Letters

Temperature dependence of calcium and magnesium induced caseinate precipitation in H 2 O and D 2 O

Description
Temperature dependence of calcium and magnesium induced caseinate precipitation in H 2 O and D 2 O
Categories
Published
of 7
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Related Documents
Share
Transcript
  Temperature dependence of calcium and magnesium induced caseinateprecipitation in H 2 O and D 2 O Francesca Cuomo, Andrea Ceglie, Francesco Lopez ⇑ Consorzio Interuniversitario per lo sviluppo dei Sistemi a Grande Interfase (CSGI), c/o Department of Food Technology (DISTAAM), Università del Molise, I-86100 Campobasso, Italy a r t i c l e i n f o  Article history: Received 23 June 2010Received in revised form 27 August 2010Accepted 4 October 2010 Keywords: CaseinateFluorescencePrecipitation temperatureDeuterium oxideCalcium a b s t r a c t Casein is a well-known and highly studied protein present in milk found in the form of large colloidalparticles called caseinmicelles. Here weassessed theinfluence of twosolvents, H 2 OandD 2 O, onthe pre-cipitation temperatures of sodium caseinate induced by calciumand magnesium ions. The intrinsic fluo-rescence of casein was correlated with the suspended and precipitated protein fractions in order toextrapolate the precipitation temperature. The precipitation temperature values were determined forseveral calciumandmagnesiumconcentrations inH 2 O, demonstratingthestrongdifferences intheasso-ciationbehaviourinducedbythetwodivalentcations.Theprecipitationtemperaturesofcaseinate, inthepresence of calcium induced by heating the samples in D 2 O, were significantly lower compared with theone performed in H 2 O. On the contrary, the precipitation temperatures of casein in the presence of mag-nesiumwereveryclosetothoseobtainedinthesamplestestedinH 2 O.Thus,calciuminteractionwiththeprotein depends on temperature and solvents, two parameters that modify the accessibility of the bind-ingsiteofthecation.Conversely,theinteractionofthedivalentmagnesiumionwithcaseinwasshowntodepend only upon the temperature. Remarkably, this evidence indicates that the two ions have differentbinding sites on the protein, suggesting that the D 2 O solvent plays an important role in the detection of the different binding sites for calciumandmagnesium. Additionally, the results obtained withthe simul-taneous presence of both cations suggest the existence of a cooperative mechanism between the twoions, in which the presence of calcium makes more sites available for the binding of magnesium.   2010 Elsevier Ltd. All rights reserved. 1. Introduction Caseins are the most representative milk proteins. These pro-teins are present in milk as large colloidal particles called caseinmicelles, held together by electrostatic and hydrophobic interac-tions (Fox & Brodkorb, 2008; Phadungath, 2005). The presence of  calcium and phosphate ions is a necessary condition for micelleintegrity(Alvarez,Risso,Gatti,Burgos,&Sala,2007;Guo,Campbell,Chen, Lenhoff, & Velev, 2003; Waugh, 1961) and stability of caseinmicellesisaffectedbypH,ionicstrengthandtemperature(HadjSa-dok, Pitkowski, Nicolai, Benyahia, & Moulai-Mostefa, 2008; Liu &Guo, 2008). Casein is made up of four main components knownas  a s1 ,  a s2 ,  b  and  j -casein; these proteins are phosphorylated onspecific serine residues and contain 8, 9–11, 5 and 1 phosphaterespectively(Farrell et al., 2004). Thepresenceof anionicphospho- serine and other anionic amino acid residues, such as carboxylatemoieties of aspartic and glutamic acids, makes caseins availablefor cations binding (Byler &Farrell, 1989). The prediction of the ef-fect induced by the addition of different cation is not easy, sincemany factors, such as the kind of cations and their concentrations,affect the association between the protein and the cations. Themoststudiedassociationistheonewithcalciumionsthatgeneratea number of important effects on casein solubility and colloidalstability. Another