Role of secondary structures in the gelation of porcine myosin at different pH values

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MEAT SCIENCE Meat Science 80 (2008) 632–639 www.elsevier.com/locate/meatsci

Role of secondary structures in the gelation of porcine myosin at different pH values Ru Liu a,b, Si-ming Zhao a, Shan-bai Xiong a,b,*, Bi-jun Xie a, Li-hong Qin c a College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei Province 430070, PR China Aquatic Product Engineering and Technology Research Center of Hubei Province, Wuhan, Hubei Province 430070, PR China c The Public Laboratory of Electron Microscope, Huazhong Agricultural University, Wuhan, Hubei Province 430070, PR China b

Received 3 December 2007; received in revised form 15 February 2008; accepted 15 February 2008

Abstract Secondary structures, gelation properties and their relationships in porcine myosin were studied by circular dichroism, dynamic rheological measurement and scanning electron microscopy. Gelling of porcine myosin involved a change in myosin conformation with protein–protein and protein–water interactions. The gelation properties were strongly pH and temperature dependent. Near the pI (pH 5.5 and 6.0), porcine myosin could spontaneously coagulate at 15 °C resulting partially from the presence of more b-sheets. Myosin at pH 6.5–9.0 began to form a gel at temperatures greater than 38 °C. Heating caused a-helices to partially turn into b-sheets and random coils. Subsequently, myosin aggregated and formed a gel network. The gel strength decreased and the water-holding capacity (WHC) increased with increasing pH. Correlation analysis indicated that both the unfolding of a-helices and the formation of b-sheets favored the gelation of porcine myosin. A high b-sheet fraction prior to heating resulted in a low WHC of resultant gel. A compact and uniform gel was also obtained at pH 6.5. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Myosin; Secondary structure; Gel property; Microstructure; pH

1. Introduction Gel formation in meat involves partial denaturation of protein followed by irreversible aggregation which results in a three dimensional network (Lanier, Carvajal, & Yongsawatdigul, 2004). Myosin is abundant in muscle protein and plays a key role in gel development in meat products. Myosin conformation is highly sensitive to changes in pH (Lin & Park, 1998) and temperature (Liu, Zhao, Xiong, Xie, & Liu, 2007; Yongsawatdigul & Sinsuwan, 2007). Disruptions in protein structure resulting from changes in * Corresponding author. Address: College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei Province 430070, PR China. Tel./fax: +86 027 87288375. E-mail addresses: [email protected] (R. Liu), zsmjx@ mail.hzau.edu.cn (S.-m. Zhao), [email protected] (S.-b. Xiong), [email protected] (B.-j. Xie), [email protected] (L.-h. Qin).

0309-1740/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2008.02.014

these variables could expose some reactive groups, which may then affect gel strength. Therefore, altering pH and heating tissues will influence the gelation properties of myosin. Numerous studies have reported the relationship between pH value and gel quality (Lesio´w & Xiong, 2003; O’Neill, Morrissey, & Mulvihill, 1993; Ruiz-Ramı´rez, Arnau, Serra, & Gou, 2005). It has been suggested that altering the pH of meat products could result in the attainment of the desired gel strength at a given temperature (Westphalen, Briggs, & Lonergan, 2005). Nevertheless, little information is available on the relationship between the gelation properties and secondary structures of myosin. Circular dichroism (CD) is a valuable spectroscopic technique for studying protein secondary structures in diluted solutions (Greenfield, 1999; Choi & Ma, 2007) whilst dynamic rheological measurements are useful in evaluating gelling behavior under certain heating conditions. In this paper, CD was employed to monitor the

R. Liu et al. / Meat Science 80 (2008) 632–639

secondary structures of porcine myosin. Gel properties were evaluated by dynamic rheological measurement, scanning electron microscopy (SEM) and the assessment of water-holding capacity (WHC). Subsequently, secondary structures, gelation properties and their relationships in porcine myosin at different pH values were studied. The aim was to provide more insight into the gelation properties of porcine myosin, allowing the manipulation of processing conditions in order to obtain products with the desired structural and textural attributes.

