Characterization of Actin and Myosin Extracts ObtainedUsing Two Improved Laboratory Methods

Sonia Fonseca & Aida Cachaldora & Javier Carballo

Received: 20 December 2012 /Accepted: 31 May 2013 /Published online: 14 June 2013# Springer Science+Business Media New York 2013

Abstract Actin and myosin are the most important contrac-tile proteins in the muscle. Numerous authors developeddifferent methods for isolation and purification of myofibril-lar proteins. The aim of this study was to obtain suitable actinand myosin extracts from post-rigor porcine muscle to beused in further studies, such as testing the proteolytic activityof microbial cultures. Actin and myosin were quantified inthe extracts using spectrophotometric methods, yielding 0.17and 0.22 mg/mL, respectively. The isolation methods pro-posed in this study provided low contaminated extracts,showing purity percentages of 74.36 % in the case of actinand 65.43 % for myosin, as determined by sodium dodecylsulfate–polyacrylamide gel electrophoresis. Subsequently,these extracts were sterilized through a 0.22-μm polyvinyli-dene difluoride filter with no significant retention observed.In conclusion, the procedures described in this work for actinand myosin isolation can be recommended for microbiolog-ical studies requiring sterilized pure muscle proteins extracts.

Keywords Muscle proteins . Actin . Myosin . Purificationprocess . SDS-PAGE

Introduction

Actin and myosin are the most important contractile proteinsin the muscle, representing 70 % of the myofibrillar fraction.Actin monomers are globular proteins, named G-actin, thatcan polymerize to form filaments, named F-actin, while my-osin, which is the major constituent of myofibrillar proteins(50 % of total), is composed of myosin heavy chains (MHC)and myosin light chains (MLC) (Kristinsson 2001). These

proteins are not only important in muscle physiology in livinganimals but also play a very important role in meat quality,since muscle texture and several functional properties, such aswater-holding capacity and the ability to form thermally in-duced gels, depend to a great extent on contractile proteins.

Numerous authors developed different methods for isola-tion and purification of myofibrillar proteins, most of themfrom pre-rigor muscle (Pardee and Spudich 1982), thoughthere are some studies using for this purpose post-rigor musclefrom different animals, such as turkey, chicken, rabbit, and pig(Dudziak and Foegeding 1988; Hasselbach and Schneider1951). Procedures for obtaining actin and myosin separatelyare based on the solubility of myosin at high ionic strength andits insolubility at low ionic strength (Perry 1955). The classicalmethod for the isolation of actin is based on the decompositionof the intermolecular linkage in F-actin and extracting it as G-actin after myosin has been solubilized and eliminated. Themost helpful agent for breaking the link without denaturingactin is acetone (Straub 1942; Szent-Györgyi 1951). Severalvariations of these methods were developed to obtain a greaterpurification degree, depending on the desired purity and thesubsequent use required for the protein (Pérez-Juan et al.2007; Syrovy 1984).

It is widely known that proteolysis is one of the biochem-ical processes with a greater contribution to the final qualitiesof meat and meat products. This proteolytic activity is con-sidered to be a result of the action of endogenous meatenzymes and bacterial proteases. Most works on microbialactivity characterization employ sarcoplasmic and myofibril-lar extracts for proteolytic activity assays. However, it may beinteresting to study this activity over specific proteins and thereal interaction between selected microbial strains and eachtype of protein.

