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Molecular forces involved in the formation and stabilization of heat-induced actomyosin gels
Meat Science 36 (1994) 407-421
Molecular Forces Involved in the Formation and Stabilization of Heat-Induced Actomyosin Gels E. O'Neill, a D. M. Mulvihill a & P. A. M o r r i s s e y , b a Department of Food Chemistry, b Department of Nutrition, University College, Cork, Ireland (Received 12 September 1992; revised version received 28 January 1993; accepted 6 February 1993)
ABSTRA CT Increasing concentrations of KCl, KF and NaCl from 0.2 to 1.0 m increased the compressive strength and cohesiveness of heat-induced actomyosin gels (0"6 m KCI p H 6.0). On the other hand, KBr, KSCN, KI, NH4CI, MgCI2 and CaCl2 decreased gel strength and cohesiveness. Gel compressive strength increased with increasing urea concentration (0.4-2.4 m). Addition of the sulphydryl blocking agent N-ethylmaleimide and the reducing agents dithiothreitol and cysteine decreased the compressive strength and cohesiveness of actomyosin gels. Overall, the results indicate that hydrophobic interactions, disulphide and hydrogen bonding contribute to the formation and stabilization of actomyosin gels.
INTRODUCTION F o r m a t i o n of a three-dimensional gel network by myosin and/or actomyosin during thermal processing contributes significantly to the textural aspects and stabil!ty of comminuted and formed meat products (Acton et al., 1983; Asghar et aL, 1985; Morrissey et al., 1987). Previous studies have shown that the properties and characteristics of myosin and actomyosin gels are influenced by protein concentration, heating temperature and rate, and the critical balance between attractive and repulsive forces (Ishioroshi et al., 1979, 1983; Acton et al., 1981; Lanier et al., 1982; Hermansson et al., 1986; Wicker et aL, 1986; Foegeding et al., 1986; 407 Meat Science 0309-1740/94/$07.00 © 1994 Elsevier Science Ltd, England. Printed in Great Britain
408
E. O'Neill, D. M. Mulvihill, P. A. Morrissey
Wu et al., 1991). Disulphide linkages (Samejima et al., 1981; Ishioroshi et al., 1982; Foegeding et al., 1987), hydrogen and hydrophobic bonding (Samejima et al., 1981; Wicker et al., 1986, 1989; Foegeding et al., 1987) are considered important in myosin gelation. However, the molecular forces involved in the formation and stabilization of actomyosin gels, which differ considerably in ultrastructure from myosin gels (Yasui et al., 1982), have not been fully characterized. In an attempt to elucidate the major molecular forces that contribute to the formation and stabilization of actomyosin gel structures (in 0-6 M KC1), the effects of added neutral salts, urea, propylene glycol, dithiothreitol (DTT), N-ethylmaleimide (NEM) and cysteine, which enhance or block specific protein-protein interactions, on the rheological properties of actomyosin gels were determined in this study.
MATERIALS AND METHODS
Preparation of actomyosin Actomyosin was prepared from rabbit skeletal muscle by the method described by Hay et al. (1972).
Preparation of actomyosin gels A stock solution of actomyosin containing 100 mg mF ~ protein was prepared in 0-6 M KC1, 0-1 N imidazole-HC1 buffer, pH 6.0. Stock solutions containing the various salts and~reagents were also prepared in 0-6 M KC1, 0.1 M imidazole-HC1 buffer, pH 6-0. To prepare the gelling solutions, the stock actomyosin solution was diluted in a 1:1 volume ratio with 0.6 M KC1, 0-1M imidazole-HC1 buffer, pH 6.0, or with stock solutions of KF, KSCN, KBr, KI, KC1, NaC1, NH4C1 , MgC12, CaC12, urea, propylene glycol, DTT, N E M or cysteine to give the desired salt or reagent concentration. Aliquots (1 ml) of the gelling solutions were transferred to glass tubes (inside diameter 6.0 mm), which had been treated with Sigrnacote (Sigma Chemical Company, St. Louis, Co.) and air-dried. The tubes containing the gelling solutions were centrifuged at low speed (500 g) to remove air bubbles and then heat-sealed to prevent evaporation. The samples were equilibrated in a water bath at 25°C for 15 min then heated to 70°C at a rate of 1.1°C min -1 and maintained a t 70°C for 30 min. The tubes were removed from the water bath, cooled in ice-water and held at 4°C for 24 h.
Formation and stabilization of heat-induced actomyosin gels
409
Measurement of gel rheological properties The gels were equilibrated at room temperature for 3 h and then removed carefully from the glass tubes. Sections 5 m m high were cut with a razoredge cutting device. Rheological properties were determined on these sections with an Instron Universal Testing Instrument (Table Model T M - M ) using an arrangement similar to that described by Mulvihill and Kinsella (1988). The top plate and base plate were set 20 m m apart. A gel section (5 m m high x 6 m m in diameter) was placed on the base plate and compressed by 65% of its original height (3.25 mm) on two successive occasions by lowering the top plate 18.25 m m from its original setting and raising it again at a speed of 10 m m per minute. The force exerted on the top plate was recorded continuously on a chart recorder operating at a speed of 500 m m min ~. The following rheological parameters were determined from the force deformation curves as described by Mulvihill and Kinsella (1988). Stress at 65% compression, units -- N m -2 Stress energy at 65% compression, units = J Cohesiveness, units -- ratio, dimensionless. All the rheological results reported are mean values calculated from a m i n i m u m of seven and up to eleven separate deformation curves obtained from different gel sections.
