research article chemical and functional characterization of sarcoplasmic proteins ... · 2019. 7....

Research ArticleChemical and Functional Characterization of SarcoplasmicProteins from Giant Squid (Dosidicus gigas) Mantle

Rosa Linda Lopez-Enriquez1 Victor Manuel Ocano-Higuera2 Wilfrido Torres-Arreola1

Josafat Marina Ezquerra-Brauer1 and Enrique Marquez-Rios1

1Departamento de Investigacion y Posgrado en Alimentos Universidad de Sonora Rosales y Ninos Heroes SN PO Box 165883000 Hermosillo SON Mexico2Departamento de Ciencias Quımico Biologicas Universidad de Sonora Rosales y Ninos Heroes SN PO Box 165883000 Hermosillo SON Mexico

Correspondence should be addressed to Enrique Marquez-Rios emarquezguayacanusonmx

Received 14 May 2015 Accepted 5 July 2015

Academic Editor Ioannis G Roussis

Copyright copy 2015 Rosa Linda Lopez-Enriquez et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Modification of pH and NaCl concentration changed the physicochemical properties of sarcoplasmic proteins (SP) from jumbosquid mantle and consequently their functional properties Better results of emulsifying activity index (EAI) and foam capacity(FC) were exhibited at pH 11 in NaCl absence due to higher solubility But better emulsifying stability index (ESI) was obtainedat pH 11 in 05M NaCl while foaming stability (FS) was better at pH near to isoelectric point (pI) These results suggest that SPfrom jumbo squid may be a promising ingredient whose functional properties can be manipulated by changing pH and NaClconcentration

1 Introduction

The giant squid (Dosidicus gigas) represents one of the mostimportant fisheries in Mexico in terms of catch volumesHowever it is an underutilized and undervalued resourcedespite its abundance and its nutritional value [1] In orderto increase its value and promote its consumption sev-eral researches have been carried out focused on obtain-ing protein concentrates from its mantle Nonetheless theconventional process for production thereof generates highvolumes of effluent with high content of soluble proteincalled sarcoplasmic proteins which can cause contaminationproblems if they are not treated before disposal [2]

Sarcoplasmic proteins represent 20 to 40 of the totalmuscle proteins These proteins are globular and exhibit sol-ubility in water and diluted salt solutions and are constitutedmainly by enzymes involved in cellularmetabolism [3]Thereare few studies about the protein composition structure andfunctionality of the sarcoplasmic proteins in comparison tomyofibrillar proteins Therefore their study could provide

information for use as food ingredientsThe above would notonly reduce pollution problems it could also make the use ofmarine resources more efficient which is the current trendnot only in fisheries but throughout the food industry

The effect of the sarcoplasmic proteins has been studiedon the textural quality of gelsmade frommyofibrillar proteinsof various species of fish obtaining favorable results [4 5]Additionally other functional properties have been exploredsuch as emulsifying and foaming property [6 7] demonstrat-ing that sarcoplasmic proteins have potential to be used asingredients in the food industry

In recent years various methods have been studied toinduce improvement of functional properties of proteinsdue to the fact that alternative processes based on extrinsicfactors such as pH shift ionic strength and temperaturemay induce conformational changes in proteins which inturn can improve their functional properties [6] In thissense Hemung et al [8] demonstrated that the sarcoplasmicproteins of sea bream (Nemipterus hexodon) treated in acidand alkaline conditions followed by neutralization exhibited

Hindawi Publishing CorporationJournal of ChemistryVolume 2015 Article ID 538721 10 pageshttpdxdoiorg1011552015538721

2 Journal of Chemistry

conformational changes which improved the emulsifyingactivity In another study Hemung et al [8] studied the effectof pH without subsequent neutralization of the sarcoplasmicproteins from the same species and found that in acidicconditions proteins exhibited conformational changes whichin turn induced the increased emulsifying activity

Nowadays there are no researches focused on the studyof sarcoplasmic proteins from giant squid mantle Conse-quently due to the importance of generating new knowledgeabout these proteins which would allow their use as afood ingredient the objective of this research is to recoversarcoplasmic proteins from giant squid mantle and evaluatethe pH and ionic strength effects on the physicochemical andfunctional properties

2 Materials and Methods

21 Raw Material Jumbo squid (D gigas) was harvested offthe coast of Kino Bay Sonora Mexico in June 2014 Tenspecimens were decapitated and gutted on site and werethen washed with freshwater at room temperature (25∘C)The mantles (experimental samples) were bagged and placedin alternating layers of ice-squid-ice in a portable coolerand transported to the laboratory Squid mantles exhibiteda length of 3785 plusmn 41 cm and a total weight of 8275 plusmn8275 g The elapsed time between capture and reaching thelaboratory did not exceed 12 h

22 Sarcoplasmic Proteins Preparation Frozen squid mantlewas thawed at 4-5∘C for 12 h minced and mixed withcold distilled water (le4∘C) at a ratio of 1 3 (mince water)Mixture was then homogenized at 1000 rpm for 1min usinga tissue homogenizer (Wisd WiseTis HG-15D Witeg Ger-many) The homogenate was centrifuged in a refrigeratedcentrifuge at 12000timesg for 20min at 4∘C (Thermo ScientificSorvall Biofuge StratosMAUSA)The supernatant obtainedwas considered as the sarcoplasmic proteins (SP) fractionand was analyzed for protein concentration according to theLowry method [9] using bovine serum albumin as standard

23 Modification of pH and NaCl Concentration TwentymL aliquots of SP were added to NaCl (0 02 and 05M)and the pH was adjusted to 30 50 70 90 or 110 Thenthe final volume of the homogenates was brought to 25mL(using cold distilled water) and stirred for 30min at 4∘CTheadjusted solutions were used to determine protein solubilityemulsifying and foam properties

24 Protein Solubility In order to determine the effect ofdifferent pH levels and NaCl concentrations on the sol-ubility of SP all solutions previously prepared were cen-trifuged at 12000timesg for 20min at 4∘C (Thermo ScientificSorvall Biofuge Stratos MA USA) Protein concentrationof the supernatants was analyzed by Lowry method [9]Protein solubility was expressed as the percentage of proteinremaining soluble after centrifugation in relation to the totalprotein present in SP Supernatants obtained were used forthe determination of protein patterns by sodium dodecyl

sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sur-face hydrophobicity (S

0-PRODAN and S

0-ANS) and total

sulfhydryl (SH) content

25 Electrophoretic Profile (SDS-PAGE) The electrophoreticprofile of each protein systemwas analyzed bymeans of poly-acrylamide gel electrophoresis (PAGE) using a dissociatingsodium dodecyl sulfate (SDS) buffer system in a discontinu-ous gel (4 stacking gel and 10 separating gel) according tothemethod of Laemmli [10] AMini-PROTEAN3CellMulti-Casting Chamber (Bio-Rad Laboratories Hercules CA) wasused Electrophoretic runs were performed at room tempera-ture (25∘C) at 80VThirty 120583g of protein was loaded into eachlane of the gels and a broad range ofmolecular weight proteinstandard solutions (Bio-Rad Laboratories Richmond CA)containing myosin (200 kDa) beta-galactosidase (116 kDa)phosphorylase b (97 kDa) bovine serum albumin (66 kDa)ovalbumin (45 kDa) and carbonic anhydrase (31 kDa) wereused After electrophoresis the gel was stained with 0125(wv) Coomassie brilliant blue R-250 in 40 (vv) methanoland 7 (vv) acetic acid and the gel was destained with 50(vv) methanol and 10 (vv) acetic acid

