Food Chemistry 170 (2015) 212–217

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Food Chemistry

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The solubility and conformational characteristics of porcine myosin asaffected by the presence of L-lysine and L-histidine

http://dx.doi.org/10.1016/j.foodchem.2014.08.0450308-8146/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Fax: +86 025 84396558.E-mail address: zqpeng@njau.edu.cn (Z.Q. Peng).

X.Y. Guo, Z.Q. Peng ⇑, Y.W. Zhang, B. Liu, Y.Q. CuiCollege of Food Science and Technology, National Center of Meat Quality and Safety Control, Nanjing Agricultural University, Nanjing 210095, China

a r t i c l e i n f o

Article history:Received 18 March 2014Received in revised form 10 July 2014Accepted 11 August 2014Available online 20 August 2014

Keywords:MyosinL-lysL-hisSolubilityConformation

a b s t r a c t

The influence of L-lys and L-his on the solubility, surface hydrophobicity, sulphydryl content and confor-mational characteristics of porcine myosin solubilised in high (0.6 M), physiological (0.15 M) and low(1 mM) ionic strength solutions were explored. The solubility of myosin was increased in the presenceof L-his and/or L-lys in all ionic strength solutions used. The presence of L-his and L-lys caused increasesin the surface hydrophobicity and reactive sulphydryl content (p < 0.05). Circular dichroism revealed asignificant decrease of a-helical content with an increase of random coils, b-turns and b-sheets in thepresence of L-his and/or L-lys. These results demonstrate that the introduction of L-lys and L-his causesthe unfolding of myosin, resulting in loss of a-helical structure, which is followed by increases in randomcoils, b-turns and b-sheets, which exposes buried hydrophobic and sulphydryl groups to the myosin sur-face, ultimately increasing the solubility of porcine myosin.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Solubility is an important functionality of myosin and is respon-sible for gelation and emulsification of myofibrillar proteins. Solu-bility is influenced by ionic composition, ionic strength, pH andtemperature (Ries-Kautt & Ducruix, 1997). The low solubility ofmyosin is a result of the spontaneous formation of filaments thatoccur in vitro or at low ionic strength, and a relatively high concen-tration of salt is required to solubilise these filaments (Craig &Woodhead, 2006; Sohn et al., 1997; Tsunashima & Akutagawa,2004; Xu, Harder, Uman, & Craig, 1996).

More than 90% of myofibrillar proteins from cod (Stefansson &Hultin, 1994) were solubilised at low ionic strength (0.002)through water washes at a neutral pH. Chicken breast(Krishnamurthy et al., 1996) and mackerel light muscles (Feng &Hultin, 1997) were also solubilised in a low ionic strength solution.To solubilise chicken breast proteins in a low ionic strength solu-tion, washing with a sodium chloride solution buffered with L-hisand dialysis against water were necessary. It has also beenreported that L-his and L-lys contribute to the solubility of myosinat low (1–300 mM) KCl concentrations (Hayakawa, Ito,Wakamatsu, Nishimura, & Hattori, 2009; Takai, Yoshizawa,Ejima, Arakawa, & Shiraki, 2013). Using transmission electronmicroscopy, it was shown that L-his causes elongation of light

meromyosin (LMM), resulting in the inhibition of native myosinfilament formation (Hayakawa, Ito, Wakamatsu, Nishimura, &Hattori, 2010). The results of Takai et al. (2013) indicated thatthe effect of L-lys on porcine myosin solubility is mediated by itsinteraction with native myosin, without structural change to themyosin. However, the reason that L-lys and L-his improve thesolubility of myosin remains unclear.

Therefore, the objective of this study was to determine theinfluence of L-lys and L-his on the solubility and conformation ofporcine myosin and also to elucidate the mechanism of solubilisa-tion of myosin at various ionic strengths in the presence of L-lysand L-his.