RESULTS AND DISCUSSION The presence of neutral salts had dramatic effects on the rheological properties of heat-induced actomyosin gels, which cannot be attributed solely to simple non-specific charge neutralization (Figs 1-6). Increasing concentrations of KC1 and K F from 0.2 to 1-0 M progressively increased stress, stress energy and gel cohesiveness relative to the control (Figs 1 3). On the other hand, KBr, K S C N and KI decreased stress, stress energy and gel cohesiveness. The relative effects of the anions on the compressive strength and cohesiveness of the heat-induced actomyosin gels was in the order: F >C1 > B r > S C N - > I With the exception of I- these anions generally behaved according to the Hofmeister series (von Hippel & Schleich, 1969), where the anions are ranked on the basis of their ability to salt out proteins. In the present study I was found to be the most effective anion in decreasing gel
410
E. O'Neill, D. M. Mulvihill, P. A. Morrissey
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strength and cohesiveness. This effect is not in line with its position between SCN- and Br- in the Hofmeister series. The effects of these salts on actomyosin gelation are probably related to their ability to 'salt out' proteins and promote coagulation. The Fand C1- anions enhance the hydrogen bonded structure of the bulk water phase and hence have a tendency to increase the driving force for hydrophobic interactions within and between proteins resulting in protein association and aggregation (Damodaran & Kinsella, 1982; Babajimopoulos et al., 1983). The positive effects of KF and KC1 on actomyosin gel strength and cohesiveness suggest that hydrophobic interactions are involved in structuring and stabilizing the gels. On the other hand, Br-, £, SCN- are large ions with low charge densities and tend to break water structure (von Hippel & Wong, 1965; Damodaran & Kinsella, 1982). These anions favour destabilization of hydrophobic interactions between protein molecules (Damodaran &
Formation and stabilization of heat-induced actomyosin gels
411
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Kinsella, 1981, 1982; Utsumi & Kinsella, 1985; Bernal et al., 1987). Thus, the decrease in gel strength and cohesiveness observed in the presence of KBr, KSCN and KI (Figs 1-3) may be due to a decrease in protein-protein hydrophobic associations within the actomyosin gel matrix. Dissociation of actomyosin to myosin and G-actin has been shown to occur at concentrations > 0-3 M KBr, KI or KSCN (Holtzer et al., 1960). Yasui et al. (1980) and Ishioroshi et al. (1980) demonstrated that dissociation of theactomyosin complex by phosphates prior to heating resulted in a decrease in gel rigidity and uniformity. Thus, part of the reduction in gel strength and cohesiveness caused by KBr, KI and KSCN may be due to dissociation of actomyosin.
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Fig. 3. Effectsof concentration of ([5]) KF, (x) KC1, (A) KBr, (A) KI or (I) KSCN on cohesiveness at 65% compression of actomyosin gels formed by heating at 70°C for 30 min at pH 6.0. The data in Figs 4-6 show that increasing the concentrations o f KC1 and NaC1 (0.2 to 1-0 M) increased stress, stress energy and gel cohesiveness c o m p a r e d with that o f the control. In general, there was no significant difference between the effects of NaC1 and KC1 on these rheological properties of actomyosin gels. Increasing concentrations o f NH4C1, MgC12 and CaC12 decreased stress, stress energy and cohesiveness. The relative effects o f the cations on the heat-induced gelling properties of actomyosin can be ranked as follows: K + ~ N a + > NH~ > M g 2+ > C a 2--. Both KC1 and NaC1 p r o m o t e and stabilize hydrophobic interactions ( D a m o d a r a n & Kinsella, 1982; Babajimopoulos et al., 1983). Thus, the positive effects o f KC1 and NaC1 on gel strength and cohesiveness m a y be accounted for by the strengthening effect o f these salts on hydrophobic protein-protein interactions within the actomyosin gel structure.