26 Surface Hydrophobicity (S0) Surface hydrophobicity (S

0)

was determined by the method reported by Alizadeh-Pasdarand Li-Chan [11] with slight modifications Protein solutionswere diluted in order to obtain protein concentrations of0 0125 025 05 and 01mgmLminus1 Four mL aliquot fromeach diluted solution was mixed with 20120583L of ANS solution(10mM phosphate buffer pH 7) or 10 120583L of PRODANsolution (14mM in methanol) ANS samples were set for10min and PRODAN samples were set in the dark for 15minSubsequently the fluorescence intensity of the samples wasmeasured using a spectrofluorometer (Agilent TechnologiesCary Eclipse CA USA) at excitationemission wavelengthof 390470 nm and 365465 for ANS and PRODAN respec-tively The surface hydrophobicity index (S

0) was calculated

from the regression slope or net relative fluorescence intensityversus protein concentration ( wv)

27 Determination of Total Sulfhydryl (TSH) Total sulfhydryl(TSH) content at different pH levels and NaCl concen-trations was determined by the method reported by Tad-pitchayangkoon et al [12] with slight modifications Proteincontent of each protein solution was adjusted to 1mgmLminus105mL aliquot of each treatment was mixed with 2mLof solubilizing buffer (02M Tris-HCl 8M urea 10mMEDTA pH 70) and 50 120583L of Ellmanrsquos reagent (10mM 551015840-dinitrobis [2-nitrobenzoic acid] 01M sodium phosphatebuffer pH 70) All sampleswere incubated at 40∘C for 25minThe absorbance was measured at 412 nm using a UV-Visspectrophotometer (Cary 50 Varian CA USA) and SHcontent was calculated using the extinction coefficient of13600Mminus1 cmminus1

28 Emulsifying Properties The emulsifying activity index(EAI) and emulsifying stability index (ESI) at differentpH levels and NaCl concentrations were determined by

Journal of Chemistry 3

turbidimetric technique of Pearce and Kinsella [13] withsome modifications Each protein solution was adjusted to05mgmLminus1 Protein solutions were mixed with canola oilat a ratio of 1 1 and homogenized at 13500 rpm for 1minusing a tissue homogenizer (Wisd WiseTis HG-15D WitegGermany) To determine EAI the emulsions phases wereallowed to separate during 1min and the lower layer wasobtained (100 120583L) and diluted 40 times with 01 of sodiumdodecyl sulphate (SDS) and the absorbance was recordedat 500 nm (119860

500) using a UV-Vis spectrophotometer (Cary

50 Varian CA USA) The EAI was calculated using thefollowing equation

120591 =2303 times 119860

500times 119865

119897

EAI (m2g2) = 2120591120601119862

(1)

where 120591 is the turbidity 119860500

is the sample absorbance119865 is the sample dilution factor (40) and 119897 (001m) is thelight path length The oil volume fractions for emulsions (120601)were 05 and the protein emulsifier concentration (119862) was05mgmLminus1

To determine emulsifying stability index (ESI) aliquotsof prepared emulsions (10mL) were placed inside of 10mLcontainers immediately after preparation At 0 and 10min fol-lowing emulsion preparation 100 120583L samples were removedand diluted 40-fold prior to turbidity measurements ESI wascalculated from

ESI (min) = 120591 times Δ119905Δ120591 (2)

where Δ120591 is the change in turbidity after a time interval Δ119905(10min)

29 Foaming Properties Foaming capacity (FC) and foamstability (FS) at different pH levels and NaCl concentrationswere determined according to the Wild and Clark method[14] with some modifications Each protein solution wasadjusted to 2mgmLminus1 Twenty milliliters of diluted proteinsolutionswas homogenized at 18000 rpm for 1min using a tis-sue homogenizer (WisdWiseTis HG-15DWiteg Germany)The solutions were transferred to a measuring cylinder Thevolume of foam at 30 s was calculated and the volumeincrease was expressed as percent foaming capacity (FC) andwas calculated from

FC () = Volume of foam (mL)Initial liquid volume (mL)

times 100 (3)

The foam stability (FS) was calculated as the percentage offoam remaining after 30min at 25∘C using the followingequation

FS ()

=Volume of foam (mL) retained after 30 minVolume of foam soon after whipping (mL)

times 100

(4)

210 Experimental Design and Statistical Analysis A factorialexperiment (5 times 3) was performed to determine the effectsof individual factors and their interactions ph with 5 levelsand NaCl concentration with 3 The response variables wereprotein solubility () total sulfhydryl content (TSH mole105 gminus1) surface hydrophobicity (S

0-ANS and S

0-PRODAN)

emulsifying activity index (EAIm2 gminus1) emulsifying stabilityindex (ESI min) foaming capacity (FC ) and foamstability (FS ) All measurements were performed intriplicate and data were presented as the mean plusmn standarddeviation In order to determine the statistical differencesamong treatments a two-way analysis of variance (ANOVA)was used to compare means with a significance level of 119875 lt005 Data were analyzed using the JMP statistical softwareversion 1000

3 Results and Discussion

31 Sarcoplasmic Protein Recovery The SP solution exhibiteda protein concentration of 659 plusmn 142mgmLminus1 and rep-resented the 1402 plusmn 421 of total protein content fromthe mantle The protein concentration of the SP extractwas in the range obtained for Pacific whiting (Merlucciusproductus) surimi wash-water (54ndash234mgmLminus1) [15 16]This latter is one of the most utilized species for the man-ufacture of surimi (Rocha-Estrada et al 2010) Regardingprotein recovery the SP fraction obtained in this studywas lower than that obtained by Sanchez-Alonso et al [17]who used a 1 5 (mantle water) ratio for the extraction ofsarcoplasmic proteins from jumbo squid mantle reporting aprotein recovery of 25ndash27 while De la Fuente-Betancourtet al [18] who used a 1 10 (mantle water) ratio reported arecovery of 961 which is lesser than that obtained in thisstudy The recovery of SP fraction from jumbo squid mantlemight be affected by factors related to protein extractionas the musclewater ratio and the centrifugation conditionsused [19] Also it might be affected by intrinsic (size andchemical composition) and extrinsic aspects (harvest seasontemperature and food availability) of the species [20]

32 Protein Solubility Solubility is a critical functional prop-erty of proteins because it is considered as prerequisitefor other functionalities including emulsifying and foamingproperties The effect of pH and NaCl concentration on thesolubility of SP is shown in Figure 1 The solubility of SP inabsence of NaCl showed the typical U-shaped curve as mostfood proteins including marine sources [12] The minimumand the maximum solubility of SP were presented at pH of5 and 11 respectively Hence the isoelectric point (pI) of SPcould be close to pH 50Thehigher solubility of SP at alkalinepH in comparison to acid pH might be due to the aminoacid ionization unfolding and hydration of protein Mostfood proteins are acidic since the sum of their acid residues(Asp and Glu) is higher than their basic residues (Lys Argand His) therefore proteins exhibit the minimal solubility atpH 4-5 (isoelectric pH) and maximum at alkaline pH values[4] In this sense the presence of higher content of acid thanbasic residues in proteins from jumbo squid (Dosidicus gigas)


0

20

40

60

80

100

120

Prot

ein

solu

bilit

y (

)

5 7 9 113pH

0M NaCl02M NaCl05M NaCl

Figure 1 Effect of pH and NaCl concentration on protein solubilityof SP from jumbo squidmantle Data represent themean plusmn standarddeviation (119899 = 3)

mantle has been reported and this fact matches with the highsolubility of SP obtained in this study at pH [21 22]

At alkaline pH the solubility was similar However atpH 30 the solubility was significantly different (119875 lt 005)It was noted that the solubility was reduced when salt wasadded obtaining the minimum with 05M of NaCl Thisbehavior could be attributed to the interaction of negativelycharged chloride ions (Clminus) with positively charged groups(NH3+ from Lys Arg and His) of SP leading to a decrease inelectrostatic repulsion and subsequently leading to the aggre-gation and precipitation [23 24] A similar phenomenonhas been reported for myofibrillar proteins from Pacificwhiting (Merluccius productus) and sarcoplasmic proteinsfrom rockfish (Sebastes flavidus) [5 24]

33 Electrophoretic Profile (SDS-PAGE) In order to evaluatethe effect of pH and NaCl concentration on the molecularweights (MW) of proteins present in sarcoplasmic fractionfrom squid mantle electrophoretic profiles were determinedSDS-PAGE of the SP is shown in Figure 2 Protein bandswith MW ranging from 30 to 637 were detected and thecorresponding bands 375 382 and 443 kDa were the mostintense When the NaCl was added to SP system super-natants obtained at pH 5ndash11 at all NaCl concentrations (0 02and 05) did not show qualitative differencesThe presence ofweak bands at pH 3 in NaCl 05M reaffirms the low proteinsolubility of SP at this condition Based on solubility resultsand SDS-PAGE patterns NaCl concentration appeared tohave a negative effect on solubility of SP from jumbo squidmantle at extreme pH But SP from jumbo squid mantlebecame more soluble and stable at neutral and at alkalinevalues than at acid pH values

The electrophoretic pattern obtained in this experimentwas similar to that obtained by Ezquerra-Brauer et al [20]

for water-soluble proteins (20ndash66 kDa) from jumbo squidmantle However De la Fuente-Betancourt et al [18] obtainedhigher MW of SP from mantle of same species (20ndash97 kDa)As described earlier the recovery of SP fraction might beaffected by intrinsic and extrinsic factors of jumbo squid aswell as by extraction conditions used [19 25]

The proteins present in sarcoplasmic fraction from fishspecies are related to aerobic metabolism of the fish cellsand have exhibited MW of 34 40 43 50 and 94 kDa whichwere attributed to glyceraldehyde phosphate dehydrogenasealdolase creatine kinase enolase and phosphorylase respec-tively [3 12 26 27] Nevertheless since jumbo squid is amollusk it has a quite different SP profile compared to fishspecies activity of arginine kinase (40ndash43 kDa) and octopinedehydrogenase (37ndash45 kDa) has been reported [28ndash31]Theseare enzymes related to the anaerobic metabolism of mollusksand exhibit MW similar to that obtained in this study forSP fraction from jumbo squid mantle However in orderto know more about the protein components of SP furtherresearch related to protein characterization is required

34 Total Sulfhydryl (SH) Content Conformational changesof proteins can lead to the exchange of SHS-SThus total SHcontent is an indicator of changes in protein conformationand flexibility [20 21] Total sulfhydryl (TSH) content ofSP varied significantly with pH and NaCl concentration(119875 lt 005) (Figure 3) The TSH content decreased whenpH was changed from 5 to 11 and from 5 to 3 the effectwas more pronounced as the NaCl concentration increasedThe unfolding of SP induced by pH shift (at both acidicand alkaline pH values) might have induced the expositionof buried SH groups and the consequent S-S formation Itwas reported that exchange reactions SHS-S are favored atalkaline pH [32 33] The results of SH content demonstratedthat at extreme pH (pH 3 and pH 11) the SH oxidation ofSP was induced Similar findings for myofibrillar proteinsfromPacificwhiting (Merluccius productus) and sarcoplasmicproteins from striped catfish (Pangasius hypophthalmus) havebeen reported [11 34] On the other hand the decrease ofTSH content by NaCl effect can be attributed to a betterprotein unfolding with the consequent exposition of internfunctional groups including the SH groups [22 35] Themajor exposition of SH groups might have allowed theexchange of SH to S-S

35 Surface Hydrophobicity (S0) Variations on surface

hydrophobicity (S0) denote changes in protein structure

conformation In this study surface hydrophobicity of SP wasdetermined at different pH levels and NaCl concentrationsbased on ANS and PRODAN probes Surface hydrophobicitybased on the ANS probe (S

0-ANS) was significantly affected

by pH and NaCl concentration (119875 lt 005) (Figure 4(a))Extremely high values of S

0-ANS were obtained at pH 3

compared to S0-PRODAN values It has been reported that

at acidic pH values (minor to pH 38) S0-ANS reflects not

only the hydrophobic interaction between ANS and proteinsbut also electrostatic interactions between anionic sulfonategroups of ANS and cationic groups (Lys Arg and His)


66

55

45

36

(kDa)

pH3 5 7 9 11 S

(a)

66

55

45

36

(kDa)

pH3 5 7 9 11 S

(b)

66

55

45

36

(kDa)

pH3 5 7 9 11 S

(c)

Figure 2 Protein patterns of SP from jumbo squid in (a) 0M NaCl (b) 02M NaCl and (c) 05M NaCl The numbers indicate pH while Sdenotes molecular weight standard

of proteins [10] Other research works reported the samebehavior of ANS at acidic pH values [11 34] Thereforein this experiment the change of surface hydrophobicity isdiscussed with more detail in function of PRODAN reagent

S0-PRODAN was also significantly affected by pH and

NaCl concentrations (119875 lt 005) (Figure 4(b)) The S0-

PRODAN values of SP in absence of NaCl increased asthe pH increased in the range from 5 to 11 The high S

0-

PRODAN values at alkaline pH (11) were attributed to ahigher exposure of buried hydrophobic groups due to theelectrostatic repulsion of negative charged residues In con-trast at pH 5 the minimum S

0-PRODAN value was showed

and this might be due to the protein aggregation becauseat this pH the minimum solubility was detected leading toa lower surface hydrophobicity of SP These results agreewith those obtained for myofibrillar proteins from Pacificwhiting (Merluccius productus) sarcoplasmic proteins fromstriped catfish (Pangasius hypophthalmus) and threadfinbream (Nemipterus sp) [6 11 34]

It was proved that a positive correlation exists betweensolubility and surface hydrophobicity of SP being highlysignificant with 1199032 of 082 091 and 096 for 00 02 and 05Mof NaCl respectivelyThis could be explained as a function ofthe unfolding of SP induced by pH and NaCl modificationand the consequent increase of both solubility and surfacehydrophobicityThe effect of NaCl concentration at pH 3 wasopposite in comparison to other pH values however thisbehavior was the same as the solubility and it could be dueto the decrease of hydrophobic groups exposed for the SPaggregation as already explained for SP solubility

36 Emulsifying Properties The effects of pH and NaClconcentration on emulsifying activity index (EAI) and emul-sifying stability index (ESI) are shown in Figure 5 The EAIvalue of crude extract of SP (pH 7 0MNaCl) obtained in thisstudy (8332 plusmn 159m2 gminus1) was lower than that obtained forsarcoplasmic proteins from threadfin bream (Nemipterus sp)


5 7 9 113pH


0

5

10

15

20

25TS

H co

nten

t (m

ole1

05

gminus1)

Figure 3 Effect of pH and NaCl concentration on total sulfhydryl(TSH) content in SP from jumbo squid mantle Data represent themean plusmn standard deviation (119899 = 3)

(sim210m2 gminus1) [6] However this was higher than thatreported for lyophilized sarcoplasmic proteins from threadfinbream (Nemipterus hexodon) because an EAI of 46m2 gminus1has been reported [6]

Figure 5(a) shows the EAI for SP as a function of pH andNaCl concentration EAI of SP was significantly affected bythe pH and NaCl concentration (119875 lt 005) showing theminimum value at pH 5 and the higher at pH 11 Since pH 5is closer to the isoelectric point the low EAI was presenteddue to aggregation and loss of solubility of SP as a resultof the minimum electrostatic repulsion [24] Yongsawatdiguland Hemung [26] also found that EAI of emulsion stabilizedby sarcoplasmic proteins of threadfin bream (Nemipterus sp)was lowest at its pI (pH 5)

At pH 5 the increase in EAI could be due to increasein solubility caused by the addition of NaCl At pH loweror higher than 5 the EAI increase can be due to a majorsolubility It was noted that at pH lower or higher than 5the EAI changes very little it remains practically unchangedwith pH 11 in absence of NaCl as exception Kristinsson andHultin [16] also found that EAI of emulsion stabilized bymyofibrillar proteins from cod (Gadus morhua) was higherat pH 11 and this result was well correlated with the increaseof surface hydrophobicity and interfacial activity In contrastHemung et al [8] found that EAI of emulsion stabilized bysarcoplasmic proteins from threadfin bream (Nemipterus sp)was higher at pH 3 (610m2 gminus1) than at pH 12 (363m2 gminus1)and these results were not correlated with S

0-PRODAN

values Hence it can be mentioned that the EAI of proteinsis species dependent

The addition of NaCl to the SP system at pH 11 decreasesthe EAI which could be related to the increase of hydropho-bic groups exposed (S

0-PRODAN) and the decrease in

solubility with the increase of NaCl concentration indicating

that the hydrophobichydrophilic balance of SP was affectedThis balance is necessary because proteins have to inter-act with both oil and water thus the high exposition ofhydrophobic groups and the possible major rigidity of SPby the disulfide bonds (S-S) formation could have inducedthe decrease of EAI of emulsion at pH 11 Yuliana et al[36] also found that EAI of emulsion stabilized by proteinsisolated from cashew nut shell (Cashew variety Venguria-4) was decreasing with the increase of NaCl concentration(0ndash2M) In contrast Zhang et al [37] found that EAI ofemulsion stabilized by protein concentrate from chickpea(Cicer arietinum L) was increased with the increase of NaClconcentration (0-1M) whichwas correlatedwith the increaseof hydrophobicity

The ESI value of crude extract of SP (pH 7) obtainedin this study was 7312 plusmn 1457min This value was higherthan those reported for sarcoplasmic proteins from rohu(Labeo rohita 52min) and from threadfin bream (Nemipterushexodon 63min) [8 38] Figure 5(b) shows ESI values forSP as a function of pH and NaCl concentration ESI wassignificantly affected by the pH and NaCl concentration (119875 lt005) The graph indicates that as pH increases ESI increasedindependently of the NaCl concentration being significantlyhigher at pH 11 The minimum ESI value was at pH 3 in theabsence of NaCl whereas the maximum value was reached atpH 11 in presence of 05M NaCl The increase of ESI at pH11 regarding SP at pH 7 was 24331 This might be due tothe large repulsion which prevents the coalesce [39] At pH 11and 05M of NaCl the highest surface hydrophobicity and thelowest SH content were present the latter indicating that themajor disulfide (SndashS) bonds formation occurred under theseconditions Therefore the SP once absorbed might stabilizeinteractions between proteins that form the interface [15 40]

37 Foaming Properties The effects of pH and NaCl concen-tration on foaming capacity (FC) and foam stability (FS) areshown in Figures 6(a) and 6(b) respectively The FC value ofcrude extract of SP (pH 7 0M NaCl) obtained in this studywas 575 plusmn 25 This value was higher than that reportedfor sarcoplasmic proteins from rohu (Labeo rohita 4133)using a solution with a protein concentration of 25mgmLminus1similar to the one used in this study (2mgmLminus1) [38] andwas lower than that reported for sarcoplasmic proteins fromthreadfin bream (Nemipterus hexodon 165) [6]

The FC of SP was significantly affected by the pH andNaCl concentration (119875 lt 005) (Figure 6(a)) The behaviorof FC as a function of pH showed a similar tendency as EAIIn absence of NaCl the FC was found to be the lowest at pH5 (2411 plusmn 365) and the highest at pH 11 (8784 plusmn 125)At pH 5 the low FC could be related to low solubility dueto the fact that at this pH low electrostatic repulsion occurscausing formation of aggregates and precipitation affectingits interfacial activity [5 12] The highest FC at pH 11 couldbe related as well as EAI to the high protein solubility andhydrophobicity of SP Krasaechol et al [6] reported that theFC and FS are related to a high hydrophobicity Yet the results