important divalent cation contained in milk toa lesser extent is magnesium. Several authors found differencesin the associationbehaviour of calciumand magnesiumwith case-ins (de la Fuente, Montes, Guerrero, &Juárez, 2003; Kull, Nylander,Tiberg, & Wahlgren, 1997; Nylander, Tiberg, & Wahlgren, 1999).The study of parameters and forces involved in the casein pre-cipitationprocessinthepresenceofdivalentionsishighlyrelevantfor biotechnological applications. Sodium caseinate, prepared bythe resolubilisation of the precipitated casein with NaOH afteracidificationatpH4.6,iscommonlyusedasaningredientinawiderange of formulated food emulsion (Dickinson, 2009; Maroziene &de Kruif, 2000) because of its physico–chemical, nutritional andfunctional properties (Raouche, Dobenesque, Bot, Lagaude, & Mar-chesseau, 2009). Sodium caseinate associates in solution formingsmall aggregates in order to shield the hydrophobic parts of casein molecules from water. Hence, the effects induced byexposure of caseins and caseinates to temperature treatmentshave been under intensive investigation for many years (Le Ray,Maubois, Gaucheron, Brule, Pronnier & Garnier, 1998; Ono, Yoshida, 0308-8146/$ - see front matter    2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.foodchem.2010.10.021 ⇑ Corresponding author. Tel.: +39 0874404634; fax: +39 0874404652. E-mail address:  lopez@unimol.it (F. Lopez).Food Chemistry 126 (2011) 8–14 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem  Tanaami, & Ohkosi, 1999). Furthermore, the study of temperature-induced changes in the physico–chemical properties of theseproteins can shed light on the general mechanisms responsiblefor the structural stability and functionality of peptides, whichare highly relevant to colloidal chemistry.The ion affinities for proteins depend on the availability of thebinding sites, which can be made more or less available by modu-lating the hydrophobic–hydrophilic balance of the protein interac-tions.Caseinsaredefinedasnativeunfoldedproteinscharacterizedby increasing structural complexity in response to extreme envi-ronmental changes, such as high temperature or extreme pH(Uversky, 2002). A further suitable strategy for altering this intri- cate equilibrium is the use of the solvent deuterium oxide (D 2 O),in comparison with the solvent water (H 2 O). D 2 O is able to modifythe above mentioned balance by enhancing the self-association of several proteins (Chakrabarti, Kim, Gupta, Barton, & Himes, 1999). Although the enhancement mechanism of protein assembly byD 2 O is not well understood, it is thought to be due to the enhance-ment of hydrophobic interactions and hydrogen bonding, whichare much stronger in D 2 O than in H 2 O. In deuterium oxide, wherethere is a greater amount of localized structures than in water,there should be a greater entropic effect and, consequently, anenhancement of hydrophobic interactions (Kresheck, Schneider, &Scheraga, 1965). In a number of cases the stabilizing effect of D 2 Ohas beenattributedto theenhancement of hydrophobicinter-actions (Parker & Clarke, 1997). In this article we performed a study on the dependence of pre-cipitation temperatures ( T  P ) of caseinate suspensions on Ca 2+ andMg 2+ concentrations by taking advantage of the different proper-ties of H 2 O and D 2 O as protein solvents. Protein precipitationwas assessed by following the intrinsic fluorescence decrease of casein. The interaction of cations withcaseinate, as well as the rel-ative induced  T  P  under different conditions, are compared and dis-cussed. In particular, the differences of binding affinities areevidenced by the experiments in deuterium oxide. 2. Materials and methods  2.1. Chemicals Sodium caseinate, sodium chloride, calcium chloride and mag-nesium chloride were purchased from Sigma. Deuterium Oxidewas from Aldrich.  