2. Materials and methods

2.1. Myosin preparations

Porcine longissimus dorsi was removed from the carcass afterthe animal was sacrificed and immediately chilled in ice. Theice-cold muscle was minced within 30 min, and the myosin wasprepared according to the method of Perry (1955) with slightmodifications. All steps were performed at 0–4 �C to minimise pro-teolysis and protein denaturation. Briefly, the minced muscle wasextracted with 3 volumes of modified Guba–Straub solution(0.3 M KCl, 0.1 M KH2PO4, 50 mM K2HPO4, 1 mM ethylenediamine-tetraacetic acid, and 4 mM sodium pyrophosphate, pH 6.5) for15 min with slight stirring. The extract was diluted with 3 volumes

X.Y. Guo et al. / Food Chemistry 170 (2015) 212–217 213

of distilled water and centrifuged at 3000g for 20 min. The super-natant was filtered through three layers of cheesecloth and dilutedwith 6.5 volumes of cold distilled water and filtered overnight.After centrifugation at 10,000g for 20 min, the precipitate wassolubilised in 0.3 M KCl, pH 7.0, and subsequently diluted with1.5 volumes of distilled water. Magnesium chloride and sodiumpyrophosphate were added to a final concentration of 5 mM.The solution was stirred vigorously for 20 min without foaming.1 volume of cold distilled water was added to adjust the ionicstrength of the solution to 0.3. The pH of the solution was adjustedto 6.6. After centrifugation at 10,000g for 20 min, the supernatantwas diluted with 9 volumes of distilled water and incubatedovernight. After centrifugation at 10,000g for 10 min, theprecipitate was resuspended in 0.6 M NaCl and 20 mM phosphatebuffer (pH 6.5) and dialysed against the same solution. Afterultracentrifugation at 110,000g for 1 h, the supernatant was usedas the myosin sample in 0.6 M NaCl, pH 6.5.

2.2. Solubilisation of myosin in high ionic strength and low ionicstrength buffers

Myosin was dialysed against high (0.6 M), physiological(0.15 M) and low (1 mM) ionic strength salt (NaCl) solutionswith/without 5 mM L-lys and/or 5 mM L-his overnight. The dia-lysed myosin suspension was centrifuged at 5000g for 15 min,and the supernatant was used as the myosin solubilised in thesample solutions. The solubility of each protein was determinedusing the protein concentrations of each dialysed suspension andsupernatant. The protein concentrations were determined by theBiuret method (Gornall, Bardawill, & David, 1949).

2.3. SDS–polyacrylamide gel electrophoresis (SDS–PAGE)

SDS–PAGE was performed according to the procedure ofLaemmli (1970) using a 12.5% acrylamide separating gel and a 4%acrylamide stacking gel in a Mini-PROTEAN� Tetra cell apparatus(Bio-Rad, Beijing, China). The gels were stained in 0.1% CoomassieBrilliant Blue R250 in 25% (v/v) methanol and 10% (v/v) acetic acidfor 1 h. The gels were destained overnight with gentle shaking in asolution of 10% (v/v) methanol and 10% (v/v) acetic acid. Themobility of the protein bands was calibrated with broad rangemolecular weight markers (Fermentas, Beijing, China) using thesoftware ‘Quantity One Ver. 4.6.2 Analysis’ (Bio-Rad LaboratoriesInc., Hercules, CA, USA).

2.4. Surface hydrophobicity

The surface hydrophobicity of the soluble myosin was deter-mined using the hydrophobic probe 8-anilino-1-naphthalene sul-phonic acid (ANS) (Sultanbawa & Li-Chan, 2001). The solublemyosin was serially diluted with its own buffering solution to afinal volume of 2 ml with the protein concentration ranging from0.01% to 0.1%. After stabilising at 20 �C, 20 ll ANS were added tothe sample solutions. The relative fluorescence was detected in aSpectramax microplate reader (Spectramax M2, Molecular Devices,Sunnyvale, CA, USA). The excitation and emission spectra weremeasured at wavelengths of 374 and 485 nm, respectively. The ini-tial slope of the fluorescence intensity against the protein concen-tration was used as the index of protein hydrophobicity (S0).