Preparations of actin and myosin are available in themarket from chemical industry, but these preparations arevery expensive, highly unstable, and not sterile. For thepurpose aimed in this study, actin and myosin preparations

S. Fonseca :A. Cachaldora : J. Carballo (*)Área de Tecnología de los Alimentos, Facultad de Ciencias deOurense, Universidad de Vigo, 32004 Ourense, Spaine-mail: carbatec@uvigo.es

Food Anal. Methods (2013) 6:1033–1039DOI 10.1007/s12161-013-9654-0

should be obtained and sterilized immediately before theiruse and by means of quick and easy methods. Several rapidmethods to purify myofibrillar proteins have been developedfor application in the industry (Hidalgo et al. 2001; Murchet al. 1992), but they resulted unsuitable when a high-purityprotein extract is necessary for further uses. The existingmethods described in the literature for obtaining myosinand actin extracts may not be suitable for microbial proteo-lytic activity assays due to several circumstances such as alow protein concentration and purity or difficulties in thesterilization of the obtained extracts. Therefore, the currentresearch was undertaken with the aim of obtaining suitableactin and myosin extracts to be used in further studies, suchas testing the proteolytic activity of microbial cultures.

Materials and Methods

Protein Isolation Procedures

Actin Isolation

The procedure described by Pérez-Juan et al. (2007) was firstlyemployed for actin isolation. However, due to low actin con-centration achieved in our laboratory when using this methodas well as the protein retention exhibited by using polyvinyli-dene difluoride (PVDF) filter, some modifications were per-formed in order to improve the results; these modificationsbasically consisted in the repetition of the extraction and elim-ination of the myosin with the Hasselbach–Schneider bufferand the suppression of a step of resuspension in buffer A andcentrifugation at the end of the process. The final protocoldeveloped and proposed was as follows (Fig. 1a): 50 g ofpost-rigor porcine biceps femoris muscle (from Landrace ×Large White pig 80 kg carcasses, 48 h after slaughtering) washomogenized with 200 mL of wash buffer pH 7.0 (0.1 M Tris–HCl; 20 mM EDTA) in a masticator blender (IUL Instruments,Konigswinter, Germany) for 3 min. The extract was centri-fuged twice at 9,150×g for 10 min, and the supernatant (sarco-plasmic proteins) was discarded. The resulting pellet (myofi-brillar proteins) was resuspended in 200 mL of Hasselbach–Schneider buffer (0.1 M KH2PO4/K2HPO4 at pH 6.4; 0.6 MKCl, 10 mM Na4P2O7·10H2O, 1 mM MgCl2, and 20 mMethylene glycol tetraacetic acid) under shaking for 30 min andcentrifuged at 11,700×g for 30 min (solubilization and discard-ing of the myosin). The resuspension and centrifugation wererepeated. After the second centrifugation, the pellet (actin) wasresuspended in 750 mL of acetone for 20 min and filtratedthrough the Whatman Grade 54 filter paper in order to prepareacetone powder that was retained in the filter. The acetonepowder retained in the filter was used to obtain G-actin; itwas resuspended in 200 mL of buffer A (2 mM Tris–HCl atpH 8.0; 0.2 mMATPNa2; 0.5 mMβ-mercaptoethanol; 0.2 mM

CaCl2 and 0.005 % NaN3) for 15 min and centrifuged at10,400×g for 20 min. The resulting pellet was again resus-pended in buffer A (80 mL) for 45 min and centrifuged in thesame conditions. Finally, the supernatant was filtered throughthe Whatman Grade 54 filter paper.

Myosin Isolation

For myosin isolation, the method described by Abdel-Mohsen et al. (2003) was employed as a basis for improve-ment because this method allows the obtaining of high-purity myosin extracts. This procedure was carried out using,in general, the steps reported by these authors except for thelast one (resuspension of the pellet in buffer B and centrifu-gation), which was replaced with the final step suggested byCrockford and Johnston (1995) (resuspension of the pellet inbuffer D and centrifugation) due to the reasons given in the“Results and Discussion.” The complete process finally pro-posed was as follows (Fig. 1b): 70 g of post-rigor porcinebiceps femoris muscle (as previously described) was dilutedwith 210 mL of Hasselbach–Schneider buffer. The extractwas stirred slowly and continuously for 15 min with occa-sional pauses, and the myosin extraction was then stopped bythe addition of 280 mL of 4 °C distilled water. The mixturewas centrifuged at 3,000×g for 10 min, and the supernatant(myosin solution) was filtrated through the Whatman Grade54 filter paper. In order to precipitate the myosin, the filtratewas next diluted with 4 °C distilled water in a ratio of 2:1v/vand was centrifuged at 10,000×g for 15 min. The resultingpellet was washed with buffer B (KH2PO4/K2HPO4 20 mMat pH 7.2; KCl 0.12 M; EDTA 1 mM and dithiothreitol(DTT) 1 mM), resuspended in 80 mL of buffer D (sodiumpyrophosphate 50 mM and DTT 1 mM at pH 7.8), andcentrifuged at 10,000×g for 10 min. Finally, the supernatantwas filtered through the Whatman Grade 54 filter paper.During all the process, myosin extract was kept at 4 °C.