Formation and stabilization of heat-induced actomyosin gels
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CaC12on stress at 65% compression of actomyosin gels formed at 70°C for 30 min at pH 6-0. The adverse effect of NH4C1 on gel properties was surprising, since NH2 is generally considered to stabilize protein protein interactions (von Hippel & Schleich, 1969). However, Wicker et al. (1989) reported that NH4C1 also adversely affected the rigidity of myosin gels and that at a protein concentration of 6.5 mg ml i gelation was completely suppressed by 0-5 M NH4C1. Both CaC12 and MgC12 (0.2-1-0 M) reduced gel strength and cohesiveness dramatically compared with the control, which is consistent with the particular effectiveness of C a 2+ and Mg 2+ at these concentrations, in dissociating non-covalently linked protein-protein aggregates (von Hippel & Wong, 1965; von Hippel & Schleich, 1969). Based on the effects of neutral salts on the theological properties of actomyosin gels we suggest that hydrophobic protein-protein interactions play a significant role in actomyosin gel formation and stabiliza-
414
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CaCI~ on stress energy of actomyosin gels formed at 70°C for 30 rain at pH 6.0. tion. This is consistent with the work of Liu e t al. (1982), who showed that 2% sodium dodecyl sulphate effectively solubilized fish actomyosin coagulum and concluded that hydrophobic interactions were involved in heat-induced actomyosin aggregation. Addition of urea (0-4-2.4 M) progressively decreased the rheological parameters relating to the compressive strength of actomyosin gels, i.e. stress and stress energy at 65% compression (Fig. 7). At urea concentrations >2-0 M, the gels were very weak and fragile and at 2.8 M urea gelation did not occur. Urea destabilizes hydrophobic interactions and hydrogen bonding due to its effect on the structural properties of water and its potential for hydrogen bonding (von Hippel & Schleich, 1969;
Formation and stabilization of heat-induced actomyosin gels
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Fig. 6. Effectsof concentration of ([~) NaC1, (x) KCI, (A) NH4C1, (A) MgCI2 or ( I ) CaC12 on cohesiveness of actomyosin gels formed at 70°C for 30 min at pH 6.0. Creighton, 1984). The adverse effect o f urea on the compressive strength o f actomyosin gels indicates that hydrophobic interactions and hydrogen bonding are critically important for the formation of a rigid actomyosin gel structure. These non-covalent intermolecular forces have been implicated in the gelation o f myosin (Foegeding et al., 1987; Wicker et al., 1986, 1989). Dissociation of actomyosin to myosin and actin occurs in 1 M urea (Stracher, 1961). Thus, the reduction in gel strength caused by urea m a y be due partly to dissociation of actomyosin. Gel cohesiveness (Fig. 7) and translucency increased relative to the control in the presence o f ___0-8 M urea. The translucent nature o f these gels in contrast to the opaque control is indicative o f a reduction in intermolecular interactions and a smaller particle size (Schmidt & Illingworth, 1978; Schmidt et al., 1978). It appears that heating actomyosin solutions containing >0.8 M urea resulted in the formation o f a uniform network
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of polypeptide chains linked together at relatively few points and hence a weak cohesive gel matrix. Propylene glycol (1-10%) increased the compressive strength of actomyosin gels relative to the control but had no significant effect on gel cohesiveness (Fig. 8). Propylene glycol reduces hydrophobic associations but it enhances the contribution of hydrogen bonds (Tanford, 1962; Utsumi & Kinsella, 1985). These results suggest that intermolecular hydrogen bonding makes a significant contribution to the compressive strength of actomyosin gels. Myosin and actin contain 42 and five sulphydryl groups per molecule, respectively (Martasoni, 1968; Buttkus, 1971; Elzinga et al., 1973; Collins & Elzinga, 1975; Hofmann & Hamm, 1978). However, the role, if any, of disulphide formation in the gelation of myofibrillar proteins is not clear. Hamm and Hofmann (1965) reported that the coagulation of myofibrillar proteins by heating up to 70°C did not involve the
Formation and stabilization of heat-induced actomyosin gels 120
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formation of disulphide bonds. In contrast, Foegeding et al. (1987) concluded that disulphide bond formation plays a significant role in the crosslinking of myosin gels formed by heating to 50°C or 70°C. Addition of N E M (5-30 mM), which acts as a sulphydryl blocking agent and DTT (1 10 mM) and cysteine (5-10 mM), which are reducing agents, significantly decreased the compressive strength and cohesiveness df actomyosin gels relative to the control gels, but did not prevent gelation (Figs 9-11). These results suggest that while disulphide bridging is not a prerequisite for the formation of a gel network during the heatinduced gelation of actomyosin, intermolecular disulphide bonds contribute significantly to the mechanical strength and cohesiveness of the gels. The present findings are consistent with those of Itoch et al. (1979, 1980a, b) who concluded that protein-protein interactions during the gelation of fish actomyosin involved intermolecular disulphide bond formation. Ishioroshi et al. (1980, 1982) and Samejima et al. (1981) proposed that
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head-to-head aggregation of myosin molecules during heating to form super junctions provides extra crosslinking and strength to the gel network. Based on the gelling properties of myosin in the presence of DTT and pchloromercuribenzoate they concluded that this aggregation process is closely associated with the oxidation of thiol groups. It is possible that a similar crosslinking mechanism may operate in actomyosin gels. Overall, the results indicate that hydrophobic interactions and hydrogen bonding are the predominant molecular forces involved in the crosslinking of polypeptide chains in heat-induced actomyosin gels (pH 6-0, 0.6 M KC1). Covalent disulphide bonding between protein molecules is not essential for gel matrix formation; however, it contributes to the overall strength and cohesiveness of the gels. The role of hydrogen and hydrophobic bonding in determining gel cohesiveness is not clear, since both urea and
420
E. O'Neill, D. M. Mulvihill, P. A. Morrissey
propylene glycol increased gel cohesiveness. These results suggest that the cohesiveness of actomyosin gels m a y be governed by the distribution of intermolecular associations rather than the specific type of bonding.