5 7 9 113pH


0

5000

10000

15000

20000

25000

30000

S0-

AN

S

(a)

5 7 9 113pH


0

1000

2000

3000

4000

5000

6000

S0-

PRO

DA

N

(b)

Figure 4 Effect of pH and NaCl concentration on the surface hydrophobicity (S0

) in SP from jumbo squid mantle using the probes (a) ANSand (b) PRODAN Data represent the mean plusmn standard deviation (119899 = 3)

5 7 9 113pH


0

25

50

75

100

125

150

EAI (

m2

gminus1)

(a)

5 7 9 113pH


0

50

100

150

200

250

ESI (

min

)

(b)

Figure 5 Effect of pH and NaCl concentration on (a) emulsifying activity index (EAI) and (b) emulsifying stability index (ESI) of SP fromjumbo squid mantle Data represent the mean plusmn standard deviation (119899 = 3)

obtained for FC in function of pH did not correlate well withsurface hydrophobicity and solubility of SP

As well as the emulsifying properties foaming propertiesof food proteins also are better at pH values away fromtheir isoelectric point (pI) especially at alkaline pH valuesSimilar results have been found by Rocha-Estrada et al [41]and Galvez-Rongel et al [42] this research found better FCvalues for proteins from jumbo squid (Dosidicus gigas)mantletreated at alkaline pH values [3]

The increment of NaCl concentration decreased the FCvalues to pH 11 possible due to the higher exposition ofhydrophobic groups and decrease of solubility with the con-sequent change of hydrophobicityhydrophobicity balance ofthe SP as occurred in EAI Rocha-Estrada et al [41] foundthat the FC of foams stabilized by proteins from jumbosquid (Dosidicus gigas) decreased with the increase of NaClconcentration (0ndash04M) Yuliana et al [36] also found thatFC stabilized by proteins isolated from cashew nut shell


5 7 9 113pH


100

90

80

70

60

50

40

30

20

10

0

FC (

)

(a)

5 7 9 113pH


120

100

80

60

40

20

0

FS (

)

(b)

Figure 6 Effect of pH and NaCl concentration on (a) foaming capacity (FC) and (b) foam stability (FS) of SP from jumbo squid mantle Datarepresent the mean plusmn standard deviation (119899 = 3)

(Cashew variety Venguria-4) decreased with the increase ofNaCl concentration (0ndash2M)

Figure 6(b) shows the results of FS (119875 lt 005) It wasdetected that FS was affected by the pH and did not followthe obtained tendency of the other functional properties(EAI ESI and FC) Furthermore all pH values presentedirregular FS values in function of NaCl concentration InNaCl absence FS was found to be the lowest at pH 3 (332 plusmn502) and the highest at pH 7 (9776plusmn085) No significantdifferences (119875 lt 005) were found at pH 5 by effect of NaClconcentrations The best results were obtained at pH 5 andpH 7 at higher or lower pH values the tendency was todecrease This indicates that pH 7 could be the best option tomake foamsOur results differ from those obtained byRocha-Estrada et al [41] who working with myofibrillar proteinsfrom jumbo squid (Dosidicus gigas) reported that alkaline pHvalues (10ndash12) presented the highest FS values when NaClwas added (0ndash04M) Nonetheless many studies have shownthat stability is better at pH close to pI With pH valuesnear to pI the absence of repulsive interactions betweenproteins promotes attractive interactions Thus it promotesthe formation of a viscoelastic layer in the interface airwater[15]

4 Conclusions

Conformational changes of sarcoplasmic proteins fromjumbo squid mantle were influenced by pH and NaClconcentration and consequently their functional propertiesProteins presented the highest solubility and hydrophobicityin absence of NaCl at pH 11 resulting in an increase ofemulsifying and foaming capacity The emulsifying stabilitywas better at pH 11 and was enhanced by the increase of NaCl

due to the hydrophobic interactions on interface oilwaterHowever foaming stability was enhanced by the absenceof electrostatic repulsions at pH near to pI These proteinshave shown that they could be used as food ingredients andtheir functional properties can be improved by changing pHandor NaCl concentration thus contributing to a better useof this fishery resource

Conflict of Interests

The authors declare that they have no conflict of interests

References

[1] M C Luna-Raya J I Urciaga-Garcıa C A Salinas-Zabala MA Cisneros-Mata and L F Beltran-Morales ldquoDiagnostico delconsumodel calamar gigante enMexico y en SonorardquoEconomıaSociedad y Territorio vol 6 pp 535ndash560 2006

[2] J W Park and M T Morrissey ldquoManufacturing and Surimifrom lightmuscle fishrdquo in Surimi and Surimi Seafood JW ParkEd pp 23ndash58 Marcel Dekker Inc New York NY USA CRCPress Basel Switzerland 2000

[3] T Nakagawa S Watabe and K Hashimoto ldquoIdentification ofthree major components in fish sarcoplasmic proteinsrdquo NipponSuisan Gakkaishi vol 54 no 6 pp 999ndash1004 1998

[4] Y S Kim J W Park and Y J Choi ldquoNew approaches for theeffective recovery of fish proteins and their physicochemicalcharacteristicsrdquo Fisheries Science vol 69 no 6 pp 1231ndash12392003

[5] Y S Kim J Yongsawatdigul J W Park and S Thawornchin-sombut ldquoCharacteristics of sarcoplasmic proteins and theirinteractionwithmyofibrillar proteinsrdquo Journal of FoodBiochem-istry vol 29 no 5 pp 517ndash532 2005


[6] N Krasaechol R Sanguandeekul K Duangmal and R KOwusu-Apenten ldquoStructure and functional properties of modi-fied threadfinbream sarcoplasmic proteinrdquoFoodChemistry vol107 no 1 pp 1ndash10 2008

[7] Y Kawai R Ohno N Inoue and H Shinano ldquoEmulsifyingactivity of heat treated sarcoplasmic protein from sardinerdquoFisheries Science vol 61 pp 852ndash855 1995

[8] B-O Hemung S Benjakul and J Yongsawatdigul ldquopH-dependent characteristics of gel-like emulsion stabilized bythreadfinbream sarcoplasmic proteinsrdquoFoodHydrocolloids vol30 no 1 pp 315ndash322 2013

[9] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo Journalof Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[10] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[11] N Alizadeh-Pasdar and E C Y Li-Chan ldquoComparison ofprotein surface hydrophobicity measured at various pH valuesusing three different fluorescent probesrdquo Journal of Agriculturaland Food Chemistry vol 48 no 2 pp 328ndash334 2000