Determination of Protein Concentration

Quantification by Bicinchoninic Acid Method

Actin concentration was measured by the bicinchoninicacid (BCA; Sigma-Aldrich, St. Louis, USA) method de-scribed by Smith et al. (1985) using bovine serum albuminto construct the standard curve. Absorbance was measuredat 562 nm in a spectrophotometer (Dinko Instruments,mod. 8500; Barcelona, Spain). Values were the means ofat least three measurements.

Quantification by Bradford Method

Myosin concentration was measured using the method de-scribed by Bradford (1976) using the commercial kit Quick

1034 Food Anal. Methods (2013) 6:1033–1039

Start Bradford Protein Assay (Bio-Rad Laboratories, Hercules,CA, USA). This method was chosen because it is compatiblewith the compounds used during protein isolation. Coloredproducts were measured at 595 nm on a spectrophotometer(Dinko Instruments, mod. 8500; Barcelona, Spain). Valueswere the means of at least three measurements.

Evaluation of Protein Purity by Sodium DodecylSulfate–Polyacrylamide Gel Electrophoresis

Sodium dodecyl sulfate–polyacrylamide gel electrophore-sis (SDS-PAGE) was performed to determine the purity ofactin and myosin extracts previously obtained. The SDS-PAGE was run according to Laemmli (1970), using 12 %acrylamide separating gel and 4 % acrylamide stacking gel.Samples for electrophoresis were prepared by mixing10 μL of each protein extract with 19 μL of Laemmli buffer(Bio-Rad Laboratories) and 1 μL of β-mercaptoethanol.

Thirty microliters of each sample were applied onto differ-ent gel wells, using 10 μL of SDS-PAGEMolecular WeightStandard Low Range (Bio-Rad Laboratories) as a marker.The electrophoresis was carried out at 220 V for about45 min. Gels were soaked in fixing solution methanol/wa-ter/acetic acid (50:43:7) for 15 min, stained in 0.05 % (w/v)Coomassie blue R-250 in methanol/acetic acid/water(45:10:45) for 2 h, and destained in a methanol/ethanol/-acetic acid/water solution (20:10:5:65) until protein bandswere clearly distinguished from the background. Afterscanning the gels using the Molecular Imager Gel DocXR System (Bio-Rad Laboratories), the molecular weightof actin, myosin, and residual proteins or peptides found ineach sample was estimated by reference to the relativemobilities of standard proteins. The results correspondingto each band were expressed as a percentage of total opticdensity, as calculated using the Quantity One software(Bio-Rad Laboratories).

Fig. 1 Steps of the improved procedures proposed for actin (a) andmyosin (b) purification from post-rigor porcine biceps femoris muscle.Steps added or modified in relation to the previous procedures aremarked; for actin extraction, 1, resuspension in Hasselbach–Schneider

buffer and centrifugation were repeated, and 2, a step of resuspension inbuffer A and centrifugation was suppressed; for myosin extraction, 3,the final resuspension in buffer B and centrifugation was replaced witha resuspension in buffer D and centrifugation

Food Anal. Methods (2013) 6:1033–1039 1035

Repeatability and Reproducibility Tests

For the procedure of the obtaining of each protein, repeat-ability tests were performed isolating consecutively eachprotein six times a day. Reproducibility test were also carried

out isolating each protein twice a day for 3 days. Significantdifferences (P<0.05) were not found between resultsobtained in either of the tests.