2.2. Sample preparation The samples were prepared with a fixed sodium caseinate con-centration (4mg/ml). Caseinate was dispersed in a 0.1M sodiumchloride, before suitable amounts of calciumand magnesiumchlo-ride were added. The same solutions were prepared in deuteriumoxide. All the solutions were prepared with ultrapure water.  2.3. Steady state fluorescence spectroscopy measurements Steady state fluorescence spectroscopy measurements wereperformed at 25  C using a Varian Eclipse spectrofluorimeterequipped with a Peltier element in a 1cm quartz cuvette. Theemission intensities were collected at 338nm, with excitation at290nm; the excitation and emission slits were 5nm and thePMT was set at 550V. The temperature scan rate was 1  C/min.All the numerical fittingprocedures wereperformedusingtheOri-gin software (Origin Lab Corporation, USA). The precipitation tem-peratures were derived following the fluorescence intensitydecrease with the temperature increase.A simple model was applied to the experimental fluorescencedata. As reported in Eq. (1) the fluorescence intensity includesthe suspended and the precipitated fractions of caseinate: I  f   ¼  K  s ½ S  þ  K  P ½ P  ð 1 Þ where  I  f   is the fluorescence intensity,  K  S  is the proportionality con-stant of the caseinate suspended fraction,  K  P  is the proportionalityconstant for the precipitated form, [ S  ] is the concentration of sus-pendedcaseinatefractionand[ P  ]istheconcentrationoftheprecipi-tated proteins. By dividing by the sum of [ S  ]+[ P  ], Eq. (1) can berewrittenasafunctionof thesuspendedandtheprecipitatedmolarfractions: I  f   ¼  K  S v S  þ  K  P v P  ð 2 Þ Considering that  v S  þ v P  ¼  1,  v S  ¼  1  v P , Eq. (2) develops into: I  f   ¼  K  S ð 1  v P Þ þ  K  P v P  ð 3 Þ AsshowninFig.1A,thefluorescenceevolvesasasigmoidalfunctionwith three characteristic regions. The first region has a linear de-crease of   I  f   withthe temperature. In this part caseins are inthe sus-pended form  ð v P  ¼  0 Þ , which means that  I  f   ¼  K  S  ¼  a þ  bT   where  a and  b  are the intercept and the slope respectively calculated of the linear fit of the first region. In the second part the fluorescenceintensity decreases sharply and casein aggregates pass from thesuspended [ S  ] to the precipitated [ P  ] form.  T  P  can be identified inthis region by considering:  v S  ¼  v P  ¼  0 : 5. The third part concernswith the precipitated fraction of proteins. Here,  v P   ¼  1 and  I  f   ¼ K  P  ¼  a 0 þ  b 0 T   where a’  istheinterceptand b’  istheslopeofthelinearfit of the thirdregion. By substitutingvalues of   K  S  and  K  P  calculatedfrom the fitting of the fluorescence data Eq. (3) becomes: v S  ¼  I  f    ð a 0 þ  b 0 T  Þð a  þ  bT  Þ  ð a 0 þ  b 0 T  Þð 4 Þ The above equation describes the experimental change of   v S , bymeans of fluorescence as a function of the temperature (seeFig. 1B). This kind of evaluation was previously used for proteinsdenaturation analysis (Acampora & Jr, 1967; Palazzo, Lopez, &Mallardi, 2010).  2.4. Dynamic light scattering experiments The average hydrodynamic diameter and the scattering of thesample were determined by means of dynamic light scatteringmeasurements using a Malvern UK commercial instrumentZetasizer-Nano ZS90 operating with a 4mW He–Ne laser (633nmwavelength). The average aggregate size values were estimatedwith a fixed detector angle of 90   by a cumulants analysis of the Fig. 1.  Precipitation of caseinate in the presence of calcium. (A) the intrinsicfluorescence intensity of caseinate, obtained by measuring the fluorescenceemissionat338nmat increasingtemperatures. (B)themolarfractionofsuspendedcaseinate as a function of temperature obtained utilizing Eq. (4) (see Section 2.3.). F. Cuomo et al./Food Chemistry 126 (2011) 8–14  9  autocorrelation function using the software provided by the man-ufacturer. The instrumentation was equipped with an attenuationsystem that allowed the avoidance of sample dilution. 