633

MHC

200 kDa 130 kDa 97.4 kDa 66.2 kDa

actin

43 kDa

2. Materials and methods

MLC

2.1. Preparation of myosin Longissimus dorsi samples (pH 6.3) were taken from pigs immediately post-exsanguination. The samples were directly frozen in liquid nitrogen and stored at 80 °C until analysis. Myosin was prepared according to the method of Park and Lanier (1989) with some modifications. All solutions used for the myosin preparation were kept cold at 4 °C to minimize proteolysis and protein denaturation. The frozen meat was powered by a grinder (FW80, Jinnan Instrument Factory, China) in liquid nitrogen and mixed with 10 volumes of solution A (0.10 M KCl, 0.02% sodium azide and 20 mM Tris–HCl buffer, pH 7.5). This was then homogenized using an inline dispersing homogenizer (Model FJ-200, Shanghai specimen and models factory, China) operating at its maximum speed setting (23,000 rpm) for 1 min. The mixture was incubated for 15 min at 4 °C and centrifuged at 3000g for 5 min in a high-speed refrigerated centrifuge (Hitachi, Japan). The sediment was suspended with five volumes of solution B (0.45 M KCl, 5 mM b-mercaptoethanol, 0.2 M Mg(COO)2, 1 mM EGTA and 20 mM Tris–maleate buffer, pH 6.8), mixed with adenosine 50 -triphosphate (ATP) to a final concentration of 10 mM and incubated for 90 min at 4 °C. The mixture was then centrifuged at 12,000g for 10 min. The resulting supernatant was diluted slowly with five volumes of 1 mM KHCO3 and kept at 4 °C for 15 min. Following this, the mixture was centrifuged at 12,000g for 10 min and the pellet was re-suspended with 2.5 volumes of solution C (0.5 M KCl, 5 mM b-mercaptoethanol and 20 mM tris–HCl buffer, pH 7.5). The re-suspended pellet was incubated at 4 °C for 10 min and diluted with 2.5 volumes of 1 mM KHCO3. The mixture was kept overnight at 4 °C prior to centrifugation at 20,000g for 15 min. The myosin pellet was dissolved in 0.5 M NaCl–20 mM Tris–HCl buffer (pH 7.0) and then centrifuged at 5000g for 10 min following which 0.02% sodium azide was added for preservation. Myosin purity was checked using sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS–PAGE) (Laemmli, 1970). Fig. 1 illustrates the electrophoretic profile of the extracted myosin. The purity of the myosin samples was greater than 90% as determined by densitometry (Gel Logic 200 Imaging System, Kodak Co., USA), although

1

2

3

Fig. 1. SDS–PAGE of extracted myosin (lane 3) from porcine longissimus dorsi. Lane 1 designates molecular weight markers. As a reference to monitor the myosin isolation and purification procedure, intact porcine muscle was denatured and applied to the gel (lane 2). Lanes 2 and 3 were loaded with 20 lg of protein, and lane 1 with 10 lg. MHC: myosin heavy chain; MLC: myosin light chain.

a little actin did remain. The samples were stored at 4 °C and used within three days of preparation. The storage period did not change its electrophoretic pattern. The protein content was determined by the Lowry method (Lowry, Rosebrough, & Randall, 1951) using bovine serum albumin (BSA) as a standard. The protein concentration was adjusted depending on the experiment. 2.2. Preparation of the thermal gel Myosin solutions (16 mg/mL) at various pH values (5.5, 6.0, 6.5, 7.0, 7.5, 8.0 and 9.0) were obtained by the addition of 1 M HCl or 1 M Tris using 25 lL micro syringes. The mixture was quickly homogenated and the pH value was measured using a pH meter (model 818, Orion Research Inc., USA). Each sample (5 mL) was placed in a 10 mL capped plastic centrifuge tube. Three replicates of each treatment were used. The tubes were heated for 60 min in a water bath held at 40 °C, followed by 30 min in a water bath at 90 °C. The tubes were immediately cooled by being held in flowing tap water, and then stored overnight at 4 °C for WHC determination and SEM observations. 2.3. Circular dichroism spectrum The CD spectrum was measured using a Jasco J-810 spectropolarimeter (Jasco Co. Ltd., Tokyo, Japan). The myosin sample was transferred to a quartz cell with a 1 cm light-path length. Molecular ellipticity was measured in the range from 200 to 250 nm, the scan rate was 100 nm/min and the temperature was regulated with a control unit. The effect of pH was assessed at 15 °C. To measure the effects of temperature, a pH of 7.0 was used and the temperature was increased at a rate of 2 °C/min from