[12] P Tadpitchayangkoon J W Park and J Yongsawatdigul ldquoCon-formational changes and dynamic rheological properties of fishsarcoplasmic proteins treated at various pHsrdquo Food Chemistryvol 121 no 4 pp 1046ndash1052 2010

[13] K N Pearce and J E Kinsella ldquoEmulsifying properties ofproteins evaluation of a turbidimetric techniquerdquo Journal ofAgricultural and Food Chemistry vol 26 no 3 pp 716ndash7231978

[14] P J Wild and D C Clark ldquoFoam formation and stabilityrdquo inMethods for Testing Protein Functionality G M Hall Ed pp110ndash148 Blackie Academic and Professional London UK 1996

[15] S Damodaran ldquoAminoacids peptides and proteinsrdquo in FoodChemistry S Damodaran K L Parkin and O R FennemaEds pp 217ndash330 CRC Press Boca Raton Fla USA 2008

[16] H G Kristinsson and H O Hultin ldquoEffect of low and high pHtreatment on the functional properties of cod muscle proteinsrdquoJournal of Agricultural and Food Chemistry vol 51 no 17 pp5103ndash5110 2003

[17] I Sanchez-Alonso M Careche and A J Borderıas ldquoMethodfor producing a functional protein concentrate from giant squid(Dosidicus gigas)musclerdquoFoodChemistry vol 100 no 1 pp 48ndash54 2007

[18] G De la Fuente-Betancourt F Garcıa-Carreno M VNavarrete-del Toro R Pacheco-Aguilar and J H Cordova-Murueta ldquoEffect of storage at 0∘C on mantle proteins andfunctional properties of jumbo squidrdquo International Journal ofFood Science and Technology vol 43 no 7 pp 1263ndash1270 2008

[19] J W Park and T M Lin ldquoSurimi manufacturing and evalua-tionrdquo in Surimi and Surimi Seafood J W Park Ed pp 35ndash98CRC Press Boca Raton Fla USA 2005

[20] J M Ezquerra-Brauer N F Haard R Ramırez-Olivas HOlivas-Burrola and C J Velazquez-Sanchez ldquoInfluence ofharvest season on the proteolytic activity of hepatopancreas andmantle tissues from jumbo squid (Dosidicus gigas)rdquo Journal ofFood Biochemistry vol 26 no 5 pp 459ndash475 2002

[21] J H Cordova-Murueta and F L Garcıa-Carreno ldquoNutritivevalue of squid and hydrolyzed protein supplement in shrimpfeedrdquo Aquaculture vol 210 no 1ndash4 pp 371ndash384 2002

[22] N Rajapakse E Mendis H-G Byun and S-K Kim ldquoPurifi-cation and in vitro antioxidative effects of giant squid muscle

peptides on free radical-mediated oxidative systemsrdquo Journal ofNutritional Biochemistry vol 16 no 9 pp 562ndash569 2005

[23] D C A Mireles J T M Lin and A Ismond ldquoWaste manage-ment utilization and challengesrdquo in Surimi and Surimi SeafoodJ W Park Ed pp 313ndash339 CRC Press New York NY USA2013

[24] SThawornchinsombut and J W Park ldquoRole of pH in solubilityand conformational changes of Pacificwhitingmuscle proteinsrdquoJournal of Food Biochemistry vol 28 no 2 pp 135ndash154 2004

[25] M Lin J W Park andM T Morrissey ldquoRecovered protein andreconditioned water from surimi processing wasterdquo Journal ofFood Science vol 50 no 1 pp 4ndash9 1995

[26] J Yongsawatdigul and B-O Hemung ldquoStructural changes andfunctional properties of threadfin bream sarcoplasmic proteinssubjected to pH-shifting treatments and lyophilizationrdquo Journalof Food Science vol 75 no 3 pp C251ndashC257 2010

[27] M Toyohara M Murata M Ando S Kubota M SakaguchiandH Toyohara ldquoTexture changes associatedwith insolubiliza-tion of sarcoplasmic proteins during salt-vinegar curing of fishrdquoJournal of Food Science vol 64 no 5 pp 804ndash807 1999

[28] J L Schrimsher and K B Taylor ldquoOctopine dehydrogenasefrom Pecten maximus steady-state mechanismrdquo Biochemistryvol 23 no 7 pp 1348ndash1353 1984

[29] G Gade and K-H Carlsson ldquoPurification and characterisationof octopine dehydrogenase from the marine nemertean Cer-ebratulus lacteus (Anopla Heteronemerta) comparison withscallop octopine dehydrogenaserdquoMarine Biology vol 79 no 1pp 39ndash45 1984

[30] A E Brown R M France and S H Grossman ldquoPurificationand characterization of arginine kinase from the Americancockroach (Periplaneta americana)rdquoArchives of Insect Biochem-istry and Physiology vol 56 no 2 pp 51ndash60 2004

[31] EMaarquez-Riaos E FMoraan-PalacioM E Lugo-SaanchezV M Ocano-Higuera and R Pacheco-Aguilar ldquoPostmortembiochemical behavior of giant squid (Dosidicus gigas) mantlemuscle stored in ice and its relation with quality parametersrdquoJournal of Food Science vol 72 no 7 pp C356ndashC362 2007

[32] J Yongsawatdigul and J W Park ldquoEffect of alkaline and acidsolubilization on gelation characteristics of rockfish proteinsrdquoFisheries Science vol 69 pp 499ndash505 2004

[33] J Kijowski ldquoMuscle proteinsrdquo in Chemical and FunctionalProperties of Food Proteins Z E Sirkorski Ed pp 233ndash270CRC Press Lancaster Pa USA 2001

[34] CAMDeWitt GGomez and JM James ldquoProtein extractionfrom beef heart using acid solubilizationrdquo Journal of FoodScience vol 67 no 9 pp 3335ndash3341 2002

[35] S Nakai and E Li-Chan Hydrophobic Interactions in FoodSystems CRC Press Boca Raton Fla USA 1988

[36] M Yuliana C T Truong L H Huynh Q P Ho and Y-H JuldquoIsolation and characterization of protein isolated fromdefattedcashew nut shell influence of pH and NaCl on solubility andfunctional propertiesrdquo LWTmdashFood Science and Technology vol55 no 2 pp 621ndash626 2014

[37] T Zhang B Jiang W Mu and Z Wang ldquoEmulsifying prop-erties of chickpea protein isolates influence of pH and NaClrdquoFood Hydrocolloids vol 23 no 1 pp 146ndash152 2009

[38] F J Monahan J B German and J E Kinsella ldquoEffect ofpH and temperature on protein unfolding and thioldisulfideinterchange reactions during heat-induced gelation of wheyproteinsrdquo Journal of Agricultural and Food Chemistry vol 43no 1 pp 46ndash52 1995