2.5. Sulphydryl content

The reactive sulphydryl (R-SH) content of the soluble myosinwas determined by the method of Ellman (1959). An aliquot(20 ll) of 5,50-dithiobis (2-nitrobenzoic acid) (DTNB) was addedto 3 ml soluble myosin. The mixture was then incubated at 25 �C

for 5 min. The amount of R-SH was measured at 412 nm using amolar extinction coefficient of 13,600 M�1 cm�1. The total contentof SH (T-SH) was estimated by the method of Hamada, Ishizaki, andNagai (1994). A sample solution with a final concentration of 6 Murea was made by adding 2.25 ml 8 M urea to 0.75 ml soluble myo-sin. The sample solution was then mixed with 20 ll DTNB and sub-sequently incubated at 25 �C for 5 min. The T-SH was determinedspectrophotometrically as described.

2.6. Measurement of circular dichroism (CD) spectra and secondarystructure calculation

The CD spectrum was measured using a Chriascan spectrometer(Applied Photophysics, Surrey, UK). The myosin samples werediluted to 0.1 mg/ml with the same solvents and transferred to aquartz cell with a 0.2 cm light-path length. The CD spectra wererecorded in the range of 200–260 nm, and the scan rate was100 nm/min. The temperature was regulated with a control unitand kept at 20 �C. The spectra were averaged over three scansand corrected for the solvent signal. A mean residue weight of110 g/mol was assumed. The percentages of a-helix, b-sheet,b-turn and random coil structures were determined using theprotein secondary structure estimation program (CDNN method)provided with the Chriascan spectrometer. All treatments weretested in triplicate. The mean values from these replicates arepresented in the data reported in this study.

2.7. Statistical analysis

The data were analysed with the Statistical Analysis System(SAS Institute Inc., Cary, NC, USA). A variance test (ANOVA) wasperformed with a significance level of p < 0.05. Duncan’s multiplerange test was used to evaluate the differences betweentreatments.

3. Results and discussion

3.1. SDS–PAGE profiling

The myosin purity was verified using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (Fig. 1). There were four mainfractions with molecular weights of 207, 20, 15 and 12 kDa, whichis consistent with the molecular weight of myosin heavy chain andmyosin light chain (Harrington & Rodgers, 1984). The polypeptidecomposition of the myosin was consistent with Hayakawa et al.(2009). The purity of the myosin was greater than 90% as deter-mined by densitometry (Quantity One� 1-D analysis software,Bio-Rad Co., Hercules, CA, USA), despite actin contamination.

3.2. Solubility of porcine myosin solution

The solubility of the porcine myosin increased with increasingionic strength buffers with/without L-his and L-lys because of thechange in the protein conformation through electrostatic andhydrophobic forces (Nakai & Li-Chan, 1988). Moreover, the resultsshowed that the solubility was significantly influenced by theintroduction of L-his and L-lys regardless of the ionic strength(p < 0.05) (Table 1). Compared to the solubility of myosin in a1 mM NaCl solution, the solubility of myosin was increased by6.21%, 8.37% and 9.95% in NaCl + 5 mM L-his solution, NaCl + 5 mML-lys solution and NaCl + 5 mM L-his + 5 mM L-lys solution at thesame ionic strength, respectively. At physiological (0.15 M) ionicstrength, the solubility was increased by 3.68%, 9.12% and 13.89%with 5 mM L-his, 5 mM L-lys and 5 mM L-his + 5 mM L-lys, respec-tively. Myosin was also more soluble in the presence of 5 mM L-his,

250 kDa

130 kDa

100 kDa

25 kDa

55 kDa

70 kDa

35 kDa

15 kDa

10 kDa

MHC

MLC-1

MLC-2

MLC-3

Actin

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

Table 1The solubility of myosin in different ionic strength solutions.

Solutions Concentrations

1 mM 0.15 M 0.6 M

NaCl 2.41 ± 1.05j 19.23 ± 1.48g 88.60 ± 1.9c

NaCl + 5 mM L-his 8.62 ± 0.88i 22.91 ± 1.19f 92.57 ± 1.15b

NaCl + 5 mM L-lys 10.78 ± 1.19h 28.35 ± 1.36e 94.67 ± 1.72a

NaCl + 5 mM L-his+ 5 mM L-lys

12.36 ± 1.73h 33.12 ± 1.36d 96.13 ± 1.19a

Note: values were mean of triplicate values ± S.D.Means with different superscripts letters differ significantly (p < 0.05).