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5 to 90 °C then held for 2 min before measurement. Spectra were averaged over three scans and corrected for the solvent signal. A mean residue weight of 110 g/mol has been assumed. The percentages of a-helix, b-sheet, b-turn and random coil structures were determined using the protein secondary structure estimation program (Yang’s method) provided with a Jasco J-810 spectropolarimeter. All treatments were tested in triplicate; mean values from these replicates are represented in the data reported. 2.4. Dynamic rheological measurement Dynamic rheological studies were performed on an AR500 dynamic rheometer (TA Co. Ltd., Manchester, England). A 40 mm parallel steel plate geometry with a 1 mm gap was used and the myosin solution (16 mg/mL) was surrounded by liquid paraffin to prevent drying. Samples were heated at a rate of 1 °C/min from 15 to 90 °C.

a 200

210

220

230

240

250

[θ] (×103 deg.cm2/dmol)→

pH 9.0 pH 8.0 pH 7.5 pH 7.0 pH 6.5 pH 6.0 pH 5.5

Wavelength (nm)

2.5. Water-holding capacity Water-holding capacity (WHC, %) was measured based on the protocol described by Kocher and Foegeding (1993). Myosin thermal gels were centrifuged at 1000g for 10 min at 4 °C. The weights (g) of the centrifuge tubes, protein samples and moisture loss were all recorded. The following formula was used to determine WHC (%): WHCð%Þ ¼

CG  ML  100 CG

ð1Þ

where ML is the amount (g) of moisture lost from the gel during centrifugation and CG is the weight (g) of the heated gel. One replicate consisted of three observations per treatment and all treatments were tested in triplicate. 2.6. Scanning electron microscopy (SEM) The thermal gels were cut into 1–2 mm cubes, fixed with 2.5% glutaraldehyde containing 0.1 M Na phosphate (pH 7.3) at 4 °C overnight, and then washed three times using 0.1 M Na phosphate (pH 7.3). The post-fixation stage involved exposure to 1% osmium tetroxide for 2 h followed by three further washes using 0.1 M Na phosphate (pH 7.3). The washed samples were dehydrated in 10% dimethyl sulphoxide (DMSO) for 6 h and then freeze-dried. Following this they were coated with platinum–palladium in an IB-5 ion coater (EIKO Ltd., Tokyo, Japan). The specimens were observed in a JSM-6390 PLV scanning electron microscope (JEOL Ltd., Tokyo, Japan) with an accelerating voltage of 10 kV. 2.7. Statistical analysis

b

α-Helix

β-Sheet

β-Turn

Random coil

The percentage of each secondary structure type was determined using the protein secondary structure estimation program (Yang’s method). The data were analyzed using Excel and Statistical Analysis System software (SAS Institute Inc., Cary, NC, USA). Duncan’s multiple range test was used to evaluate the differences between treatments. Pearson correlation analysis was performed to explore the relationship between gel properties and secondary structures prior to heating. The differences were considered significant at P < 0.05.

100

Secondary structure (%)

The oscillation stress was 0.6 Pa, and the oscillation frequency was 0.1 Hz. Elastic modulus (G0 ), viscous modulus (G00 ) and phase angle (d) were recorded. All treatments were tested in triplicate.

80

60

40

20

0

3. Results and discussion 5.5

6.0

6.5

7.0

7.5

8.0

9.0

pH Fig. 2. CD spectra (a) and secondary structures (b) of myosin at various pH values (15 °C).

3.1. Circular dichroism of porcine myosin solution Myosin consists of two globular head regions, each having two non-covalently bonded light chains, and a rod-like tail portion that is a coiled-coil of a-helix (Harrington &