REFERENCES Acton, J. C., Hanna, M. A. & Satterlee, L. D. (1981). J. Food Biochem., 5, 101. Acton, J. C., Ziegler, G. R. & Burge, D. L. (1983). CRC Crit. Rev. Food Sci. Nutr., 18, 99. Asghar, A., Samejima, K. & Yasui, T. (1985). CRC Crit. Rev. Food Sci. Nutr., 22, 27. Babajimopoulos, M., Damodaran, S., Rizvi, S. S. H. & KinseUa, J. E. (1983). J. Agric. Food Chem., 31, 1270. Bernal, V. M., Smajda, C. H., Smith, J. L. & Stanley, D. W. (1987). J. Food Sci., 52, 1121. Buttkus, H. (1971). Can. J. Biochem., 49, 97. Collins, J. H. & Elzinga, M. (1975). J. Biol. Chem., 250, 5915. Creighton, T. E. 1984). In Proteins. Structure and Molecular Principles, ed. T. E. Creighton. W. H. Freeman & Co., New York, p. 265. Damodaran, S. & Kinsella, J. E. (1981). J. Biol. Chem., 256, 3394. Damodaran, S. Kinsella, J. E. (1982). In Food Protein Deterioration Mechanisms and Functionality, ed. J. P. Cherry. American Chemical Society, Washington DC, p.327. Elzinga, M., Collins, J. H., Kuehl, W. M. & Adelstein, R. S. (1973). Proc. Natl Acad. Sci. USA, 70, 2687. Foegeding, E. A., Allen, C. E. Dayton, W. R. (1986). J. Food Sci., 51, 104. Foegeding, E. A., Dayton, W. R. & Allen, C. E. (1987). J. Agric. Chem., 35, 559. Hamm, R. & Hofmann, K. (1965). Nature, 207, 1269. Hay, J. D., Currie, R. W. & Wolfe, F. H. (1972). J. Food Sei., 37, 346. Hermansson, A.-M., Harbitz, O. & Langton, M. (1986). J. Sci. Food Agrie., 37, 69. Hofmann, K. & Hamm, R. (1978). Adv. Food Res., 24, 1. Holtzer, A., Wang, T. Y. & Noelken, M. E. (1960). Biochim. Biophys. Acta, 42, 453. Ishioroshi, M., Samejima, K. & Yasui, T. (1979). J. Food Sci., 44, 1280. Ishioroshi, M., Samejima, K., Arie, Y. & Yasui, T. (1980). Agric. Biol. Chem., 44, 2185. Ishioroshi, M. Samejima, K. & Yasui, T. (1982). J. Food Sci., 44, 114. Ishioroshi, M., Samejima, K. & Yasui, T. (1983). Agric. Biol. Chem., 47, 2809. Itoch, Y., Yoshinaka, R. & Ikeda, S. (1979). Bull. Jap. Soc. Sci. Fish., 45, 1019. Itoch, Y., Yoshinaka, R. & Ikeda, S. (1980a). Bull. Jap. Soe. Sci. Fish., 46, 617. Itoch, Y., Yoshinaka, R. & Ikeda, S. (1980b). Bull. Jap. Soc. Sci. Fish., 46, 621. Lanier, T. C., Lin, T. S., Liu, Y. M. & Hamann, D. D. (1982). J. Food Sci., 47, 1921. Liu, Y. M., Lin, T. S., & Hamann, D. D. (1982). J. Food Sei., 47, 1916. Martonosi, A. (1968). Arch. Biochem. Biophys., 123, 29.
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Morrissey, P. A., Mulvihill, D. & O'Neill, (1987). In Developments in Food Proteins--5, ed. B. J. F. Hudson. Elsevier Applied Science Publishers, London, p. 195. Mulvihill, D. M. & Kinsella, J. E. (1988). J. FoodSci., 53, 231. Samejima, K., Ishioroshi, M. Yasui, T. (1981). J. Food Sei., 46, 1412. Schmidt, R. H. & Illingworth, B. L. (1978). Food Product Dev., 12(10), 60. Schmidt, R. H., Illingworth, B. L., Ahmed, E. M. & Richert, R. L. (1978). J Food Proc. Pres., 2, 111. Stracher, A. (1961). J. Biol. Chem., 236, 2467. Tanford, C. (1962). J Am. Chem. Soc., 84, 4240. Utsumi, S. & Kinsella, J. E. (1985). J. Food Sci., 50, 1278. von Hippel, P. H. & Schleich, T. (1969). In Strueture and Stability of Biological Maeromolecules, ed. S. N. Timashaff & G. D. Fasman. Marcel Dekker Inc., New York, p. 417. von Hippel, P. H. & Wong, K. Y. (1965). J. Biol. Chem., 240, 3909. Wicker, L., Lanier, T. C., Hamann, D. D. & Akahane, T. (1986). J. Food Sei., 51, 1540. Wicker, L., Lanier, T. C., Knopp, J. A. & Hamann, D. D. (1989). J. Agric. Food Chem., 37, 18. Wu, J. Q, Hamann, D. D. & Foegeding, E. A. (1991). J. Agric. Food Chem., 39, 229. Yasui, T., Ishioroshi, M. & Samejima, K. (1980). J. Food Bioehem., 4, 61. Yasui, T., Ishioroshi, M. & Samejima, K. (1982). Agric. Biol. Chem., 46, 1049.
Molecular Forces Involved in the Formation and Stabilization of Heat-Induced Actomyosin Gels E. O'Neill, a D. M. Mulvihill a & P. A. M o r r i s s e y , b a Department of Food Chemistry, b Department of Nutrition, University College, Cork, Ireland (Received 12 September 1992; revised version received 28 January 1993; accepted 6 February 1993)
ABSTRA CT Increasing concentrations of KCl, KF and NaCl from 0.2 to 1.0 m increased the compressive strength and cohesiveness of heat-induced actomyosin gels (0"6 m KCI p H 6.0). On the other hand, KBr, KSCN, KI, NH4CI, MgCI2 and CaCl2 decreased gel strength and cohesiveness. Gel compressive strength increased with increasing urea concentration (0.4-2.4 m). Addition of the sulphydryl blocking agent N-ethylmaleimide and the reducing agents dithiothreitol and cysteine decreased the compressive strength and cohesiveness of actomyosin gels. Overall, the results indicate that hydrophobic interactions, disulphide and hydrogen bonding contribute to the formation and stabilization of actomyosin gels.