214 X.Y. Guo et al. / Food Chemistry 170 (2015) 212–217

5 mM L-lys and 5 mM L-his + 5 mM L-lys at high (0.6 M) ionicstrength (p < 0.05). These results indicate that L-lys and L-his con-tribute to the solubilisation of myosin, which is somewhat similarwith the study by Hayakawa et al. (2009, 2010), who found thatL-his contributed to the solubility of chicken breast muscle myosin atboth low (1 mM) and physiological (0.15 M) ionic strength butnot high (0.6 M) ionic strength. Moreover, myosin was more solubleat low ionic strength with 5 mM L-his. This may be because of thedifferent animal species and muscle fibre type (Choi et al., 2010;Gill, Chan, & Paulson, 1992; Vesessanguan, Ogawa, Nakai, & An,2000). Notably, L-lys contributed more to the solubility of myosinthan the L-his (p < 0.05), suggesting that the greater net positivecharge at pH 6.5 played a role (Takai et al., 2013). The weakenedsolubilisation effect of L-lys and L-his and the increased ionicstrength indicate that the effect of L-lys and L-his may be overwhelmedby the effect of ionic strength on filament depolymerisation.

The question remains as to the role of L-lys and L-his in the sol-ubilisation of myosin. Takai et al. (2013) showed that the marginalsolubilisation effect of lys might be because of the strong tendencyof myosin to self-associate into filaments at low NaCl concentra-tion, and a specific interaction between lys and myosin may dis-rupt the electrostatic interactions and prevent filamentformation. Krishnamurthy et al. (1996) suggested that L-his wasimportant for neutralising the low ionic strength solution and didnot have a specific effect on the solubilisation of myofibrillar pro-teins. Contrary to the Krishnamurthy et al. (1996) study, Hayakawa

et al. (2009) found that L-his directly affected the solubility of myo-sin in a low (1 mM) ionic strength solution rather than observing apH effect. Further studies showed that possible reason for this wasbecause of elongation of LMM region, contributing to myosin fila-ment weakening and the dissociation of myosin in a low ionicstrength solution (Hayakawa et al., 2010). However, the mecha-nism of myosin dissociation by LMM elongation remains unclear.

To clarify the role of L-lys and L-his in the solubilisation of por-cine myosin, the conformational changes of myosin wereidentified.

3.3. Surface hydrophobicity

ANS-S0 is useful for determining surface aromatic hydrophobic-ity (Hayakawa & Nakai, 1985; Leblanc & Leblanc, 1992). The ANS-S0 was increased with ionic strengths with/without L-his and L-lys,which was primarily because of the disruption of electrostaticinteractions by NaCl (Supawan & Jae, 2005). The results alsoshowed that the surface hydrophobicity of porcine myosinincreased with the introduction of L-his and L-lys despite the ionicstrengths (Fig. 2). Compared to the ANS-S0 of myosin solubilised inthe same ionic strength NaCl solution, the ANS-S0 increased from1120 to 1202.6 at low (1 mM) ionic strength, from 1602 to2012.8 at physiological (0.15 mM) strength, and from 2640 to2947.7 at high (0.6 M) ionic strength in the presence of L-his. Sim-ilar to the effect of L-his, the ANS-S0 increased from 1120 to 1586.3at low (1 mM) ionic strength, from 1602 to 2191.6 at physiological(0.15 mM) ionic strength, and from 2640 to 2902.2 at high (0.6 M)ionic strength when L-lys was introduced. This effect was more sig-nificant with the presence of L-his and L-lys (p < 0.05). The resultssuggest that the introduction of L-his and L-lys destroy the proteinstructure, causing the exposure of aromatic acids. At pH 6.5, lysand his were both positively charged, whereas myosin gained anet negative charge. The lys and his were likely to bind to thecharged residues of myosin because of the electrostatic effect,thereby disrupting intra and inter molecular ionic linkages to alterthe protein structure and lead to the exposure of hydrophobicgroups. Similar to the solubility behaviour, L-lys had a greater con-tribution to the hydrophobicity than L-his.