R. Liu et al. / Meat Science 80 (2008) 632–639

Rodgers, 1984). The fraction of each secondary structure present depends, amongst other things, on the myosin’s linear amino acid sequence (primary structure), pH condition and temperature. Fig. 2 shows the CD spectra and secondary structure fractions of myosin solutions under different pH conditions at 15 °C. At neutral pH (pH 7.0), the CD spectrum exhibited two minima at around 208 and 222 nm, suggesting the predominant presence of a-helix structures (Greenfield, 1999). The a-helix structure is mainly stabilized by hydrogen bonds between the carbonyl oxygen (–CO) and amino hydrogen (NH–) of a polypeptide chain (Sano, Ohno, Otsuka-Fuchino, Matsumoto, & Tsuchiya, 1994; Damodaran, 1996). Electrostatic interactions between amino acids also contribute to stability of secondary structures (Damodaran, 1996; Satoh, Nakaya, Ochiai, & Watabe, 2006). Shifting pH away from neutral significantly decreased the negative ellipticity values at 208 and 222 nm, denoting a loss of a-helix structures. As pH fell from 7.0 to 5.5, a-helical content markedly decreased from 87.4% to 15.8% (Fig. 2b). The isoelectric point (pI) of myosin is at about pH 5.5 (Foegeding & Lanier, 1996), implying that it is negatively charged at neutral pH. Reducing the pH might enhance the electrostatic interactions in the protein via charge neutralization, and influence the stability of the hydrogen bonds (Damodaran, 1996). The changes in electrostatic interactions and hydrogen bond stability could in turn contribute to the loss of a-helix content under acidic conditions. As the pH increased into the alkaline range, a net increase in negative charges produced abundant repulsion which promoted the extension of the myosin chain. Alkaline treatments also decreased the ahelix content, although there was no marked change in content at pH in the range 7.5–9.0. The b-sheet fraction declined rapidly from 41.2% to 5.1% when pH was increased from 5.5 to 7.0, and continued to decrease further but at a reduced rate with further increases in pH (Fig. 2b).

α-helix

β-sheet

β-turn

random coil

Secondary structure (%)

100

80

60

40

20

0

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90

Temperature (ºC) Fig. 3. Effect of temperature on secondary structures of myosin at pH 7.0. Temperature effects were carried out for myosin at pH 7.0 at a rate of 2 °C/min from 5 to 90 °C and held for 2 min before the measurement.

635

b-Turn and random coil fractions tended to increase when the pH was moved away from neutral. The CD data demonstrated the sensitivity of myosin’s conformation to pH and that the secondary structures were more sensitive to acidification than exposure to alkali. In order to explore the conformational changes during heating, the effect of temperature on myosin secondary structures at pH 7.0 was also measured by CD. The a-helix fraction gradually decreased from 87.7% to 36.0% with increasing temperature from 5 to 90 °C (Fig. 3). The bsheet fraction was considerably higher in the range 55– 90 °C than in the range 5–50 °C, whereas the b-turn fraction exhibited a slight decrease above 70 °C. In general, the random coil fraction tended to increase with increasing temperature. 3.2. Dynamic rheological properties of porcine myosin solution In this paper, we studied the dynamic rheological properties of myosin by measuring the elastic modulus (G0 ), viscous modulus (G00 ) and phase angle (d) at different pH values during heating. Tan d, a ratio of G0 /G00 , gives a relative measurement of the associated energy loss versus energy stored in a cyclical deformation. A d of 90° thus represents a fully viscous material whilst a fully elastic material has a d of 0°. The rheological parameters of myosin at pH 5.5–9.0 are illustrated in Fig. 4. The sample at pH 5.5 exhibited an increase in G0 and an extremely low d at 15 °C, suggesting the formation of aggregates. A sharp decrease in G0 and a considerable increase in d were observed at 19.3 °C. The G0 and d values of myosin at pH 5.5 reached their respective minimum and maximum at 26.4 °C. It is well accepted that the negative and positive charges between protein molecules are approximately equal at a pH value close to pI. At this point, the repulsion between protein molecules is minimized, promoting aggregation. However, primarily non-covalent, short term intermolecular interactions are involved in this process (Van Camp, Messens, Cle´ment, & Huyghebaert, 1997). In the experiment, disentanglement occurred and the increased molibility of the myosin molecules as a result of the deformation and breaking of protein-protein bonds (Chen, Dickinson, & Edwards, 1999), caused a decline in G0 . Subsequently, the formation of new bonds produced a permanent network structure, as indicated by an increase in G0 up to 90 °C. Similar to the sample at pH 5.5, the G0 value at pH 6.0 began to increase at 15 °C. The d of the sample at pH 6.0 was very low (20.6°) at the beginning of heating. Taking these findings into consideration, it is suggested that porcine myosin at pH 6.0 could also coagulate spontaneously in cold conditions (15 °C). Fretheim, Egelandsdal, Harbitz, and Samejima (1985) reported that myosin solution formed gels at 5 °C when the pH slowly decreased to within the range 2.5–5.5. It was likely that conformational changes during acidification initiated the protein–protein interactions and, as a consequence, a gel matrix was