INTRODUCTION F o r m a t i o n of a three-dimensional gel network by myosin and/or actomyosin during thermal processing contributes significantly to the textural aspects and stabil!ty of comminuted and formed meat products (Acton et al., 1983; Asghar et aL, 1985; Morrissey et al., 1987). Previous studies have shown that the properties and characteristics of myosin and actomyosin gels are influenced by protein concentration, heating temperature and rate, and the critical balance between attractive and repulsive forces (Ishioroshi et al., 1979, 1983; Acton et al., 1981; Lanier et al., 1982; Hermansson et al., 1986; Wicker et aL, 1986; Foegeding et al., 1986; 407 Meat Science 0309-1740/94/$07.00 © 1994 Elsevier Science Ltd, England. Printed in Great Britain
408
E. O'Neill, D. M. Mulvihill, P. A. Morrissey
Wu et al., 1991). Disulphide linkages (Samejima et al., 1981; Ishioroshi et al., 1982; Foegeding et al., 1987), hydrogen and hydrophobic bonding (Samejima et al., 1981; Wicker et al., 1986, 1989; Foegeding et al., 1987) are considered important in myosin gelation. However, the molecular forces involved in the formation and stabilization of actomyosin gels, which differ considerably in ultrastructure from myosin gels (Yasui et al., 1982), have not been fully characterized. In an attempt to elucidate the major molecular forces that contribute to the formation and stabilization of actomyosin gel structures (in 0-6 M KC1), the effects of added neutral salts, urea, propylene glycol, dithiothreitol (DTT), N-ethylmaleimide (NEM) and cysteine, which enhance or block specific protein-protein interactions, on the rheological properties of actomyosin gels were determined in this study.
MATERIALS AND METHODS
Preparation of actomyosin Actomyosin was prepared from rabbit skeletal muscle by the method described by Hay et al. (1972).
Preparation of actomyosin gels A stock solution of actomyosin containing 100 mg mF ~ protein was prepared in 0-6 M KC1, 0-1 N imidazole-HC1 buffer, pH 6.0. Stock solutions containing the various salts and~reagents were also prepared in 0-6 M KC1, 0.1 M imidazole-HC1 buffer, pH 6-0. To prepare the gelling solutions, the stock actomyosin solution was diluted in a 1:1 volume ratio with 0.6 M KC1, 0-1M imidazole-HC1 buffer, pH 6.0, or with stock solutions of KF, KSCN, KBr, KI, KC1, NaC1, NH4C1 , MgC12, CaC12, urea, propylene glycol, DTT, N E M or cysteine to give the desired salt or reagent concentration. Aliquots (1 ml) of the gelling solutions were transferred to glass tubes (inside diameter 6.0 mm), which had been treated with Sigrnacote (Sigma Chemical Company, St. Louis, Co.) and air-dried. The tubes containing the gelling solutions were centrifuged at low speed (500 g) to remove air bubbles and then heat-sealed to prevent evaporation. The samples were equilibrated in a water bath at 25°C for 15 min then heated to 70°C at a rate of 1.1°C min -1 and maintained a t 70°C for 30 min. The tubes were removed from the water bath, cooled in ice-water and held at 4°C for 24 h.
Formation and stabilization of heat-induced actomyosin gels
409
Measurement of gel rheological properties The gels were equilibrated at room temperature for 3 h and then removed carefully from the glass tubes. Sections 5 m m high were cut with a razoredge cutting device. Rheological properties were determined on these sections with an Instron Universal Testing Instrument (Table Model T M - M ) using an arrangement similar to that described by Mulvihill and Kinsella (1988). The top plate and base plate were set 20 m m apart. A gel section (5 m m high x 6 m m in diameter) was placed on the base plate and compressed by 65% of its original height (3.25 mm) on two successive occasions by lowering the top plate 18.25 m m from its original setting and raising it again at a speed of 10 m m per minute. The force exerted on the top plate was recorded continuously on a chart recorder operating at a speed of 500 m m min ~. The following rheological parameters were determined from the force deformation curves as described by Mulvihill and Kinsella (1988). Stress at 65% compression, units -- N m -2 Stress energy at 65% compression, units = J Cohesiveness, units -- ratio, dimensionless. All the rheological results reported are mean values calculated from a m i n i m u m of seven and up to eleven separate deformation curves obtained from different gel sections.
RESULTS AND DISCUSSION The presence of neutral salts had dramatic effects on the rheological properties of heat-induced actomyosin gels, which cannot be attributed solely to simple non-specific charge neutralization (Figs 1-6). Increasing concentrations of KC1 and K F from 0.2 to 1-0 M progressively increased stress, stress energy and gel cohesiveness relative to the control (Figs 1 3). On the other hand, KBr, K S C N and KI decreased stress, stress energy and gel cohesiveness. The relative effects of the anions on the compressive strength and cohesiveness of the heat-induced actomyosin gels was in the order: F >C1 > B r > S C N - > I With the exception of I- these anions generally behaved according to the Hofmeister series (von Hippel & Schleich, 1969), where the anions are ranked on the basis of their ability to salt out proteins. In the present study I was found to be the most effective anion in decreasing gel
410
E. O'Neill, D. M. Mulvihill, P. A. Morrissey
50
~-~ 40 E z
30
20
10
0 0.2
• 0.4
0.6
0.8
1.0
Salt concentration (M)
Fig. 1. Effects of concentration of (E3) KF, (X) KC1, (A) KBr, (A) KI or (11) KSCN on stress at 65% compression of actomyosin gels formed by heating at 70°C for 30 rain at pH 6.0.