3. Results and discussion  3.1. Estimation of casein T  P   by T-scan experiments Thefluorescenceofproteinsisknowntocomefromtryptophan,tyrosine and phenylalanine residues. These amino acids are gener-ally located in a hydrophobic portion of the proteins, which arenormally self-assembled to shield the hydrophobic parts fromwater. The binding of divalent cations on casein leads to aggrega-tion phenomena enhanced by the temperature increase. The pre-cipitation temperature ( T  P ) of caseinate, defined as thetemperaturevaluethatcausesthesharpformationofbiggeraggre-gates (which precipitate as a slow process), was calculated bymeans of T-scan experiments. In particular, the fluorescence of tryptophan ( k max  338nm) was used as a signal to follow the pro-tein fluorescence (Vivian & Callis, 2001) while the temperature was continuously increased at a fixed scan rate. In Fig. 1A a typicalT-scanexperimentillustratingtheprecipitationofcaseinate(4mg/ml) in the presence of Ca 2+ (7.5mM) as a function of the tempera-ture is shown. In order to investigate the  T  P  of casein, the differ-ences between the suspended and the precipitated fractions of caseins were taken into account (see experimental section), andthe dependence of   I  f   on the temperature was rewritten as a func-tion of the suspended and the precipitated molar fractions. Thus,inFig.1B(obtainedbyapplyingtheEq.(4)reportedinSection2.3.), values of   v S  as a function of the temperature are reported. Thiskind of elaboration, based on the tryptophan fluorescence emis-sion, represents in our opinion a suitable tool to investigate the T  P  of casein by means of   v S  values.When caseins are heated they are supplied with energy thatmodifies the hydrophilic–hydrophobic balance and the electro-static interactions that rule out aggregation. This energy supplycauses the casein to rearrange with progressive exposure of thehydrophobic domains to a more polar milieu. The fluorescenceemission of the casein as the temperature increases shows anintensity decrease with a shift toward the red of the spectrum(data not shown). This red shift, together with the intensity de-crease,revealsthefluorescentaminoacidswhicharefacingamorepolar microenvironment. The amino acid rearrangement does notchangethesampleaggregation, justasshownbythedynamiclightscattering measurements.  3.2. T  P   in the presence of Ca  2+ and Mg   2+ in H   2 O It is convenient to call attention to the starting conditions of alltheexperiments.Thecaseinateconcentrationwasfixedat4mg/mlin 0.1M NaCl in order to avoid the occurring of association phe-nomena between the suspended particles (Pitkowski, Nicolai, &Durand, 2009). Fig. 2A shows the T-scan experiments performed on caseinate dissolved in H 2 O in the presence of various calciumconcentrations. The T-dependence analysis highlighted that the T  P   is strongly dependent on Ca 2+ concentration (see also Table 1).As mentioned above the driving force for the precipitation processis strongly related to the hydrophobicity of protein fractions; thefractions  a s1  and  a s2  present the alternation of hydrophilic andhydrophobic blocks,  b -casein is characterized by higher hydropho-bicity, j -casein with its glycosidic residues is the most hydrophilicfraction. In the caseinate structures the hydrophobic parts areturned into the core of the aggregates to shield the interactionswith the water solvent, this means the fraction of   b -casein ismainlylocatedintheinteriorofthestructures, while j -caseinpre-dominantly covers the surface of the aggregates (Dalgleish, Horne,& Law, 1989). The heating treatment on caseins by forcing theexposure of hydrophobic domains to the solvent water, allowedthe  a s1 ,  a s2  and  b  fractions to be arranged on the particle surface.The change of protein conformation leads to an increase of thenumber of phosphoserine residues available for the interactionwith calcium. This condition allows the progress of the neutraliza-tion process, between the native negative surface charge of theprotein and the positively charged divalent ion, which causes theparticles aggregation.A T-scan experiment, achieved by loading the samples withMg 2+ , is reportedinFig. 