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a

100000 10000

G' (Pa)

1000 100 pH5.5 pH6.0 pH6.5 pH7.0 pH7.5 pH8.0 pH9.0

10 1 0.1 0.01 15

30

45

60

75

90

T (ºC)

b

100

pH5.5 pH6.0

80

pH6.5 pH7.0

60

δ (˚)

pH7.5 pH8.0 40

pH9.0

3.3. Water-holding capacity of porcine myosin gel

20

0

15

30

45

60

75

interactions at low temperatures (Lanier et al., 2004). The slight decrease in G0 probably arose from the increased molibility of myosin molecules with heating whilst the second increase in G0 could have resulted from stronger interactions between denatured myosin molecules. As pH increased from 6.5 to 9.0, there was a slight decrease in Tg1 (the onset temperature of the first increase in G0 ) and Tg2 (the onset temperature of the second increase in G0 ). This may have been due to a reduction in stability of myosin with increasing pH (Wright & Wilding, 1984; Raghavan & Kristinsson, 2008). The rate of gelation from 60 to 75 °C, as indicated by the slope of G0 versus temperature, decreased from 49.9 Pa/°C at pH 5.5 to 4.6 Pa/°C at pH 9.0. This result was consistent with the report on porcine myofibrillar protein (Westphalen et al., 2005) which was potentially attributable to the closer association of proteins at low pH or the increased strength of hydrophobic interactions and disulfide bonds (Westphalen et al., 2005). Riebroy, Benjakul, Visessanguan, Erikson, and Rustad (2007) also reported the formation of disulfide bonds and increased hydrophobicity in myosin during acidification. By the end of heating (90 °C), G0 was seen to decline with increasing pH. The d value of all samples at 90 °C was lower than 4° (Fig. 4), characterising of the formation of an elastic gel structure.

90

T (ºC) Fig. 4. Dynamic rheological properties of myosin solution at various pH values. Myosin solutions containing 16 mg/mL, 0.5 M NaCl were heated at a rate of 1 °C/min from 15 to 90 °C at various pH values.

formed. However, porcine myofibrils at pH 5.6 and 6.0 have been found to start forming a gel beyond 40 °C (Westphalen et al., 2005). The difference probably resulted from other myofibrillar proteins (actin, titin, tropomyosin, troponin and nebulin) that were suspected to have a strong influence on rheological properties by influencing the formation of the myosin gel matrix. The samples at pH 6.5–9.0 began to form a gel after reaching 38 °C, as indicated by increasing G0 (38 °C) and decreasing d (35 °C) values (Fig. 4). This gelation continued until the temperature reached approximately 51 °C where a weakening of gel took place. Upon further heating, the gel strength increased at about 55 °C. In contrast to G0 , d decreased with increasing temperature. In general, heating caused myosin molecules to partially unfold (Fig. 3), such that the inner hydrophobic regions became exposed (Lanier et al., 2004). This denaturation was followed by aggregation, and finally a gel network was formed. The first increase in G0 may be attributed to protein–protein

The gelling process entails the association of myosin chains which produce a continuous three dimensional network in which water is trapped. The water-holding capacity (WHC) can indicate a protein’s ability to bind water and is generally used to objectively evaluate the quality and yield of meat and meat products (Trout, 1988; Rosenvold & Andersen, 2003). As shown in Fig. 5, the WHC of porcine myosin gel increased significantly (P < 0.05) from 31.2% to 73.0% as pH increased from 5.5 to 7.0, and the maximum WHC (72–73%) was achieved at pH 7.0–9.0. The WHC of porcine myofibril gel significantly increased 80

73.0a 72.0a 72.1a 72.5a 63.0b

60

WHC (%)

636

40

31.3d

35.2c

20

0

5.5

6.0

6.5

7.0

7.5

8.0

9.0

pH Fig. 5. Water-holding capacity of myosin gel at various pH values. Error bars indicate mean values ± standard deviations of three replicates, and mean values and different characters on the top of each column (a, b, c and d) indicate significant difference (P < 0.05).