strength and cohesiveness. This effect is not in line with its position between SCN- and Br- in the Hofmeister series. The effects of these salts on actomyosin gelation are probably related to their ability to 'salt out' proteins and promote coagulation. The Fand C1- anions enhance the hydrogen bonded structure of the bulk water phase and hence have a tendency to increase the driving force for hydrophobic interactions within and between proteins resulting in protein association and aggregation (Damodaran & Kinsella, 1982; Babajimopoulos et al., 1983). The positive effects of KF and KC1 on actomyosin gel strength and cohesiveness suggest that hydrophobic interactions are involved in structuring and stabilizing the gels. On the other hand, Br-, £, SCN- are large ions with low charge densities and tend to break water structure (von Hippel & Wong, 1965; Damodaran & Kinsella, 1982). These anions favour destabilization of hydrophobic interactions between protein molecules (Damodaran &
Formation and stabilization of heat-induced actomyosin gels
411
200[ 150
-
×
~ lO0 -
o
I
I
I
I
0.2
0.4
0.6
0.8
~ 1.0
Salt concentration (M) Fig. 2. Effects of concentration of ([Z]) KF, (X) KC1, (A) KBr, (A) KI or ( 1 ) KSCN on stress energy at 65% compression of actomyosin gels formed by heating at 70°C for 30 min at pH 6.0.
Kinsella, 1981, 1982; Utsumi & Kinsella, 1985; Bernal et al., 1987). Thus, the decrease in gel strength and cohesiveness observed in the presence of KBr, KSCN and KI (Figs 1-3) may be due to a decrease in protein-protein hydrophobic associations within the actomyosin gel matrix. Dissociation of actomyosin to myosin and G-actin has been shown to occur at concentrations > 0-3 M KBr, KI or KSCN (Holtzer et al., 1960). Yasui et al. (1980) and Ishioroshi et al. (1980) demonstrated that dissociation of theactomyosin complex by phosphates prior to heating resulted in a decrease in gel rigidity and uniformity. Thus, part of the reduction in gel strength and cohesiveness caused by KBr, KI and KSCN may be due to dissociation of actomyosin.
412
E. O'Neill, D. M. Mulvihill, P. A. Morrissey
08/ 0.7
-
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0.5
o.4
I
I
I
I
I
0.2
0.4
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Salt concentration (M)
Fig. 3. Effectsof concentration of ([5]) KF, (x) KC1, (A) KBr, (A) KI or (I) KSCN on cohesiveness at 65% compression of actomyosin gels formed by heating at 70°C for 30 min at pH 6.0. The data in Figs 4-6 show that increasing the concentrations o f KC1 and NaC1 (0.2 to 1-0 M) increased stress, stress energy and gel cohesiveness c o m p a r e d with that o f the control. In general, there was no significant difference between the effects of NaC1 and KC1 on these rheological properties of actomyosin gels. Increasing concentrations o f NH4C1, MgC12 and CaC12 decreased stress, stress energy and cohesiveness. The relative effects o f the cations on the heat-induced gelling properties of actomyosin can be ranked as follows: K + ~ N a + > NH~ > M g 2+ > C a 2--. Both KC1 and NaC1 p r o m o t e and stabilize hydrophobic interactions ( D a m o d a r a n & Kinsella, 1982; Babajimopoulos et al., 1983). Thus, the positive effects o f KC1 and NaC1 on gel strength and cohesiveness m a y be accounted for by the strengthening effect o f these salts on hydrophobic protein-protein interactions within the actomyosin gel structure.
Formation and stabilization of heat-induced actomyosin gels
413
40
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25 E z
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I 0.2
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[ 1.0
Effects o f concentration o f (V]) NaCI, ( x ) KC1, (A) NH4C], (A) MgC12 or (m)
CaC12on stress at 65% compression of actomyosin gels formed at 70°C for 30 min at pH 6-0. The adverse effect of NH4C1 on gel properties was surprising, since NH2 is generally considered to stabilize protein protein interactions (von Hippel & Schleich, 1969). However, Wicker et al. (1989) reported that NH4C1 also adversely affected the rigidity of myosin gels and that at a protein concentration of 6.5 mg ml i gelation was completely suppressed by 0-5 M NH4C1. Both CaC12 and MgC12 (0.2-1-0 M) reduced gel strength and cohesiveness dramatically compared with the control, which is consistent with the particular effectiveness of C a 2+ and Mg 2+ at these concentrations, in dissociating non-covalently linked protein-protein aggregates (von Hippel & Wong, 1965; von Hippel & Schleich, 1969). Based on the effects of neutral salts on the theological properties of actomyosin gels we suggest that hydrophobic protein-protein interactions play a significant role in actomyosin gel formation and stabiliza-
414
E. O'Neill, D. M. Mulvihill, P. A. Morrissey
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80
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~'£ 60' × >.