Table 1 Actin and myosin concentration (in milligram per milliliter), assessed using BCA and Bradford methods, respectively, of the extractsobtained following the previous methods and those proposed in the present work (mean ± standard deviation of 36 determinations)

Protein concentration (mg/mL) Sign.

Before 0.22 μm filtration After 0.22 μm filtration

Actin

Method of Pérez-Juan et al. (2007)a 0.20±0.04 0.12±0.01 *

Method proposed in this workb 0.17±0.04 0.16±0.01 n.s.

Sign. n.s. **

Myosin

Method of Abdel-Mohsen et al. (2003)c 0.05±0.00 0.01±0.00 ***

Method proposed in this workd 0.22±0.01 0.21±0.01 n.s.

Sign. *** ***

n.s. not significant

*P<0.05; ***P<0.001a Actin extract obtained following the method proposed by Pérez-Juan et al. (2007)b Actin extract obtained following the method proposed in this workcMyosin extract obtained following the method proposed by Abdel-Mohsen et al. (2003)dMyosin extract obtained combining the method proposed by Abdel-Mohsen et al. (2003) and the final step suggested by Crockford and Johnston(1995), as proposed in the present work

MLC

Act

D

TtTr

Ti

MkDa

20711478

53

35

28

19

7

A B C D

Fig. 2 SDS-PAGE gels of the purification process of actin. Lane Mmolecular weight standard [myosin (207 kDa), β-galactosidase(114 kDa), bovine serum albumin (78 kDa), ovalbumin (53 kDa), carbonicanhydrase (35 kDa), soybean trypsin inhibitor (28 kDa), lysozyme(19 kDa), and aprotinin (7 kDa)]. Lane A actin extract obtained as de-scribed by Pérez-Juan et al. (2007) before 0.22 μm filtration (D desmin,Act actin, Tt troponin T, Tr tropomyosin, MLC myosin light chain, Titroponin I). Lane B actin extract obtained as described by Pérez-Juan et al.(2007) after 0.22 μm filtration. Lane C actin extract obtained using theproposed method before 0.22 μm filtration. Lane D actin extract obtainedusing the proposed method after 0.22 μm filtration

7

MkDa

20711478

53

35

28

19

A B C D

MHC

α-act

D

Act

TtTr

MLC

Tc

Fig. 3 SDS-PAGE gels of the purification process of myosin. Lane Mmolecular weight standard [myosin (207 kDa), β-galactosidase(114 kDa), bovine serum albumin (78 kDa), ovalbumin (53 kDa),carbonic anhydrase (35 kDa), soybean trypsin inhibitor (28 kDa), lyso-zyme (19 kDa), and aprotinin (7 kDa)]. Lane Amyosin extract obtainedas described by Abdel-Mohsen et al. (2003) before 0.22 μm filtration(MHCmyosin heavy chain, α-act alpha-actinin, D desmin, Act actin, Tttroponin T, Tr tropomyosin, MLC myosin light chain, Tc troponin C).Lane B myosin extract obtained as described by Abdel-Mohsen et al.(2003) after 0.22 μm filtration. Lane Cmyosin extract obtained follow-ing the improved method proposed in the present work before 0.22 μmfiltration. Lane D myosin extract obtained following the improvedmethod proposed in the present work after 0.22 μm filtration

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Preparation of Protein Extracts for Further Use

The concentration of actin and myosin extracts was stan-dardized in order to employ the same protein content in eachassay. With the aim of providing a suitable carbon source formicroorganisms’ growth, 1 % dextrose (w/v) was added.Extracts were filter sterilized through a PVDF 0.22-μm filter(Millipore, Bedford, USA) in order to obtain low proteinbinding, as confirmed by subsequent quantification andSDS-PAGE analysis. Sterility was confirmed by determiningthe absence of bacterial growth in plate count agar (PCA)(Oxoid, Basingstoke, UK). The actin and myosin extractsobtained and sterilized using the proposed methods weresuccessfully used in the assessment of proteolytic activityof Staphylococcus strains (Cachaldora et al. 2013).