R. Liu et al. / Meat Science 80 (2008) 632–639

wi th increases in pH from 5.4 to 7.0 (Bertram, Kristensen, & Andersen, 2004). Similarly, chicken-breast muscle gels have been found to retain water better at high pH values (7.0–7.4) compared to low (6.4–6.8) (Kristinsson & Hultin, 2003). As pH increased away from the pI, the net increase in negative charge caused electrostatic repulsion between myosin molecules in the gel network, providing more binding sites for water, and increasing the surface available for hydration. 3.4. Microstructure of porcine myosin gel Scanning electron micrographs of myosin gels were observed at pH 5.5, 6.5, 7.0 and 8.0. Myosin solutions at pH 5.5 formed a coarse and disordered gel network arranged in clusters (Fig. 6), possibly since the molecules randomly aggregated in the cold (15 °C) before unfolding. A compact gel was obtained at pH 6.5, which consisted of small, uniform, bead-like particles. It was presumed that the net negative charge at pH 6.5 facilitated the unfolding of the myosin chains before orderly aggregation occurred, leading to a fine gel network and a high WHC (63.0%). The results agree with Hermansson (1979) who suggested that gel properties in proteins depend on the relative speeds of unfolding and aggregation. When the rate of protein aggregation is slower than denaturation, heat-denatured proteins are allowed to align in an ordered fashion to form a fine gel network. In contrast, a coarse gel network would be formed if the rate of aggregation was faster than dena-

637

turation. The results showed a decrease in three dimensional ordering and uniformity with further increases in pH. Gel strength also exhibited a decreasing trend with increasing pH (Fig. 4). This could be ascribed to strong electrostatic repulsion between molecules which prevented the interactions required to form a gel matrix (Ma & Holme, 1982), although Kristinsson and Hultin (2003) suggested that electrostatic repulsion of muscle proteins is a major driving force behind gel formation and WHC. The results confirmed that the physical integrity of a gel is maintained by hydration (Westphalen, Briggs, & Lonergan, 2006; Damodaran, 1996) and the counter-balancing attraction and repulsion forces between the protein molecules (Zayas, 1997). 3.5. Relationship between secondary structures and gel properties The a-helix fraction of porcine myosin gradually decreased with increasing temperature, whereas the b-sheet fraction tended to increase (Fig. 3). This indicates that the formation of b-sheets occurred simultaneously with the unfolding of a-helical structures during the gelation process. Increases in b-sheet content have been observed during thermal gelling of Alaska pollock surimi (Sa´nchezGonza´lez et al., 2008) and Pacific whiting surimi (Bouraoui, Nakai, & Li-Chan, 1997) by Raman spectroscopy. Some limitations of this study were that CD was performed at a very low protein concentration, whereas gelation takes

Fig. 6. Scanning electron micrographs of myosin gel at various pH values. (a) pH 5.5; (b) pH 6.5; (c) pH 7.0; (d) pH 9.0.

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place at high protein concentrations. However, Choi and Ma (2007) studied the conformation of globulin from common buckwheat using CD (at a low concentration of 0.01%) and Raman spectroscopy (at a high concentration of 5%), and suggested that the CD results were mostly consistent with the Raman data. We therefore suggest that both the formation of b-sheets and the unfolding of a-helices aided the gelling of porcine myosin. It has been reported that b-sheets also play an important role in the gelation of globular proteins (Meng, Ma, & Phillips, 2003; Choi & Ma, 2007). The role of a-helices in the thermal gelling of myosin was reported in our preceding paper (Liu et al., 2007). In this paper, we investigated the relationship between gel properties and the secondary structures of porcine myosin prior to heating. Table 1 shows the correlation coefficient between the abundance of secondary structures and gel properties at various pH values. b-Sheets and b-turns exhibited significant and positive effects on G0 at 15 °C (Table 1), indicating that they assisted the aggregation of myosin in the cold. It has been reported that b-sheets are an important conformational component of the aggregated-state secondary structure of globulin derived from common buckwheat (Choi & Ma, 2007). The correlation coefficient between the a-helix content and d at 15 °C was 0.76 (P < 0.05) whilst that between the b-sheet content and d at 15 °C was 0.92 (P < 0.01). It is suggested that samples with a higher a-helix content are more viscous, whilst those with a higher b-sheet content are more elastic. Greater elasticity could be attributed to the weak fluidity of the myosin molecules resulting from stronger protein–protein interactions. The presence of more b-sheets and fewer a-helices prior to heating would increase gel strength (Table 1). It was possible that the reduced hydration of b-sheets compared to a-helices promoted protein–protein interactions. The secondary structures of porcine myosin prior to heating had no significant influence on the d value at 90 °C. In addition, the WHC of myosin gel was negatively correlated (P < 0.05) with the b-sheet fraction prior to heating (Table 1). This agrees with Choi and Ma (2007) who suggested that water hydration strength of bsheets is weaker than of a-helices.