0~
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Fig. 5. Effects of concentration of (D) NaC1, (x) KCI, (zX)NH4CI, (A) MgC12 or ( I )
CaCI~ on stress energy of actomyosin gels formed at 70°C for 30 rain at pH 6.0. tion. This is consistent with the work of Liu e t al. (1982), who showed that 2% sodium dodecyl sulphate effectively solubilized fish actomyosin coagulum and concluded that hydrophobic interactions were involved in heat-induced actomyosin aggregation. Addition of urea (0-4-2.4 M) progressively decreased the rheological parameters relating to the compressive strength of actomyosin gels, i.e. stress and stress energy at 65% compression (Fig. 7). At urea concentrations >2-0 M, the gels were very weak and fragile and at 2.8 M urea gelation did not occur. Urea destabilizes hydrophobic interactions and hydrogen bonding due to its effect on the structural properties of water and its potential for hydrogen bonding (von Hippel & Schleich, 1969;
Formation and stabilization of heat-induced actomyosin gels
415
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Fig. 6. Effectsof concentration of ([~) NaC1, (x) KCI, (A) NH4C1, (A) MgCI2 or ( I ) CaC12 on cohesiveness of actomyosin gels formed at 70°C for 30 min at pH 6.0. Creighton, 1984). The adverse effect o f urea on the compressive strength o f actomyosin gels indicates that hydrophobic interactions and hydrogen bonding are critically important for the formation of a rigid actomyosin gel structure. These non-covalent intermolecular forces have been implicated in the gelation o f myosin (Foegeding et al., 1987; Wicker et al., 1986, 1989). Dissociation of actomyosin to myosin and actin occurs in 1 M urea (Stracher, 1961). Thus, the reduction in gel strength caused by urea m a y be due partly to dissociation of actomyosin. Gel cohesiveness (Fig. 7) and translucency increased relative to the control in the presence o f ___0-8 M urea. The translucent nature o f these gels in contrast to the opaque control is indicative o f a reduction in intermolecular interactions and a smaller particle size (Schmidt & Illingworth, 1978; Schmidt et al., 1978). It appears that heating actomyosin solutions containing >0.8 M urea resulted in the formation o f a uniform network
E. O'Neill, D. M. Mutvihill, P. A. Morrissey
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Effects o f urea concentrations on (A) stress ( k N m 2), ( 0 ) stress energy (J x 105) and ( O ) cohesiveness at 65% compression o f actomyosin gels.
of polypeptide chains linked together at relatively few points and hence a weak cohesive gel matrix. Propylene glycol (1-10%) increased the compressive strength of actomyosin gels relative to the control but had no significant effect on gel cohesiveness (Fig. 8). Propylene glycol reduces hydrophobic associations but it enhances the contribution of hydrogen bonds (Tanford, 1962; Utsumi & Kinsella, 1985). These results suggest that intermolecular hydrogen bonding makes a significant contribution to the compressive strength of actomyosin gels. Myosin and actin contain 42 and five sulphydryl groups per molecule, respectively (Martasoni, 1968; Buttkus, 1971; Elzinga et al., 1973; Collins & Elzinga, 1975; Hofmann & Hamm, 1978). However, the role, if any, of disulphide formation in the gelation of myofibrillar proteins is not clear. Hamm and Hofmann (1965) reported that the coagulation of myofibrillar proteins by heating up to 70°C did not involve the
Formation and stabilization of heat-induced actomyosin gels 120
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Effects of propylene glycol concentration on ( 0 ) stress (kN m-2), (A) stress
energy (J × 105)and (O) cohesivenessat 65% compression of actomyosin gels.
formation of disulphide bonds. In contrast, Foegeding et al. (1987) concluded that disulphide bond formation plays a significant role in the crosslinking of myosin gels formed by heating to 50°C or 70°C. Addition of N E M (5-30 mM), which acts as a sulphydryl blocking agent and DTT (1 10 mM) and cysteine (5-10 mM), which are reducing agents, significantly decreased the compressive strength and cohesiveness df actomyosin gels relative to the control gels, but did not prevent gelation (Figs 9-11). These results suggest that while disulphide bridging is not a prerequisite for the formation of a gel network during the heatinduced gelation of actomyosin, intermolecular disulphide bonds contribute significantly to the mechanical strength and cohesiveness of the gels. The present findings are consistent with those of Itoch et al. (1979, 1980a, b) who concluded that protein-protein interactions during the gelation of fish actomyosin involved intermolecular disulphide bond formation. Ishioroshi et al. (1980, 1982) and Samejima et al. (1981) proposed that
E. O'Neill, D. M. Mulvihill, P. A. Morrissey
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Formation and stabilization of heat-induced actomyosin gels
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0.66
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Effects of concentration of (D) N-ethylmaleimide, (A) dithiothreitol or (N) cysteine on cohesiveness at 65% compression of actomyosin gels.
head-to-head aggregation of myosin molecules during heating to form super junctions provides extra crosslinking and strength to the gel network. Based on the gelling properties of myosin in the presence of DTT and pchloromercuribenzoate they concluded that this aggregation process is closely associated with the oxidation of thiol groups. It is possible that a similar crosslinking mechanism may operate in actomyosin gels. Overall, the results indicate that hydrophobic interactions and hydrogen bonding are the predominant molecular forces involved in the crosslinking of polypeptide chains in heat-induced actomyosin gels (pH 6-0, 0.6 M KC1). Covalent disulphide bonding between protein molecules is not essential for gel matrix formation; however, it contributes to the overall strength and cohesiveness of the gels. The role of hydrogen and hydrophobic bonding in determining gel cohesiveness is not clear, since both urea and
420
E. O'Neill, D. M. Mulvihill, P. A. Morrissey
propylene glycol increased gel cohesiveness. These results suggest that the cohesiveness of actomyosin gels m a y be governed by the distribution of intermolecular associations rather than the specific type of bonding.
