molecules form soluble oligomers at 0.30 M NaCl and bind to soluble filaments most likely via the myosin heads.
4. Discussion
Fig. 7. Thermal unfolding of myosin monitored by CD 222 nm intensity change. The continuous curve indicates a theoretical line calculated on the basis of the twostate thermal unfolding equation. (Inset) Far-UV CD spectra of myosin in 0.30 M NaCl, 20 mM sodium phosphate buffer (pH 7.5) were measured at 25 ◦ C or 98 ◦ C.
The overall changes in myosin solution are depicted schematically in Fig. 9. Soluble myosin samples were prepared by 1.0 M NaCl at 25 ◦ C followed by dilution to different salt concentrations. The diluted solutions consistently contained both myosin monomers and soluble filaments. The filament size decreased with increasing salt concentration and temperature (Fig. 4). High temperatures above Tm led to dissociation of soluble filaments and unfolding followed by formation of soluble oligomers and a complex between the oligomers and soluble filaments.
M. Shimada et al. / International Journal of Biological Macromolecules 73 (2015) 17–22
21
Fig. 8. TEM observations and graphical representations of myosin in 0.30 M NaCl incubated at 25 ◦ C or 55 ◦ C for 90 min. (a, b) 25 ◦ C. (c, d) 55 ◦ C. The scale bars indicate 200 nm.
Fig. 9. Schematic illustration of 1.0 M salt solubilization, dilution, and heating of porcine myosin.
4.1. Structural properties of soluble myosin filaments in high-salt solution The findings of this study indicated the coexistence of soluble myosin filaments and myosin monomers in the presence of NaCl above 0.30 M (Fig. 2, also schematic illustration in Fig. 9). It has been shown that myosin forms (1) insoluble filaments in physiological saline solution [1,2], (2) monomers at high salt concentrations (typically >0.3 M) [10], and (3) soluble short filaments induced by dilution or dialysis of high-salt-dissociated monomeric myosin in low-salt solution (typically <0.2 M) [5,18–21]. However, this study showed that high salt concentration stabilized soluble myosin filaments below the melting temperature (46 ◦ C). The presence of soluble myosin filaments was confirmed by systematic DLS measurements discussed here as well as by electrophoresis (data not shown). In addition, TEM experiments also confirmed the presence of both monomeric and filamentous myosin molecules above 0.30 M NaCl. We observed filamentous myosin in the presence of salt, which may have implications for heat-induced myosin aggregation. In
the previous aggregation model, it was assumed that the soluble fraction contains only monomeric myosin below the melting temperature. Above the melting temperature, the myosin monomers unfold and form aggregates by head–head interaction [10], followed by tail–tail cross-linking [22] with a ball-like structure. In agreement with this model, we also observed a similar balllike structure (Fig. 8). Unlike the previous model, however, we observed stable soluble myosin filaments as well as monomers below the melting temperature in the presence of NaCl above 0.30 M. At around the melting temperature, soluble myosin filaments dissociated into smaller filaments or monomers due to thermal unfolding. Above 55 ◦ C, myosin molecules unfolded and formed soluble oligomers, which bound to the persistent soluble filaments. 4.2. Implications of myosin gelation We propose a new potential mechanism of heat-induced gelation of soluble myosin in the presence of NaCl at high concentrations. High temperature plays a major role in unfolding,
22
M. Shimada et al. / International Journal of Biological Macromolecules 73 (2015) 17–22
while high salt concentration dissociates myosin filaments within a short period during DLS experiments (1 min). Upon prolonged incubation, both parameters, i.e., temperature and salt concentration, affect aggregation of unfolded and dissociated unfolded myosin filaments. Heat-induced gelation of myosin has classically been defined as either aggregate-type gel derived from myosin monomers in high-salt solution above 0.3 M [10] or strand-type gel derived from myosin filaments in low-salt solution below 0.3 M [11]. However, the results of the present study suggest that heatinduced myosin gel may be composed of both myosin oligomers and myosin filaments. The composition of oligomers and filaments depends on the solution conditions. TEM showed spherical aggregates bound by the filaments, consistent with the myosin network in low-salt solution [11,23]. We have recently demonstrated that insoluble myosin can be solubilized in physiological salt solution using arginine as an additive [12]. In addition, arginine causes protein aggregates to adopt a more compact morphology [14]. Thus, it is possible that arginine improves the elasticity of the heat-induced myosin gel. Our study provided useful information for investigating heat-induced myosin aggregation with gel formation in physiological salt solution containing arginine. In conclusion, myosin in high salt solution above 0.3 M consistently contained both myosin monomers and soluble filaments. Above melting temperature, the soluble filament dissociated and unfolded, resulting in formation of soluble oligomers and binding to the persistently present soluble filament. It would be a new potential mechanism of heat-induced gelation of myosin in high salt solution.
Acknowledgments The authors are grateful to Dr. Katsuhiro Yamamoto and Dr. Yasuhiro Funatsu for helpful discussions. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]
[14] [15] [16] [17] [18] [19] [20] [21] [22] [23]
A.D. McLachlan, J. Karn, Nature 299 (1982) 226–231. Y. Tsunashima, T. Akutagawa, Biopolymers 75 (2004) 264–277. J.E. Godfreyt, W.F. Harrington, Biochemistry 580 (1969) 886–893. J.E. Godfreyt, W.F. Harrington, Biochemistry 581 (1969) 894–908. I. Katsura, H. Noda, J. Biochem. 69 (1971) 219–229. B.L. Eaton, F.A. Pepe, J. Mol. Biol. 82 (1974) 421–423. T. Fukazawa, Y. Hashimoto, T. Yasu, J. Food Sci. 26 (1961) 541–549. K. Samejima, Y. Hashimoto, T. Yasui, T. Fukazawa, J. Food Sci. 34 (1969) 242–245. E. Puolanne, M. Halonen, Meat Sci. 86 (2010) 151–160. K. Yamamoto, J. Biochem. 108 (1990) 896–898. K. Yamamoto, K. Samejima, Agric. Biol. Chem. 52 (1988) 1803–1811. E. Takai, S. Yoshizawa, D. Ejima, T. Arakawa, K. Shiraki, Int. J. Biol. Macromol. 62 (2013) 647–651. P.K. Smith, R.I. Krohn, G.T. Hermanson, A.K. Mallia, F.H. Gartner, M.D. Provenzano, E.K. Fujimoto, N.M. Goeke, B.J. Olson, D.C. Klenk, Anal. Biochem. 150 (1985) 76–85. S. Tomita, H. Yoshikawa, K. Shiraki, Biopolymers 95 (2011) 695–701. J.K. Amisha Kamal, D.V. Behere, Biochemistry 41 (2002) 9034–9042. C. Louis-Jeune, M.A. Andrade-Navarro, C. Perez-Iratxeta, Proteins 80 (2012) 374–381. M. Ishioroshi, K. Samejima, T. Yaw, J. Food Sci. 44 (1979) 1280–1284. E. Reisler, C. Smith, G. Seegax, J. Mol. Biol. 143 (1980) 129–145. E. Reisler, P. Cheung, C. Oriol-Audit, J.A. Lake, Biochemistry 21 (1982) 701–707. K.M. Trybus, S. Lowey, Cell Biol. 105 (1987) 3007–3019. J.H. Sinard, W.F. Stafford, T.D. Pollard, Cell Biol. 109 (1989) 1537–1547. M. Ishioroshi, K. Samejima, T. Yasui, J. Food Sci. 46 (1981) 1412–1418. C. Boyer, S. Joandel, A. Ouali, J. Culioli, J. Food Sci. 61 (1996) 1138–1143.