Table 1 Correlation coefficient between secondary structure and gel properties at different pH values (n = 21) Gel properties

a-Helix (%)

b-Sheet (%)

b-Turn (%)

Random coil (%)

G0 at 15 °C d at 15 °C G0 at 90 °C d at 90 °C WHC

0.93** 0.76* 0.92** 0.41a 0.71a

0.91** 0.92** 0.98*** 0.02a 0.91**

0.89** 0.59a 0.82* 0.58a 0.52a

0.35a 0.03a 0.21a 0.74a 0.05a

a

NS, not significant. P < 0.05. ** P < 0.01. *** P < 0.001. *

4. Conclusion CD analysis showed that porcine myosin possessed ahelix, b-sheet, b-turn and random coil structures. Gelling of porcine myosin involved changes in myosin conformation, and protein–protein and protein–water interactions. The gelation properties were strongly pH and temperature dependent as well as being affected by the myosin secondary structures. The a-helix fraction declined with increasing temperature and movement of the pH away from 7.0, whilst the b-sheet fraction increased with decreasing pH and was relatively high at above 55 °C. Near the pI (pH 5.5 and 6.0), porcine myosin could spontaneously coagulate in the cold (15 °C) which was partially attributable to the presence of more b-sheets. A gel began to form at pH 6.5–9.0 at temperatures greater than 38 °C. Heating caused the partial transformation of a-helices into b-sheets and random coils. Subsequently, myosin aggregated and formed a gel network structure. As pH increased, the gel strength decreased and the WHC increased. Correlation analysis indicated that both the unfolding of a-helices and the formation of bsheets played an important role in the gelation process. A high b-sheet fraction prior to heating decreased the WHC of the thermal gel. A compact and uniform gel was also obtained at pH 6.5 whilst there was a decrease in three dimensional ordering and an increase in particle size when pH was moved away from 6.5. Acknowledgement The authors are grateful to Qun-ying Xu (Wuhan Polytechnic University) for her technical advice and support. This research is funded by ‘‘National Key Technology R&D Program in Eleventh-Five-Year Plan”. The accession number is 2006BAD05A18. References Bertram, H. C., Kristensen, M., & Andersen, H. J. (2004). Functionality of myofibrillar proteins as affected by pH, ionic strength and heat treatment – A low-field NMR study. Meat Science, 68, 249–256. Bouraoui, M., Nakai, S., & Li-Chan, E. (1997). In situ investigation of protein structure in Pacific whiting surimi and gels using Raman spectroscopy. Food Research International, 30(1), 65–72. Chen, J., Dickinson, E., & Edwards, M. (1999). Rheology of acid-induced sodium caseinate stabilized emulsion gels. Journal of Texture Studies, 30, 377–396. Choi, S. M., & Ma, C. Y. (2007). Structural characterization of globulin from common buckwheat (Fagopyrum esculentum Moench) using circular dichroism and Raman spectroscopy. Food Chemistry, 102, 150–160. Damodaran, S. (1996). Amino acids, peptides, and proteins. In O. R. Fennema (Ed.), Food chemistry (3rd ed.) (pp. 321–429). New York: Marcel Dekker. Foegeding, E. A., & Lanier, T. C. (1996). Characteristics of edible muscle tissues. In O. R. Fennema (Ed.), Food chemistry (3rd ed.) (pp. 879–942). New York: Marcel Dekker. Fretheim, K., Egelandsdal, B., Harbitz, O., & Samejima, K. (1985). Slow lowering of pH induces gel formation of myosin. Food Chemistry, 18, 169–178.

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