Statistical Analysis

All statistical analyses were performed using the IBM SPSSStatistics 19 software (IBM, Armonk, USA). Significantdifferences were determined using one-way analysis of var-iance. Tukey’s test was employed to determine any signifi-cant difference among samples. Values were consideredsignificantly different when P<0.05.

Results and Discussion

The average values of protein extracts concentration obtainedfollowing the procedures described in this study are shown inTable 1. The actin extract obtained in our laboratory accordingto the method proposed by Pérez-Juan et al. (2007) had aprotein concentration of 0.20±0.04 mg/mL before 0.22 μmfiltration. These values were lower than those reported byPérez-Juan et al. (2007), who obtained a protein concentrationof 0.86 mg/mL and a recovery of 2.5 mg of G-actin per g ofmeat. A more aged muscle with more actin susceptible of

extraction was possibly used by Pérez-Juan et al. (2007)since it is well-known that the extractability of the actinincreases as the postmortem time increases. These differ-ences could also be due to a less purified extract obtainedby these authors, since contaminant peptides are alwaysquantified together with the protein of interest, actin inthis case, when using spectrometric quantification methods.In addition, significant protein retention (P<0.05) could beobserved after 0.22 μm filtration when following themethod proposed by these authors (Fig. 2, lanes A andB). Regarding the actin extract obtained following theimproved method proposed in this work, a protein con-centration of 0.17±0.04 mg/mL was obtained when mea-sured before 0.22 μm filtration, meaning a recovery of0.27 mg of G-actin per g of meat (post-rigor porcinemuscle). After 0.22 μm filtration of this protein extract,no significant (P>0.05) differences in concentration werefound, demonstrating the suitability of PVDF filters to beused in the sterilization of protein extracts due to their lowprotein binding. This behavior can be observed in Fig. 2(lanes C and D), where the intensity of the actin band, aswell as the other bands in the extract, remained stableafter 0.22 μm filtration. Since the purpose of the study ofPérez-Juan et al. (2007) was not the obtaining of sterilizedextracts to be used in microbiological studies, the actinextract recovered by these authors was not subjected tofilter sterilization in their work. That is why the valuesobtained in the present work after 0.22 μm filtration couldnot be compared with the study of these authors as shownin Table 1. The proposed modifications with regard to themethod of Pérez-Juan et al. (2007) allowed us to increasethe elimination of the impurities of myosin and to obtainthe actin solubilized in a smaller final volume. The de-creased actin retention in a PVDF filter in the proposedmethod with regard to the method of Pérez-Juan et al.(2007) could be due to differences in the contaminants ofthe final extract.

Table 2 Protein components (inpercent) of actin and myosinsterile extracts obtained follow-ing the new methods proposed inthis work, as calculated by den-sitometric analysis of SDS-PAGE gels (mean ± standard de-viation of 36 determinations)

NI not identified

Molecular weight (kDa) Protein component Actin extract (%) Myosin extract (%)