Fig. 2. Surface hydrophobicity (ANS-S0) of porcine myosin solubilised in differentsalt solutions.

Fig. 4. Reactive sulphydryl content of porcine myosin solubilised in different saltsolutions. 1 = 1 mM; 2 = 0.15 M; 3 = 0.6 M.

X.Y. Guo et al. / Food Chemistry 170 (2015) 212–217 215

3.4. Sulphydryl content

The total content of SH groups (T-SH) in the porcine myosin(mole/105 g protein) solubilised in different solutions are shownin Fig. 3. The T-SH content was not affected by the introductionof L-his and L-lys (p > 0.05), indicating that L-his and L-lys did notcause the disruption or formation of disulphide bonds. Conversely,an increase in R-SH content, indicating exposed SH groups on thesurface of the protein, was observed with the introduction ofL-his and L-lys at each ionic strength (p < 0.05) (Fig. 4). Comparedto the R-SH content of the myosin solubilised in the same ionicstrength NaCl solution, the R-SH increased from 1.71 to 2.18 atlow ionic (1 mM) strength, from 1.89 to 2.31 at physiological(0.15 mM) ionic strength and from 2.6 to 2.92 at high (0.6 M) ionicstrength with the introduction of 5 mM L-his. Similar to the effectof L-his, in presence of 5 mM L-lys, the R-SH content increased from1.71 to 2.18 at low ionic strength, from 1.89 to 2.31 at physiologicalionic strength, and from 2.6 to 3.05 at high ionic strength.

Fig. 3. Total sulphydryl content of porcine myosin solubilised in different saltsolutions. 1 = 1 mM; 2 = 0.15 M; 3 = 0.6 M.

Moreover, the increased R-SH content was more significant withthe introduction of 5 mM L-his + 5 mM L-lys (p < 0.05). This resultindicates that more SH groups are exposed to the protein surface.This result also shows that R-SH is increased with increasing ionicstrength regardless of the introduction of L-his and L-lys, which isconsistent with Tein, who found that the T-SH content wasconstant and that the R-SH content increased with increasing KClconcentration (Tein & Jae, 1998).

The observation that the T-SH content was constant and theR-SH increased with the introduction of L-his and L-lys suggeststhat they do not disrupt or form disulphide bond, and expose themasked SH to the protein surface. This indicates that L-his andL-lys do not change the primary structure but effect the secondaryor tertiary structure instead.

3.5. Circular dichroism of porcine myosin solution

The secondary structure of the porcine myosin was determinedusing circular dichroism and was significantly affected by theintroduction of L-his and L-lys (Fig. 5). The CD spectrum exhibitedtwo minima at approximately 208 and 222 nm, showing the pre-dominance of a-helix structures (Greenfield, 1999). With the intro-duction of L-his and L-lys, there was a significant loss of a-helixstructures regardless of the ionic strength (Fig. 5a–c). It wasobserved that the a-helix content significantly decreased from58.61% to 53.27%, 51.72% and 46.44%, followed by an increase ofother secondary structures, particularly random coil structures,with the introduction of 5 mM L-his, 5 mM L-lys and 5 mML-his + 5 mM L-lys at 1 mM ionic strength, respectively (Fig. 5f). At0.15 M ionic strength, the a-helix content decreased from 68.45%in a NaCl solution to 56.58% in NaCl + 5 mM L-his solution,56.06% in NaCl + 5 mM L-lys solution and 47.16% in NaCl + 5 mML-his + 5 mM L-lys solution (Fig. 5e). The same trend was observedat 0.6 M ionic strength. This result shows that the secondary struc-tures were influenced by L-his and L-lys, which was different fromTakai et al. (2013) study, who found that L-lys caused no structuralchanges at 200 and 300 mM NaCl but the interaction with nativemyosin. It might be due to the different wavelength range of CDspectra and salt concentrations. Because the myosin was treatedat pH 6.5, the lys and his likely to bind to the charged residues ofmyosin because of the electrostatic effect, which disrupts intraand inter molecular ionic linkages contributing to the stability of