REFERENCES Acton, J. C., Hanna, M. A. & Satterlee, L. D. (1981). J. Food Biochem., 5, 101. Acton, J. C., Ziegler, G. R. & Burge, D. L. (1983). CRC Crit. Rev. Food Sci. Nutr., 18, 99. Asghar, A., Samejima, K. & Yasui, T. (1985). CRC Crit. Rev. Food Sci. Nutr., 22, 27. Babajimopoulos, M., Damodaran, S., Rizvi, S. S. H. & KinseUa, J. E. (1983). J. Agric. Food Chem., 31, 1270. Bernal, V. M., Smajda, C. H., Smith, J. L. & Stanley, D. W. (1987). J. Food Sci., 52, 1121. Buttkus, H. (1971). Can. J. Biochem., 49, 97. Collins, J. H. & Elzinga, M. (1975). J. Biol. Chem., 250, 5915. Creighton, T. E. 1984). In Proteins. Structure and Molecular Principles, ed. T. E. Creighton. W. H. Freeman & Co., New York, p. 265. Damodaran, S. & Kinsella, J. E. (1981). J. Biol. Chem., 256, 3394. Damodaran, S. Kinsella, J. E. (1982). In Food Protein Deterioration Mechanisms and Functionality, ed. J. P. Cherry. American Chemical Society, Washington DC, p.327. Elzinga, M., Collins, J. H., Kuehl, W. M. & Adelstein, R. S. (1973). Proc. Natl Acad. Sci. USA, 70, 2687. Foegeding, E. A., Allen, C. E. Dayton, W. R. (1986). J. Food Sci., 51, 104. Foegeding, E. A., Dayton, W. R. & Allen, C. E. (1987). J. Agric. Chem., 35, 559. Hamm, R. & Hofmann, K. (1965). Nature, 207, 1269. Hay, J. D., Currie, R. W. & Wolfe, F. H. (1972). J. Food Sei., 37, 346. Hermansson, A.-M., Harbitz, O. & Langton, M. (1986). J. Sci. Food Agrie., 37, 69. Hofmann, K. & Hamm, R. (1978). Adv. Food Res., 24, 1. Holtzer, A., Wang, T. Y. & Noelken, M. E. (1960). Biochim. Biophys. Acta, 42, 453. Ishioroshi, M., Samejima, K. & Yasui, T. (1979). J. Food Sci., 44, 1280. Ishioroshi, M., Samejima, K., Arie, Y. & Yasui, T. (1980). Agric. Biol. Chem., 44, 2185. Ishioroshi, M. Samejima, K. & Yasui, T. (1982). J. Food Sci., 44, 114. Ishioroshi, M., Samejima, K. & Yasui, T. (1983). Agric. Biol. Chem., 47, 2809. Itoch, Y., Yoshinaka, R. & Ikeda, S. (1979). Bull. Jap. Soc. Sci. Fish., 45, 1019. Itoch, Y., Yoshinaka, R. & Ikeda, S. (1980a). Bull. Jap. Soe. Sci. Fish., 46, 617. Itoch, Y., Yoshinaka, R. & Ikeda, S. (1980b). Bull. Jap. Soc. Sci. Fish., 46, 621. Lanier, T. C., Lin, T. S., Liu, Y. M. & Hamann, D. D. (1982). J. Food Sci., 47, 1921. Liu, Y. M., Lin, T. S., & Hamann, D. D. (1982). J. Food Sei., 47, 1916. Martonosi, A. (1968). Arch. Biochem. Biophys., 123, 29.
Formation and stabilization of heat-induced actomyosin gels
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Morrissey, P. A., Mulvihill, D. & O'Neill, (1987). In Developments in Food Proteins--5, ed. B. J. F. Hudson. Elsevier Applied Science Publishers, London, p. 195. Mulvihill, D. M. & Kinsella, J. E. (1988). J. FoodSci., 53, 231. Samejima, K., Ishioroshi, M. Yasui, T. (1981). J. Food Sei., 46, 1412. Schmidt, R. H. & Illingworth, B. L. (1978). Food Product Dev., 12(10), 60. Schmidt, R. H., Illingworth, B. L., Ahmed, E. M. & Richert, R. L. (1978). J Food Proc. Pres., 2, 111. Stracher, A. (1961). J. Biol. Chem., 236, 2467. Tanford, C. (1962). J Am. Chem. Soc., 84, 4240. Utsumi, S. & Kinsella, J. E. (1985). J. Food Sci., 50, 1278. von Hippel, P. H. & Schleich, T. (1969). In Strueture and Stability of Biological Maeromolecules, ed. S. N. Timashaff & G. D. Fasman. Marcel Dekker Inc., New York, p. 417. von Hippel, P. H. & Wong, K. Y. (1965). J. Biol. Chem., 240, 3909. Wicker, L., Lanier, T. C., Hamann, D. D. & Akahane, T. (1986). J. Food Sei., 51, 1540. Wicker, L., Lanier, T. C., Knopp, J. A. & Hamann, D. D. (1989). J. Agric. Food Chem., 37, 18. Wu, J. Q, Hamann, D. D. & Foegeding, E. A. (1991). J. Agric. Food Chem., 39, 229. Yasui, T., Ishioroshi, M. & Samejima, K. (1980). J. Food Bioehem., 4, 61. Yasui, T., Ishioroshi, M. & Samejima, K. (1982). Agric. Biol. Chem., 46, 1049.