2B. As shownbyloadingthe caseinatesus-pension with 2.5mM MgCl 2 , the precipitation does not occur,whereas by loading with 10mM, the precipitation takes place ata temperature higher than the calcium containing caseinate solu-tion (42 instead of 37  C). These results indicate that the tempera-ture values derived from the interaction with Mg 2+ cations aredifferent from the ones gained with Ca 2+ cations.The temperature dependence of caseinate precipitation in thepresence of calcium and magnesium was also examined monitor-ing the light scattering intensity of the suspensions during T-Scanexperiments. Using this technique, the ability of the solutions toscatterthelightat90   wasmeasured.AsindicatedinFig.3,asharpincrease in light scattering intensity as a function of the temperature Fig. 2.  The measurements have been plotted as the molar fraction of suspendedcasein as a function of temperature. (A) precipitation profiles of caseinate loadedwith several concentrations of Ca 2+ . (B) precipitation profiles of caseinate loadedwith Mg 2+ .  Table 1 T  P  values obtained by means of steady state fluorescence T-scan experiments. T  P /  CCa 2+ (H 2 O) Mg 2+ (H 2 O) Ca 2+ (D 2 O) Mg 2+ (D 2 O)2.5mM 83 – a 66 – a 5mM 60 76 48 727.5mM 46 53 36 5310mM 37 42 32 422.5mM Ca 2+ +2.5mMMg 2+ 64 58 a Precipitation does not occur.10  F. Cuomo et al./Food Chemistry 126 (2011) 8–14  is observed at a certain temperature when caseins are loadedwithcations.Thetemperaturecorrespondingtothelightscatteringintensity increase was found to coincide with the temperature va-lue determined in the protein fluorescence study, confirming thatthe emission decrease is due to protein precipitation. The lightscattering study demonstrated that the two divalent ions influ-encedtheprecipitationprocessindifferentways. Infact theaggre-gation induced by Ca 2+ (Fig. 3A) causes a more opaque suspensioncomparedwiththe aggregation inducedbyMg 2+ (Fig. 3B). This dif-ference can be easily observed by visual inspection of the samples.In the inset of  Fig. 3B a picture of caseinate samples after the heat-ing process, containing either 7.5mM Ca 2+ (left) or Mg 2+ (right), isshown.The light scattering experiments on caseinate gave evidence of bimodal distributions with the two aggregate sizes of about 30nmand 200nm. The smaller sized portion disappeared when the lightscattering intensity reached its maximum, whereas the biggeraggregates increased their size up to about 400nm. In Fig. 4, thesize distribution of samples free from cations and loaded with10mM calcium at three different temperatures are shown. Ascan be seen, the size distribution of caseinate without cations isunaffected by the heating treatment. Moreover, for this systemthe light scattering intensity remains unchanged (black points inFig. 3) and the suspension does not precipitate (black points inFig. 2). Caseinate loaded with 10mM calcium has a bimodal sizedistribution at 20  C and evolves into a monomodal distributionat higher temperatures. The smaller peak completely disappearedat 37  C, while at 43  C the only peak observed has a diameter of about 430nm. These results seem to be in accordance with themodel of smaller micelles incorporated into a bigger micelle re-ported by Ono et al. (1999).  3.3. T  P   in the presence of Ca  2+ and Mg   2+ in D  2 O It is obviously a matter of some importance to establish theinteractionbehaviourbetweenthedivalentionsandcaseinate.De-spite the evidence that the different aggregation temperature in-duced by the two cations at the same concentration can be dueto the differences in the dimensions of their hydrated ionic radius(Le Ray et al., 1998), we argue that this issue needs further inves-tigation. As reported by several authors, the effect of pH variationonthestructureofcaseinsismainlybasedontheformationofpar-tially folded intermediate structures (Chakraborty & Basak, 2007;Uversky, 2002). Here we will analyze the