[39] D J McClements Food Emulsions Principles Practices andTechniques CRC Press Boca Raton Fla USA 2005

[40] S Tcholakova N D Denkov I B Ivanov and B CampbellldquoCoalescence in 120573-lactoglobulin-stabilized emulsions effects ofprotein adsorption and drop sizerdquo Langmuir vol 18 no 23 pp8960ndash8971 2002

[41] J G Rocha-Estrada J H Cordova-Murueta and F L Garcıa-Carreno ldquoFunctional properties of protein from frozen mantleand fin of jumbo squid Dosidicus gigas in function of pH andionic strengthrdquo Food Science and Technology International vol16 no 5 pp 451ndash458 2010

[42] A Galvez-Rongel J M Ezquerra-Brauer V M Ocano-Higueraet al ldquoMethods to obtain protein concentrates from jumbosquid (Dosidicus gigas) and evaluation of their functionalityrdquoFood Science and Technology International vol 20 no 2 pp109ndash117 2014

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


conformational changes which improved the emulsifyingactivity In another study Hemung et al [8] studied the effectof pH without subsequent neutralization of the sarcoplasmicproteins from the same species and found that in acidicconditions proteins exhibited conformational changes whichin turn induced the increased emulsifying activity

Nowadays there are no researches focused on the studyof sarcoplasmic proteins from giant squid mantle Conse-quently due to the importance of generating new knowledgeabout these proteins which would allow their use as afood ingredient the objective of this research is to recoversarcoplasmic proteins from giant squid mantle and evaluatethe pH and ionic strength effects on the physicochemical andfunctional properties

2 Materials and Methods

21 Raw Material Jumbo squid (D gigas) was harvested offthe coast of Kino Bay Sonora Mexico in June 2014 Tenspecimens were decapitated and gutted on site and werethen washed with freshwater at room temperature (25∘C)The mantles (experimental samples) were bagged and placedin alternating layers of ice-squid-ice in a portable coolerand transported to the laboratory Squid mantles exhibiteda length of 3785 plusmn 41 cm and a total weight of 8275 plusmn8275 g The elapsed time between capture and reaching thelaboratory did not exceed 12 h

22 Sarcoplasmic Proteins Preparation Frozen squid mantlewas thawed at 4-5∘C for 12 h minced and mixed withcold distilled water (le4∘C) at a ratio of 1 3 (mince water)Mixture was then homogenized at 1000 rpm for 1min usinga tissue homogenizer (Wisd WiseTis HG-15D Witeg Ger-many) The homogenate was centrifuged in a refrigeratedcentrifuge at 12000timesg for 20min at 4∘C (Thermo ScientificSorvall Biofuge StratosMAUSA)The supernatant obtainedwas considered as the sarcoplasmic proteins (SP) fractionand was analyzed for protein concentration according to theLowry method [9] using bovine serum albumin as standard

23 Modification of pH and NaCl Concentration TwentymL aliquots of SP were added to NaCl (0 02 and 05M)and the pH was adjusted to 30 50 70 90 or 110 Thenthe final volume of the homogenates was brought to 25mL(using cold distilled water) and stirred for 30min at 4∘CTheadjusted solutions were used to determine protein solubilityemulsifying and foam properties

24 Protein Solubility In order to determine the effect ofdifferent pH levels and NaCl concentrations on the sol-ubility of SP all solutions previously prepared were cen-trifuged at 12000timesg for 20min at 4∘C (Thermo ScientificSorvall Biofuge Stratos MA USA) Protein concentrationof the supernatants was analyzed by Lowry method [9]Protein solubility was expressed as the percentage of proteinremaining soluble after centrifugation in relation to the totalprotein present in SP Supernatants obtained were used forthe determination of protein patterns by sodium dodecyl

sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sur-face hydrophobicity (S

0-PRODAN and S

0-ANS) and total

sulfhydryl (SH) content

25 Electrophoretic Profile (SDS-PAGE) The electrophoreticprofile of each protein systemwas analyzed bymeans of poly-acrylamide gel electrophoresis (PAGE) using a dissociatingsodium dodecyl sulfate (SDS) buffer system in a discontinu-ous gel (4 stacking gel and 10 separating gel) according tothemethod of Laemmli [10] AMini-PROTEAN3CellMulti-Casting Chamber (Bio-Rad Laboratories Hercules CA) wasused Electrophoretic runs were performed at room tempera-ture (25∘C) at 80VThirty 120583g of protein was loaded into eachlane of the gels and a broad range ofmolecular weight proteinstandard solutions (Bio-Rad Laboratories Richmond CA)containing myosin (200 kDa) beta-galactosidase (116 kDa)phosphorylase b (97 kDa) bovine serum albumin (66 kDa)ovalbumin (45 kDa) and carbonic anhydrase (31 kDa) wereused After electrophoresis the gel was stained with 0125(wv) Coomassie brilliant blue R-250 in 40 (vv) methanoland 7 (vv) acetic acid and the gel was destained with 50(vv) methanol and 10 (vv) acetic acid

26 Surface Hydrophobicity (S0) Surface hydrophobicity (S

0)

was determined by the method reported by Alizadeh-Pasdarand Li-Chan [11] with slight modifications Protein solutionswere diluted in order to obtain protein concentrations of0 0125 025 05 and 01mgmLminus1 Four mL aliquot fromeach diluted solution was mixed with 20120583L of ANS solution(10mM phosphate buffer pH 7) or 10 120583L of PRODANsolution (14mM in methanol) ANS samples were set for10min and PRODAN samples were set in the dark for 15minSubsequently the fluorescence intensity of the samples wasmeasured using a spectrofluorometer (Agilent TechnologiesCary Eclipse CA USA) at excitationemission wavelengthof 390470 nm and 365465 for ANS and PRODAN respec-tively The surface hydrophobicity index (S

0) was calculated

from the regression slope or net relative fluorescence intensityversus protein concentration ( wv)

27 Determination of Total Sulfhydryl (TSH) Total sulfhydryl(TSH) content at different pH levels and NaCl concen-trations was determined by the method reported by Tad-pitchayangkoon et al [12] with slight modifications Proteincontent of each protein solution was adjusted to 1mgmLminus105mL aliquot of each treatment was mixed with 2mLof solubilizing buffer (02M Tris-HCl 8M urea 10mMEDTA pH 70) and 50 120583L of Ellmanrsquos reagent (10mM 551015840-dinitrobis [2-nitrobenzoic acid] 01M sodium phosphatebuffer pH 70) All sampleswere incubated at 40∘C for 25minThe absorbance was measured at 412 nm using a UV-Visspectrophotometer (Cary 50 Varian CA USA) and SHcontent was calculated using the extinction coefficient of13600Mminus1 cmminus1