200 Myosin HC – 65.43±9.89

102 α-Actinin – 3.19±2.47

66 NI 3.59±1.25 –

55 Desmin 2.06±0.93 2.68±1.47

45 Actin 74.36±7.00 13.13±5.47

37 Troponin T 4.40±2.60 4.24±1.99

35 Tropomyosin 2.46±1.92 2.44±1.06

25 Myosin LC 5.16±2.02 2.43±1.15

24 Troponin I 7.97±3.22 –

20 Troponin C – 3.07±1.15

12 NI – 3.90±2.20

Food Anal. Methods (2013) 6:1033–1039 1037

The first approach used in this study to isolate myosin fromporcine muscle was carried out following the method pro-posed by Abdel-Mohsen et al. (2003). These authors isolatedmyosin from porcine meat, obtaining high-purity myosinextracts. However, the low myosin concentration obtainedfor this extract in our laboratory (0.05±0.00 mg/mL), togetherwith the high protein retention showed by the PVDF filter(Table 1), leads us to the replacement of the final step in thisisolation process with the final step suggested by Crockfordand Johnston (1995). The proposed modifications with regardto the method of Abdel-Mohsen et al. (2003) allowed us tosolubilize a higher quantity of myosin which was contained ina smaller final volume. This new method provided a moreconcentrated myosin extract with no significant retention inthe sterilization filter. The decrease in myosin retention in thePVDF filter could be attributed to the differences in thecomposition of the buffer in which myosin was finally resus-pended. This improvement can be also observed by SDS-PAGE analysis (Fig. 3), where the band of the myosinextracted according to the method proposed in this work(lanes C and D) resulted to be more intense compared to theoriginal method proposed by Abdel-Mohsen (lanes A and B).In addition, the low retention exhibited when myosinextracted according to the new proposed method was steril-ized by filtration can be clearly observed in Fig. 3, lane D.

Table 2 gives the percentage of protein components foundin actin and myosin sterile extracts obtained by the newproposed methods based on densitometric analysis of SDS-PAGE gels, and Figs. 2 and 3 show the band profilesobtained and its distribution according to their molecularweight. The protein profile of actin extract consisted of74.36 % actin (45 kDa). The sample also contained contam-ination from desmin (55 kDa), troponin T (37 kDa), tropo-myosin (35 kDa), myosin light chain (25 kDa), troponin I(24 kDa), and an unknown 66 kDa peptide. The extractobtained Pérez-Juan et al. (2007) also showed contaminantbands, mainly below 25 kDa, when isolating actin from post-rigor porcine skeletal muscle. The protein profile of myosinextract consisted of 65.43 % myosin (200 kDa) containingsubstantial contamination from actin (13.13 %). The samplealso contained other contaminant proteins such as α-actinin(102 kDa), desmin (55 kDa), troponin T (37 kDa) and C(20 kDa), tropomyosin (35 kDa), myosin light chain(25 kDa), and an unknown 12 kDa peptide (Porzio andPearson 1977). Kristinsson (2001) compared the purity ofmyosin samples obtained by four different isolation methodsfrom cod muscle. This author concluded that a slightlymodified version of the method proposed by Martone et al.(1986) was the most successful, obtaining a myosin extractwith a purity of 83.2 %. Nevertheless, results obtained in thepresent study cannot be compared to this latter due to thetype of animal species used with very different muscle struc-tures and compositions.

Conclusions

The method proposed by Pérez-Juan et al. (2007) for theisolation of actin, after the modifications proposed in thiswork, provides a high-purity extract with a suitable con-centration to be employed for testing the proteolytic activityof microbial cultures. In addition, the modifications per-formed on the method proposed by Abdel-Mohsen et al.(2003) for the isolation of myosin may be recommended toobtain a high-purity and more concentrated myosin extract.It should be also pointed out that according to the resultsobtained in this study, PVDF filters provided sterilizedprotein extracts with no significant differences from thenonfiltered extracts in concentration or purity.

Acknowledgments The authors thank the University of Vigo forthe financial support (Contracts Program with Reference ResearchGroups, Call 2009, Reference 09VIA12). Sonia Fonseca acknowl-edges the financial support from the Spanish Ministry of Scienceand Innovation through a predoctoral FPU fellowship (AP2008-03385).

Conflict of Interest Sonia Fonseca declares that she has no conflictof interest. Aida Cachaldora declares that she has no conflict ofinterest. Javier Carballo declares that he has no conflict of interest.This article does not contain any studies with human or animalsubjects.

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