Fig. 5. CD spectra (a,b,c) and secondary structures (d,e,f) of myosin. (a) CD spectra of myosin solubilised in different solutions at high (0.6 M) ionic strength; (b) CD spectra ofmyosin solubilised in different solutions at physiological (0.15 M) ionic strength; (c) CD spectra of myosin solubilised in different solutions at low (1 mM) ionic strength; (d)secondary structures of myosin solubilised in different solutions at high ionic strength. 1 = 0.6 M NaCl solution; 2 = 0.6 M NaCl + 5 mM L-his solution; 3 = 0.6 M NaCl + 5mML-lys solution; 4 = 0.6 M NaCl + 5 mM L-his + 5 mM L-lys solution; (e) secondary structures of myosin solubilised in different solutions at physiological ionic strength.1 = 0.15 M NaCl solution; 2 = 0.6 M NaCl + 5mM L-his solution; 3 = 0.15 M NaCl + 5 mM L-lys solution; 4 = 0.15 M NaCl + 5 mM L-his + 5 mM L-lys solution; (f) secondarystructures of myosin solubilised in different solutions at low ionic strength. 1 = 1 mM NaCl solution; 2 = 0.6 M NaCl + 5 mM L-his solution; 3 = 1 mM NaCl + 5 mM L-lyssolution; 4 = 1 mM NaCl + 5 mM L-his + 5 mM L-lys solution.

216 X.Y. Guo et al. / Food Chemistry 170 (2015) 212–217

secondary structures (Damodaran, 1996; Satoh, Nakaya, Ochiai, &Watabe, 2006). This results in the unfolding of the helical tail por-tion of myosin and the exposure of hydrophobic groups. This isconsistent with the report of Jiang et al. (2011), who found thatthe surface hydrophobicity increased with a decrease of a-helixcontent. It is hypothesised that the introduction of L-his and L-lyschange the myosin secondary structure, releasing the monomerfrom aggregates (Hamm, 1960; Huxley, 1963) and subsequentlyimproving myosin solubility.

It was also observed that the secondary structure compositionwas constant despite the ionic strength and presence of L-his and

L-lys. The a-helix was the predominant structure, followed by ran-dom coils, b-turns and -sheets. Moreover, the a-helix contentincreased with increasing ionic strength (1–150 mM) with/withoutL-his and L-lys. This is consistent with Ralston and Dunbar (1979),who showed that low ionic strength solutions of spectrin had adecreased apparent helix content. This may be due to the effectof salts disrupting the electrostatic interactions through bothnon-specific binding (Debye–Hückel) and through specific ionbinding to the protein (Goto, Takahashi, & Fink, 1990). Contraryto Krishnamurthy et al. (1996) and Hayakawa et al. (2009), wehypothesise that the mechanism of solubility enhancement by

X.Y. Guo et al. / Food Chemistry 170 (2015) 212–217 217

the amino acids is that L-his and L-lys cations are able of bindingnegative charged residues of myosin through electrostatic interac-tion, thereby disrupting the intra and inter molecular ionic link-ages, which causes transformation of the myosin conformation,resulting in a loss of a-helical structure and the exposure of hydro-phobic groups and masked SH groups to the surface. Subsequently,the depolymerisation of the myosin filament is induced, ultimatelyincreasing the solubility of myosin.

4. Conclusions

In 1 mM, 0.15 M and 0.6 M ionic strength solutions, L-his andL-lys decreased a-helix content and increased the content of ran-dom coils, b-turns and b-sheets. The hydrophobicity and reactiveSH content were increased at 1 mM–0.6 M NaCl and wereincreased more with 5 mM his and 5 mM lys. Lys and his increasedthe solubility of porcine myosin, regardless of the ionic strength,and lys had a greater contribution to solubility than his. Lys andhis caused a transformation of the porcine myosin conformation,which may contribute to the depolymerisation of myosin filamentsand expose masked hydrophobic and SH groups, resulting inincreased solubility.

Acknowledgements

This work is supported by the Earmarked fund for modernagro-industry technology research system (No: nycytx-38).

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