28 Emulsifying Properties The emulsifying activity index(EAI) and emulsifying stability index (ESI) at differentpH levels and NaCl concentrations were determined by





120591 =2303 times 119860

500times 119865

119897

EAI (m2g2) = 2120591120601119862

(1)




ESI (min) = 120591 times Δ119905Δ120591 (2)




times 100 (3)


FS ()


times 100

(4)


0-ANS and S

0-PRODAN)






0

20

40

60

80

100

120

Prot

ein

solu

bilit

y (

)

5 7 9 113pH




















66

55

45

36

(kDa)

pH3 5 7 9 11 S

(a)

66

55

45

36

(kDa)

pH3 5 7 9 11 S

(b)

66

55

45

36

(kDa)

pH3 5 7 9 11 S

(c)






0-







5 7 9 113pH


0

5

10

15

20

25TS

H co

nten

t (m

ole1

05

gminus1)





0-PRODAN










5 7 9 113pH


0

5000

10000

15000

20000

25000

30000

S0-

AN

S

(a)

5 7 9 113pH


0

1000

2000

3000

4000

5000

6000

S0-

PRO

DA

N

(b)



5 7 9 113pH


0

25

50

75

100

125

150

EAI (

m2

gminus1)

(a)

5 7 9 113pH


0

50

100

150

200

250

ESI (

min

)

(b)






5 7 9 113pH


100

90

80

70

60

50

40

30

20

10

0

FC (

)

(a)

5 7 9 113pH


120

100

80

60

40

20

0

FS (

)

(b)




4 Conclusions





References























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





120591 =2303 times 119860

500times 119865

119897

EAI (m2g2) = 2120591120601119862

(1)




ESI (min) = 120591 times Δ119905Δ120591 (2)




times 100 (3)


FS ()


times 100

(4)


0-ANS and S

0-PRODAN)






0

20

40

60

80

100

120

Prot

ein

solu

bilit

y (

)

5 7 9 113pH




















66

55

45

36

(kDa)

pH3 5 7 9 11 S

(a)

66

55

45

36

(kDa)

pH3 5 7 9 11 S

(b)

66

55

45

36

(kDa)

pH3 5 7 9 11 S

(c)






0-







5 7 9 113pH


0

5

10

15

20

25TS

H co

nten

t (m

ole1

05

gminus1)





0-PRODAN










5 7 9 113pH


0

5000

10000

15000

20000

25000

30000

S0-

AN

S

(a)

5 7 9 113pH


0

1000

2000

3000

4000

5000

6000

S0-

PRO

DA

N

(b)



5 7 9 113pH


0

25

50

75

100

125

150

EAI (

m2

gminus1)

(a)

5 7 9 113pH


0

50

100

150

200

250

ESI (

min

)

(b)






5 7 9 113pH


100

90

80

70

60

50

40

30

20

10

0

FC (

)

(a)

5 7 9 113pH


120

100

80

60

40

20

0

FS (

)

(b)




4 Conclusions





References























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


0

20

40

60

80

100

120

Prot

ein

solu

bilit

y (

)

5 7 9 113pH




















66

55

45

36

(kDa)

pH3 5 7 9 11 S

(a)

66

55

45

36

(kDa)

pH3 5 7 9 11 S

(b)

66

55

45

36

(kDa)

pH3 5 7 9 11 S

(c)






0-







5 7 9 113pH


0

5

10

15

20

25TS

H co

nten

t (m

ole1

05

gminus1)





0-PRODAN










5 7 9 113pH


0

5000

10000

15000

20000

25000

30000

S0-

AN

S

(a)

5 7 9 113pH


0

1000

2000

3000

4000

5000

6000

S0-

PRO

DA

N

(b)



5 7 9 113pH


0

25

50

75

100

125

150

EAI (

m2

gminus1)

(a)

5 7 9 113pH


0

50

100

150

200

250

ESI (

min

)

(b)






5 7 9 113pH


100

90

80

70

60

50

40

30

20

10

0

FC (

)

(a)

5 7 9 113pH


120

100

80

60

40

20

0

FS (

)

(b)




4 Conclusions





References























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


66

55

45

36

(kDa)

pH3 5 7 9 11 S

(a)

66

55

45

36

(kDa)

pH3 5 7 9 11 S

(b)

66

55

45

36

(kDa)

pH3 5 7 9 11 S

(c)






0-







5 7 9 113pH


0

5

10

15

20

25TS

H co

nten

t (m

ole1

05

gminus1)





0-PRODAN










5 7 9 113pH


0

5000

10000

15000

20000

25000

30000

S0-

AN

S

(a)

5 7 9 113pH


0

1000

2000

3000

4000

5000

6000

S0-

PRO

DA

N

(b)



5 7 9 113pH


0

25

50

75

100

125

150

EAI (

m2

gminus1)

(a)

5 7 9 113pH


0

50

100

150

200

250

ESI (

min

)

(b)






5 7 9 113pH


100

90

80

70

60

50

40

30

20

10

0

FC (

)

(a)

5 7 9 113pH


120

100

80

60

40

20

0

FS (

)

(b)




4 Conclusions





References























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


5 7 9 113pH


0

5

10

15

20

25TS

H co

nten

t (m

ole1

05

gminus1)





0-PRODAN










5 7 9 113pH


0

5000

10000

15000

20000

25000

30000

S0-

AN

S

(a)

5 7 9 113pH


0

1000

2000

3000

4000

5000

6000

S0-

PRO

DA

N

(b)



5 7 9 113pH


0

25

50

75

100

125

150

EAI (

m2

gminus1)

(a)

5 7 9 113pH


0

50

100

150

200

250

ESI (

min

)

(b)






5 7 9 113pH


100

90

80

70

60

50

40

30

20

10

0

FC (

)

(a)

5 7 9 113pH


120

100

80

60

40

20

0

FS (

)

(b)




4 Conclusions





References























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


5 7 9 113pH


0

5000

10000

15000

20000

25000

30000

S0-

AN

S

(a)

5 7 9 113pH


0

1000

2000

3000

4000

5000

6000

S0-

PRO

DA

N

(b)



5 7 9 113pH


0

25

50

75

100

125

150

EAI (

m2

gminus1)

(a)

5 7 9 113pH


0

50

100

150

200

250

ESI (

min

)

(b)






5 7 9 113pH


100

90

80

70

60

50

40

30

20

10

0

FC (

)

(a)

5 7 9 113pH


120

100

80

60

40

20

0

FS (

)

(b)




4 Conclusions





References























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


5 7 9 113pH


100

90

80

70

60

50

40

30

20

10

0

FC (

)

(a)

5 7 9 113pH


120

100

80

60

40

20

0

FS (

)

(b)




4 Conclusions





References























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

research article chemical and functional characterization of sarcoplasmic proteins ... · 2019. 7....

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