effects of solvent changeonthestructureof caseinintermsof   T  P  inducedbythecationscal-cium and magnesium, respectively. To investigate the differencesin T  P  inducedbycalciumandmagnesium,alltheexperimentsweredonebyexchangingwatersolventwithdeuteriumoxidesolvent.Itis well known that aggregation phenomena for some proteins areinfluenced by D 2 O. For example, the deuterated solvent stabilizedthe oligomeric form of halophilic malate dehydrogenase (Bonnete,Madern, & Zaccai, 1994) and is also known to affect the stability of some proteins, such as  b -lactoglobulin (Verheul, Roefs, & de Kruif,1998).Fig. 5 shows the precipitation profiles induced by Ca 2+ on case-inate in both the solvents (deuterated and protonated water). Ascan be seen, for each of the Ca 2+ concentrations used (2.5, 5, 7.5and 10mM), the precipitation temperatures in D 2 O are found low-er than in H 2 O (see Table 1), indicating that the exposure of theinteraction sites for the cation binding occurs at lower tempera-tures compared to the samples analyzedin H 2 O. Recalling the con-cept of energy supply, it can be thought that less energy issufficient to change the binding sites arrangement since differentprotein folding occurs in D 2 O.Evidence of the effect of deuterium oxide on caseins is high-lighted by the fluorescence emission spectra. The higher startingvalues of fluorescence emission in D 2 O indicated that the caseinswere in a more compact form, which influenced the availabilityof the sites for the calciumbinding (see inset of  Fig. 5). This aspectoutlined that the microenvironment around the fluorescent resi-dues is more polar than that provided by H 2 O. This hypothesis issupported by the lower zero-point vibrational energy of the D–O Fig. 3.  Variation of dynamic light scattering intensity in the presence of Ca 2+ (A)andMg 2+ (B).Intheinsetapictureoftwocuvettescontainingprecipitatedcaseinatein the presence of Ca 2+ (left) and Mg 2+ (right) heated at 60  C. Fig. 4.  Size distributions of caseinate without cations and with 10mM Ca 2+ at 20,37 and 43  C. F. Cuomo et al./Food Chemistry 126 (2011) 8–14  11  bond, whichleadstoanhigherstabilityoftheD–Obondcomparedto the H–O bond (Efimova, Haemers, Wierczinski, Norde, & vanWell, 2007). D 2 O is a poor solvent for the apolar protein residuescompared to H 2 O; hydrophobic effects bury the nonpolar aminoacids in the interior of the polypeptide structures, making the pro-tein structure more compact and less flexible (Cioni & Strambini,2002; Efimova et al., 2007). The influence of D 2 O on caseinate sta-bilityinthepresenceofMg 2+ isshowninFig.6.Surprisingly,asob-servedinthiscomparativepanel,theprecipitationprofilesinducedbyMg 2+ indeuteriumoxide areveryclose to theprecipitationpro-files reported for the samples prepared in H 2 O. This evidence iscorroborated in Table 1, where all the  T  P  obtained for each of thecation concentrations are summarized. Considering this result,the different aggregation behaviour induced by magnesium, withrespect to samples treated with calcium, seems to indicate thepresence of at least two binding sites.Furthermore, by conducting experiments in the presence of both the calcium and magnesium ions, new insight can be Fig. 5.  The measurements have been plotted as the molar fraction of suspended casein as a function of temperature. Comparison of the precipitation profiles induced by 2.5,5, 7.5 and 10mM Ca 2+ on caseinate suspensions in water and deuterium oxide. In the inset of the figure the initial fluorescence spectra of caseinate in protonated anddeuterated water are shown. Fig. 6.  The measurements have been plotted as the molar fraction of suspended casein as a function of temperature. Comparison of the precipitation profiles induced by 2.5,5, 7.5 and 10mM Mg 2+ on caseinate suspensions in water and deuterium oxide.12  F. Cuomo et al./Food Chemistry 126 (2011) 8–14
Search
Similar documents
View more...
Related Search
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks