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Phase Separation and Association of Globular Protein Aggregates in the Presence of Polysaccharides: 1. Mixtures of Preheated β-Lactoglobulin and K-Carrageenan at Room Temperature Philippe Croguennoc, Dominique Durand, and Taco Nicolai* Polyme` res, Colloı¨des, Interfaces UMR CNRS, Universite´ du Maine, 72085 Le Mans Cedex 9, France
Allan Clark Unilever Research Colworth, Sharnbrook, Bedford MK441LQ, U.K. Received November 30, 2000. In Final Form: March 24, 2001 Mixtures of globular protein (β-lactoglobulin) aggregates and polysaccharides (κ-carrageenan) were studied using dynamic and static light scattering, size exclusion chromatography, and optical microscopy. Above a critical κ-carrageenan concentration the protein aggregates phase separate and form spherical microdomains, which slowly sediment. The effect of concentration and size of the protein aggregates was investigated. The phase separation is initially reversible upon dilution, but the microdomains become progressively more stable until after several days they can no longer be redissolved.
Introduction It is well documented that mixtures of globular proteins and polysaccharides with the same charge sign may phase separate into a protein-rich phase and a polysacchariderich phase.1 Generally, phase separation occurs at relatively high volume fractions of the components. However, if instead of native proteins, protein aggregates are used, phase separation occurs at much lower volume fractions. Syrbe studied this phenomenon for whey protein aggregates by preheating the protein solutions before mixing with a variety of polysaccharides.2 He observed, using optical microscopy, in some conditions the formation of relatively well defined, protein-rich spherical domains with a diameter in the range of 1-10 µm. The microdomains are initially liquid but become gel-like after some time. Recently, Tuinier et al.3 studied in detail the phase separation of small whey protein aggregates with zaverage radius of gyration Rgz ≈ 20 nm when mixed with an exopolysaccharide. They observed that the initial stage of the phase separation was similar to that observed for spinodal decomposition of a simple binary liquid. They assumed that there are no specific interactions between the protein aggregates and the exopolysaccharide and that the phase separation is induced by depletion of the polysaccharides from the region between neighboring protein aggregates. Such depletion leads to an effective attractive force between the protein aggregates and may induce phase separation.3,4 The purpose of the present paper is to further investigate the phase separation of protein aggregates and polysaccharides at room temperature. Our motivation for studying (1) Grinberg, V. Y.; Tolstoguzov, V. B. Food Hydrocolloids 1997, 11, 145. (2) Syrbe, A. Thesis, Munich Technical University, Munich, Germany, 1997. (3) Tuinier, R.; Dhont, J. K. G.; De Kruif, K. G. Langmuir 2000, 16, 1497. (4) Poon, W. C. K. Curr. Opin. Colloid Interface Sci. 1998, 3, 593.
these systems is not only because they are important in industrial applications but also because we believe they are good model systems to study the effect of flexible polymers on colloid aggregation. We will address a number of issues that remain outstanding, such as, the influence of the average size and the size distribution of the protein aggregates, the structure of the microdomains, and the reversibility of the phase separation. In part 2 we report on an investigation of mixtures of native proteins and polysaccharides heated above the denaturation temperature of the proteins. In that case protein aggregation and phase separation occur simultaneously. The particular system we study is β-lactoglobulin (βlg), which is the main component of whey, and κ-carrageenan (κ-car), which is a polysaccharide extracted from blue algae. Both components are widely used in the food industry. At pH 7 both κ-car and β-lg have a net negative charge and are fully compatible in the concentration domain used in this study. We have chosen the conditions (temperature, type, and concentration of counterions) such that κ-car always has a random coil configuration. β-lg aggregates were prepared by heating the native proteins in solution for some time above the temperature for denaturation and subsequently cooling to room temperature. Aggregates of various sizes, but with the same structure, were obtained by varying the heating temperature or duration. Experimental Section Materials. The κ-carrageenan used for this study was an alkali-treated extract from Eucheuma cottonii supplied by SKW Biosystems (Baupte, France). The solutions were prepared by as follows: A freeze-dried sample of κ-carrageenan in the sodium salt form was dissolved while stirring for a few hours in hot Millipore water (70 °C). The pH was adjusted to 9 to eliminate the risk of hydrolysis during the preparation. The solution was first dialyzed against Millipore water at pH 7 to eliminate excess salt and subsequently against 0.1 M NaCl. A 200 ppm NaN3 (0.003 M) solution was added to avoid bacterial growth. The solutions always contained a small amount of large aggregates
10.1021/la001674q CCC: $20.00 © 2001 American Chemical Society Published on Web 06/12/2001
Mixtures of Protein Aggregates and Polysaccharides which perturbed the light scattering results so these aggregates were removed by filtration through 0.2 µm pore size Anotop filters. The absence of aggregated material and other spurious scatterers was checked by dynamic light scattering. κ-car shows a reversible coil-helix transition when lowering the temperature below a critical value (Tc).5,6 The value of Tc depends on the concentration and type of ions in the systems. In the helix conformation κ-car usually aggregates and forms a self-supporting gel if the concentration is sufficient. For the present study we used κ-car with sodium counterions and we adjusted the ionic strength with NaCl. Under these conditions κ-car has the coil configuration at temperatures above 11 °C. The κ-car used in this study was characterized using size exclusion chromatography (SEC) and static (SLS) and dynamic (DLS) light scattering. The following characteristics were obtained: weight average molar mass Mw ) 4.3 × 105 g/mol, polydispersity index Mw/Mn ≈ 2, z-average radius of gyration Rgz ) 72 nm, and z-average hydrodynamic radius Rhz ) 30 nm.6 The β-lg used in this study was a gift from Lactalis (batch no. 754). High-performance liquid chromatography shows that the sample consists of equal fractions of genetic variants A and B. Solutions were prepared by dialyzing against distilled and deionized water at pH 7. The solutions were filtered through 0.2 µm or 0.45 µm pore size Anatope filters depending on the concentration. The concentrations were determined after filtration by SEC with refractive index detection using the refractive index increment dn/dc ) 0.189 g/mL for β-lg7 and dn/dc ) 0.145 g/mL for κ-car. The protein concentration was also determined using UV absorption and the extinction coefficient 0.96 g/(L/cm).8 Both methods gave the same results within a few percent. Small aggregates with a hydrodynamic radius of 20 nm were prepared by heating a solution containing 20 g/L β-lg without salt for 10 h at 75 °C. Larger aggregates were prepared by heating solutions containing 60 g/L β-lg and 0.1 M NaCl between 34 and 46 min at 70 °C. The solutions were characterized using size exclusion chromatography and light scattering. Details of the method of characterization are given in refs 9 and 10. The aggregates formed by heating β-lg at pH 7 are polydisperse and have a self-similar structure characterized by a fractal dimension df ) 2. They are formed by association of small relatively monodisperse primary aggregates with a hydrodynamic radius of about 15 nm and containing about a 100 proteins.11 Methods. SLS and DLS measurements were made using an ALV-5000 multibit multitau correlator and a solid-state laser (Spectra Physics, Millenium II) operating with vertically polarized light with wavelength λ ) 532 nm. The range of scattering wave vectors covered was 3.0 × 10-3 < q < 3.5 × 10-2 nm-1. The temperature was controlled by a thermostat bath set at 20 ( 0.1 °C. The electric field autocorrelation function (g1(t)) is related to the normalized measured intensity autocorrelation function (g2(t)) as: g2(t) ) 1 + g1(t)2.12 g1(t) was analyzed in terms of a distribution of relaxation times: g1(t) ) ∫A(log τ) d log τ. SEC experiments were carried out at room temperature with a TSK PW 5000 + PW 6000 column set (30 cm + 60 cm) in series. We used a combination of refractive index detection and UV detection at 278 nm. The columns were eluted with a 0.1 M NaNO3 solution at a flow rate of 1 mL/min, 200 ppm NaN3 being added as a bacteriostatic agent. The injected volume was 300 µL, and the injected concentration was approximately 0.1%. (5) Rochas, C.; Landry, S. Carbohydr. Polym. 1987, 7, 435. (6) Meunier, V.; Nicolai, T.; Durand, D. Macromolecules 2000, 33, 2497. (7) Perlmann, G. E.; Longsworth, L. G. J. Am. Chem. Soc. 1948, 70, 2719. (8) Townend, R.; Winterbottom, R. J.; Timasheff, S. N. J. Am. Chem. Soc. 1960, 82, 3161. (9) Gimel, J.-C.; Durand, D.; Nicolai, T. Macromolecules 1994, 27, 583. (10) Le Bon, C.; Nicolai, T.; Durand, D. Int. J. Food Sci. Technol. 1999, 34, 451. (11) Aymard, P., Gimel, J.-C.; Nicolai, T.; Durand, D. J. Chim. Phys. 1996, 93, 987. (12) Berne, B. J.; Pecora, P. Dynamic Light Scattering with Applications to Chemistry, Biology and Physics; John Wiley & Sons: New York, 1976.
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Figure 1. State diagram of mixed systems of κ-car and β-lg aggregates at two sizes indicated in the figure. The filled symbols indicate the lowest concentrations tested at which the systems are phase separated, while the open symbols indicate the highest concentrations tested at which the systems are in one phase. Optical microscopy experiments were done in collaboration with D. Ausserre (PSPI, Universite´ du Maine, France) using a new visualization method.
Results At pH 7, native β-lg is fully compatible with κ-car in the range of concentrations used for this study. Static and dynamic light scattering showed that the interaction between the two macromolecules is negligible, at least at low concentrations. The scattered intensity autocorrelation function of a mixture containing 0.5 g/L β-lg and 2.4 g/L κ-car is simply the sum of that of the individual components. The scattering intensity of the mixture equals the sum of the contributions of the individual components. Mixing κ-car and β-lg aggregates in sufficient quantities causes an increase of the turbidity followed by a macroscopic phase separation. We have studied this phase separation systematically for mixtures of β-lg aggregates with z-average radius of gyration Rgz ) 165 nm and κ-car and obtained the state diagram shown in Figure 1. It is obvious that the determining factor for phase separation is the κ-car concentration. The dependence on the β-lg concentration is very weak, at least at low concentrations, and we have used a logarithmic scale for the β-lg concentration to show this more clearly. The solution of β-lg aggregates contained 60% β-lg that had not yet aggregated. However, as we will show below, unaggregated β-lg plays no role in the phase separation. Throughout this article therefore we will give only the concentration of the aggregates. The κ-car concentration needed for phase separation (Cps(κ-car)) decreases with increasing size of the aggregates. Figure 1 shows the results obtained for very large aggregates with Rgz > 1 µm. We can only give a lower limit for the size of these aggregates as their scattering intensity has a power law q dependence over the whole accessible q range. For the smallest β-lg aggregates with Rgz ) 20 nm, we observed phase separation for κ-car concentrations above approximately 8 g/L. In all cases Cps(κ-car) depends weakly on the concentration of β-lg aggregates at least in the range investigated here. On the other hand, Cps(κ-car) depends on the time of observation. This is illustrated in Figure 2 where we have plotted the time (tps) needed to observe the onset of the increase in turbidity as a function of the concentration of κ-car and β-lg aggregates with Rgz ) 35 nm. In practice,
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Figure 2. Time elapsed before the onset of the phase separation for mixtures at fixed κ-car concentration as a function of the β-lg aggregate concentration (top) and at fixed β-lg aggregate concentration as a function of the κ-car concentration (bottom). The z-average radius of gyration of the β-lg aggregates was 35 nm. The solid lines represent results of linear least-squares fits.
tps was determined by monitoring the scattering intensity at 90°. The increase in turbidity corresponds to a decrease of the scattering intensity. Over the narrow range investigated, the rate of the phase separation increases approximately exponentially with the concentration of both κ-car and β-lg, but the dependence on κ-car is much stronger than on β-lg. It follows that the state diagram shown in Figure 1 depends on the time of observation. However, the dependence of tps on the κ-car concentration is so strong that Cps(κ-car) is roughly the same whether we wait for a few minutes or a few days. Structure of the Phase-Separated Mixture. The turbidity of the phase-separated system decreases with decreasing concentration of β-lg. Therefore, at low enough concentrations we can study the system using static and dynamic light scattering without the interference of multiple scattering. Figure 3a compares the intensity autocorrelation functions of the scattered light for a solution containing 0.1 g/L β-lg aggregates with Rgz165 nm with that of a mixture with 3.6 g/L κ-car. The measurement was done 1 day after mixing but well before a macroscopic phase separation appears. The correlation function of the β-lg aggregates is characterized by a single relaxation distribution, while two relaxation processes characterize the correlation function of the mixture. Both modes are q2 dependent and are due to a process of translational diffusion. We have used the so-called generalized exponential to describe the broader relaxation distribution that characterizes the fast mode:13 A(log τ) ) k(τ/τg)p exp[-(τ/τg)s], where k is a normalization constant and τg is the characteristic relaxation time. This versatile function has (13) Nicolai, T.; Gimel, J.-C.; Johnsen, R. J. Phys. II 1996, 6, 697.
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Figure 3. (a) Comparison of intensity autocorrelation functions of a solution containing 0.1 g/L β-lg aggregates with Rgz ) 165 nm and a mixture with, in addition, 3.6 g/L κ-car. The mixture was studied before the sedimentation was noticeable. The solid lines represent results of nonlinear least-squares fits; see text. (b) Hydrodynamic radius distribution of the β-lg aggregates before mixing (dashed line) and of the mixture (solid line).
two parameters (p,s) to describe a wide variety of distributions such as the Schultz-Zimm and the Pearson distribution. The values of p and s are sensitive to noise on the data, but the general shape of the distribution is robust. The slow mode is narrower and was fitted to a log-normal relaxation time distribution. The experimental correlation functions could be accurately fitted to the sum of these two relaxation time distributions; see solid lines through the data in Figures 3a and 6a. If the relaxation is due to diffusion of individual particles, the relaxation time distribution may be transformed into a distribution of hydrodynamic radii using the Stokes-Einstein relation12
D ) kT/6πηRh
(1)
where k is Boltzmann’s constant, T is the absolute temperature, η is the viscosity, and D is the diffusion coefficient which is determined from the relaxation time as: D ) 1/(q2τ). Figure 3b shows the hydrodynamic radius distribution corresponding to the correlograms shown in Figure 3a. Note that the contribution of κ-car to the scattering intensity is small but that it increases the viscosity by a factor 10. We attribute the slow mode to the diffusion of the protein-rich microdomains observed by Syrbe.2 Indeed, we also observe spherical microdomains for mixtures of β-lg aggregates and κ-car with optical microscopy; see Figure 4. The fast relaxation mode is due to the diffusion of β-lg aggregates that are smaller than before mixing as is seen more clearly with SEC; see below. Notice that the peak corresponding to the fast mode has a tail that extends
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Figure 4. Micrograph of protein-rich microdomains obtained with optical microscopy for a mixture of 5.7 g/L β-lg aggregates with Rgz ) 20 nm and 9.2 g/L κ-car.
Figure 5. Double logarithmic representation of the q dependence of the relative excess scattering intensity for a solution containing 0.1 g/L β-lg aggregates (squares) with Rgz ) 165 nm and a mixture with, in addition, 3.6 g/L κ-car (triangles). The circles indicate the q dependence of the phase-separated microdomains. The solid line has slope -4.
to very small values of Rh, which is due to the cooperative diffusion of κ-car which contributes very weakly to the scattering intensity. The q dependence of the scattered light intensity relative to that of toluene (Ir) is shown in Figure 5 for the initial aggregates and the mixture. Compared to the initial aggregates, the mixture shows much stronger scattering at small q values and somewhat weaker scattering at large q values. Neglecting the small contribution of κ-car, the scattering of the mixture contains contributions from the microdomains and the residual aggregates outside the microdomains. The contribution of each component to the relaxation time distribution obtained from DLS is proportional to its scattering intensity. We can therefore determine the scattering intensity of each component by multiplying the total intensity with the relative amplitude of each mode in the relaxation time distribution. The resulting q dependence of the scattering intensity of the microdomains is shown in Figure 5. The straight line has slope -4, which is expected for homogeneous domains with sharp interfaces. The data deviate from this power law behavior for q < 0.02 nm-1, which means that the microdomains are homogeneous down to a length scale of about 50 nm. The q dependence of the other component is small, because the remaining aggregates are on average smaller than the initial aggregates. It is significant that
Figure 6. (a) Comparison of intensity autocorrelation functions of a mixture containing 6.5 g/L β-lg aggregates with Rgz ) 165 nm and 7.1 g/L κ-car at different times after dilution. The solid lines represent results of nonlinear least-squares fits; see text. (b) Hydrodynamic radius distribution of the mixture at different times after dilution.
the residual aggregates have approximately the same size as the length scale beyond which the microdomains are homogeneous. Aging of the Microdomains. For more concentrated systems we need to dilute the system if we want to study it with standard light scattering techniques. If we dilute the system just after mixing the β-lg aggregates with κ-car, we only observe the initial β-lg aggregates. On the other hand, if we wait for some time after mixing, we observe at first two relaxation processes, but the slow mode disappears with time and after some time we observe again only the initial β-lg aggregates. The rate at which the slow mode disappears decreases with increasing waiting time after mixing. This is illustrated in Figure 6a, where we show correlograms at different times after dilution for a solution containing 6.5 g/L β-lg aggregates and 7.1 g/L κ-car, 1 day after mixing. The corresponding hydrodynamic radius distributions, see Figure 6b, show that after dilution the microdomains slowly decrease in size and scattering amplitude. The fast mode is due to residual β-lg aggregates that have not phase separated. These residual aggregates are at first smaller than the initial β-lg aggregates, but simultaneously with the decrease in size and amplitude of the microdomains, the β-lg aggregates increase in size and become on average even larger than the initial aggregates. Then, once all the microdomains have dispersed, the size of the aggregates decreases again. This second stage of the dispersion is more clearly seen for a system containing less κ-car (3.2 g/L). For this system the microdomains are much larger (several tens of
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Figure 7. Double logarithmic representation of the q dependence of the relative excess scattering intensity of a mixture containing 6.5 g/L β-lg aggregates with Rgz ) 165 nm and 3.2 g/L κ-car at different times after dilution. The straight line has slope -1.7.
micrometers). The system was diluted 2 days after mixing. Just after dilution we observe the microdomains, but they are too large for characterization by light scattering. After about an hour they have disappeared and we observe a single relaxation mode. The scattering intensity has a power law q dependence over the whole accessible range: Ir ∝ q-R with exponent R ) 1.7; see Figure 7. Clearly, the microdomains have dispersed into large self-similar β-lg aggregates. The power law exponent is equal to the fractal dimension (df) if the aggregates are not extremely polydisperse.14 If, on the other hand, the number of aggregates with molar mass M has a power law dependence on M: N(M) ∝ M-τ, with τ > 2, then R ) df(3 - τ).15 This means that it is also possible that the microdomains disperse to form aggregates with the same fractal dimension as the initial aggregates, but with polydispersity exponent τ ) 2.2.15 This value of τ is in fact expected for percolating clusters. The experimental results do not allow us to distinguish between these possibilities. At later times the large aggregates break up to form smaller and smaller aggregates until we finally reach the size of the aggregates used to produce the system. However, for all systems investigated the microdomains are stable if we wait long enough (about a week) before diluting the system. Sedimentation of the Microdomains. The increase of the turbidity caused by the formation of the microdomains is very rapid except close to Cps(κ-car). On the other hand, the sedimentation of the microdomains is slow and depends on the composition of the mixture. For one system we have studied the sedimentation of the microdomains in more detail. The system is a mixture of 2 g/L β-lg aggregates with Rgz > 1 µm and various concentrations of κ-car. Above about 1.5 g/L κ-car we observe an increase of the turbidity after mixing indicating the formation of microdomains. Initially, the microdomains are spread over the whole volume, but after some time we clearly observe two phases. In Figure 8a we have plotted the height of the bottom phase, which contains the phase-separated microdomains, as a function of the time after mixing. For the two higher κ-car concentrations, the decrease of the height of the bottom phase can be explained by simple sedimentation of the microdomains with constant velocity;
Figure 8. (a) Height of the turbid bottom phase as a function of time after mixing 2 g/L β-lg aggregates with Rgz > 1 µm and different κ-car concentrations indicated in the figure. The solid lines are fits assuming constant velocity and in the case of 1.7 g/L an induction time of 50 min. (b) Same data as in part a after correction for the viscosity of the κ-car solutions.
see solid lines through the data. This is not the case for the system with C(κ-car) ) 1.7 g/L which is close to Cps(κ-car). This sample becomes increasingly grainy and then separates into two phases over a very short period of time. We observed similar features for mixtures with other sizes and concentrations of the β-lg aggregates. Always for C(κcar) well above Cps(κ-car) we observed a slow continuous decrease of the height of the bottom phase and for C(κcar) close to Cps(κ-car) delayed sedimentation. It has been suggested that delayed sedimentation for weakly associating colloids is caused by the formation of a transient gel.16 With time, the transient gel restructures and becomes less well connected until it no longer supports its own weight and collapses. We may speculate that such a process occurs in the mixtures close to Cps(κ-car) for which optical microscopy shows that the microdomains are much larger than at higher C(κ-car) (see Figure 9). At higher κ-car concentrations light scattering experiments showed that well-defined, unconnected microdomains are formed very shortly after mixing. For those mixtures the height of the bottom phase is, at least initially, controlled by the sedimentation velocity of the microdomains. The sedimentation velocity (v) is determined by the excess density over the solvent (∆F), the radius (R) of the microdomains, and the solvent viscosity (η)
v)
g ∆F 2R2 9η
(2)
with g the gravitation constant. In the derivation of eq 2 (14) Nicolai, T.; Durand, D.; Gimel, J. C. In Light Scattering. Principles and Developments; Brown, W., Ed.; Clarendon Press: Oxford, 1996. (15) Stauffer, D., Aharony, A. Introduction to Percolation Theory; Taylor and Francis: London, 1992.
(16) Poon, W. C. K.; Starrs, L.; Meeker, S. P.; Moussaı¨d, A.; Evans, R. M. L.; Pusey, P. N.; Robins, M. M. Faraday Discuss. 1999, 112, 143.
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Figure 9. Micrographs of redispersed sediment obtained for mixtures of 4.8 g/L β-lg aggregates with Rgz ) 100 nm and different amount of k-car as indicated in the micrographs. We observed delayed sedimentation only for the lowest concentration of κ-car.
it is assumed that the microdomains are dilute. The final volume fraction of the bottom phase is less than 5% for the samples we are considering so that we may neglect the influence of the finite volume fraction for the initial part of the sedimentation process. However, when the height of the bottom phase approaches its final value, the volume fraction of the microdomains becomes important.
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This leads to a reduction of the sedimentation velocity and explains the deviation from the solid lines in Figure 8. The “solvent” in which the microdomains sediment is in fact a semidilute solution of κ-car plus a residual fraction of small β-lg aggregates. Nevertheless, on the length scale of the microdomains, we may consider this solution as a homogeneous solvent with density close to that of water, but with a viscosity that strongly increases with the κ-car concentration. The dependence of η on C(κ-car) explains partially the decrease of v with increasing C(κ-car) as is shown in Figure 8b where we have plotted the data as a function of time corrected for the solvent viscosity. The remaining difference is caused by the difference in the density and/or the size of the microdomains. The density of fractal objects is related to their size via the so-called fractal dimension (df): F ∝ Rdf-3. This means that vη ∝ Rdf-1, which explains why we do not observe sedimentation of the initial aggregates for which df ) 2 even if they are very big. (Notice that the equilibrium height is very small both for the microdomains and for large β-lg aggregates.) The actual value of v depends on the local structure of the microdomains. It is reasonable to suppose that the local structure of the microdomains is equal to that of the β-lg aggregates and is homogeneous only above a given length scale L*. This means that D is independent of R and proportional to 1/L* and so vη ∝ R2/L*. The increase of vη with decreasing κ-car concentration can thus be explained by either an increase of R or a decrease of L*. We have seen only a modest increase of R with decreasing C(κ-car) if the latter is much larger than Cps(κ-car). On the other hand one might expect that L* decreases with increasing C(κ-car) as smaller and smaller β-lg aggregates participate to the formation of the microdomains. After redispersion of the sediment it precipitates again, but with a much greater velocity. This is due to the fact that the sediment cannot be redispersed on the level of individual microdomains. Microscopy of the redispersed samples shows mainly clusters of microdomains; see Figure 9. Consequently microdomains sediment much more rapidly after redispersion than in the initial mixture. Also delayed sedimentation is no longer observed for the systems close to Cps(κ-car), presumably because dispersed clusters of microdomains do not form a transient gel. Composition of the Supernatant. We have analyzed the composition of the supernatant using size exclusion chromatography with combined detection of the refractive index and the UV absorption at 278 nm. κ-car does not absorb at 278 nm while the extinction coefficient of both native and aggregated β-lg is 0.96 g/(L/cm). This means that we measure only β-lg with UV absorption, which enables us to deduce the κ-car concentration by subtracting the contribution of β-lg from the refractive index signal. In all cases we found that the κ-car concentration in the supernatant was the same as in the mixture within the experimental error. One might expect an increase of C(κcar) in the supernatant due to the exclusion of κ-car from the microdomains, but the volume fraction of the microdomains is too small for this effect to be significant. In all cases investigated here the volume fraction of the precipitate does not exceed 10%. Figure 10a shows chromatograms using UV absorption detection of a mixture of 4.8 g/L β-lg aggregates with Rgz ) 100 nm and various concentrations of κ-car between 0 and 9.2 g/L. The mixtures also contain 7.2 g/L residual unaggregated proteins which leave the column at elution volume Ve ) 30 mL. It is evident that the unaggregated proteins are not influenced by the presence of κ-car in the
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Figure 10. (a) Chromatograms of the supernatant of phaseseparated mixtures containing 5.7 g/L β-lg aggregates with Rgz ) 100 nm and various amounts of κ-car indicated in the figure. The inset shows a zoom of the area corresponding to the aggregates. The chromatograms were normalized by the total concentration of β-lg in the mixtures. (b) Chromatograms of the supernatant of phase-separated mixtures containing 5.7 g/L κ-car and various amounts of β-lg aggregates with Rgz ) 100 nm indicated in the figure. The inset shows a zoom of the area corresponding to the aggregates. For comparison we included the chromatogram of the initial β-lg solution before mixing. The chromatograms were normalized by the total concentration of β-lg in the mixtures.
concentration range investigated here. This is the reason we may ignore the unaggregated β-lg and consider only the concentration of aggregates. It is also clear that larger aggregates phase separate preferentially, which explains why we observed with light scattering only smaller residual aggregates in the mixture. At a given κ-car concentration, only aggregates larger than a particular size phase separate and this critical size decreases with increasing κ-car concentration. Figure 10b shows that for a fixed κ-car concentration the influence of the concentration of β-lg aggregates (0.7-12 g/L) is modest in accordance with the weak influence on the macroscopically observed phase separation. Note that in Figure 10b the chromatograms are normalized by the total β-lg concentration. After this normalization the peak corresponding to the native proteins is not modified, which demonstrates again that the native proteins are not influenced by κ-car. Discussion The present results show clearly that β-lg aggregates and κ-car are incompatible above a given concentration of the latter. Both β-lg and κ-car are negatively charged at pH 7 and there is no complex formation. A possible origin for the phase separation is depletion of κ-car chains, which results in an effective attraction between the β-lg aggregates.3,4 The depletion phenomenon is well understood for the case of large homogeneous spheres in the
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presence of smaller flexible polymers that do not overlap.17 The potential is given in terms of the radius of the spheres and of the polymers. Recently, it was found experimentally that almost the same functional form of the attractive potential could be used also if the polymers overlap.18 In this case the radius of the polymers needs to be replaced by an effective radius that decreases with increasing concentration. It has been proposed in the literature that for semidilute polymer solutions the relevant length scale is the correlation length (ξ) of the polymer segments and that the effect of depletion becomes important once ξ decreases below the radius of the spheres. The concentration dependence of the correlation length is reported in ref 19. ξ has a power law concentration dependence for C > 1.5 g/L (ξ ∝ C-0.83), but below 1 g/L the correlation length depends only weakly on the concentration. The overlap concentration may be estimated as C* ) 3Mw/(Na4πRgz3) and is about 0.5 g/L. For all systems reported here the phase separation is observed for C > C*, but there is no obvious relation between ξ values at Cps(κ-car) and Rgz of the β-lg aggregates apart from the general trend that smaller aggregates phase separate at smaller ξ. In all cases ξ at Cps(κ-car) is much smaller than Rgz of the β-lg aggregates. To quantitatively explain the phase separation in terms of the depletion potential, one would need to know the effect of the fractal structure and the large polydispersity of the aggregates. It has been observed that depletion by polydisperse spheres leads to fractionation, with the larger spheres preferentially in the dense phase.20 The effect can be understood qualitatively in terms of the depletion potential, but a quantitative treatment has only been given for narrow distributions.21 The fractal structure means that there is more space for the κ-car chains between two approaching aggregates than between two homogeneous spheres of the same size, which reduces the depletion effect. In addition, one needs to consider that for charged systems phase separation leads to an entropically unfavorable confinement of counterions. Although depletion may play a role, it should be remembered that different macromolecules are almost always more or less incompatible. A small incompatibility may go unnoticed for small molar masses but may nevertheless lead to phase separation at high molar masses. Thus the fact that no noticeible interaction is observed between the native proteins and the polysaccharide does not mean that depletion is the only reason that protein aggregates phase separate as was assumed by Tuinier et al.3 Whatever the details of the interaction, it is clear that we are initially observing a reversible liquid-liquid phase separation. In most cases the phase separation is rapid and occurs by spinodal decomposition as described by Tuinier et al.,3 but close to Cps(κ-car) we observe very slow phase separation due to nucleation and growth in the metastable regime. At the low protein concentrations studied here, phase separation leads to the formation of spherical, protein-rich microdomains that have relatively sharp interfaces and are homogeneous. The length scale (17) Asakura, S.; Oosawa, F. J. Polym. Sci. 1958, 33, 183. (18) Verma, R.; Crocker, J. C.; Lubensky, T. C.; Yodh, A. G. Macromolecules 2000, 33, 177. (19) Croguennoc, P.; Meunier, V.; Durand, D.; Nicolai, T. Macromolecules 2000, 33, 7471. (20) Bibette, J. J. Colloid Interface Sci. 1991, 147, 474. (21) Evans, R. M.; Fairhurst, D. J.; Poon, W. C. K. Phys. Rev. Lett. 1998, 81, 1326.
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down to which the microdomains are homogeneous is smaller than the average size of the aggregates that phase separated, which implies that the aggregates interpenetrate each other and/or contract. During this initial stage of the phase separation the microdomains dissolve when diluted. However, we have seen that the dissolution takes more time the longer we wait before diluting the microdomains. In addition, the microdomains do not fuse as might be expected if they were truly liquid. These observations imply that the aggregates in the microdomains associate through physical bonds that are weak initially but which become stronger with time. The strengthening of the bonds occurs probably by restructuring so that, with time, more and more proteins are involved in the bonding. We observed that if diluted samples were heated, the microdomains dissolved much more rapidly, which renders hydrophobic interactions less likely as the origin for the association. Possibly, hydrogen bonding or electrostatic interactions are involved. The outcome of this association process is the formation of stable spherical microgels, which tend to stick together, but do not fuse. Association of preheated β-lg aggregates at room temperature also occurs in the absence of κ-car, although much more slowly.22 The average size of aggregates increases approximately logarithmically with time. The association rate at room temperature decreases with decreasing concentration, and for dilute samples (C < 2 g/L) we no longer observe it. The aggregates formed at room temperature and at elevated temperatures have the same structure, but while the latter are stable upon dilution, aggregates formed at room temperature are initially not stable. However, they become more and more stable the longer we wait before diluting just like the microdomains. Probably the forces involved in the aggregation at room temperature are the same with and without κ-car except that inside the phase-separated microdomains the protein concentration is higher so that the rate of aggregation is higher. Recently, it was shown that disulfide bridges are formed very slowly between β-lg aggregates even at room temperature.23 They are not involved in the phase separation itself, which is often very rapid, but they possibly contribute to the stabilization of microdomains. The reason for the slow rate of the aggregation at room temperature could be that only a limited number of proteins are in a configuration which allows bonding, i.e., all the unaggregated (native) proteins and most of the aggregated proteins have an inert conformation. In heated samples there is a continuous influx of denatured proteins and therefore of sites amenable to physical bonding. But, even for heated samples the growth of aggregates slows down dramatically once most of the proteins have aggregated and the influx of denatured proteins has stopped.24 (22) Croguennoc, P. Thesis, Universite´ du Maine, Le Mans, France, 2000. (23) Alting, A. C.; Hamer, R. J.; de Kruif, C. G.; Visschers, R. W. J. Agric. Food Chem. 2000, 48, 5001.
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Syrbe2 investigated heated mixtures of whey proteins (70% β-lg) and pectin. We will discuss the effect of mixing first and heating next for the present system in part 2. It is, however, relevant for the discussion here that, in certain conditions, Syrbe observed the formation of microdomains similar to those we observed. The size of the microdomains depended on the heating rate and the pectin concentration but not on the protein concentration. At higher heating rate or higher pectin concentration, the microdomains were smaller. Analysis of the microdomains showed that they contained negligible amounts of pectin. He suggested that the size of the microdomains is controlled by a competition of aggregation and phase separation. At low heating rate the phase-separated microdomains have more time to grow before they become stuck in their configuration by gelation. At higher polysaccharide concentrations the protein concentration in the microdomains is larger, which increases the rate of association and gelation. In addition, the viscosity of the system increases rapidly with increasing polysaccharide concentration, which inhibits the growth of the microdomains. For most systems investigated here the diameter of the microdomains is a few micrometers. It is only weakly dependent on the protein concentration, but it increases with decreasing C(κ-car); see Figure 9. Close to Cps(κ-car) the diameter becomes much larger (a few tens of micrometers). These large microdomains are also more deformed and show some signs of partial fusing in addition to clustering. Again we may invoke the competition between aggregation and phase separation to rationalize these findings. Close to Cps(κ-car) phase separation is weak and the density of preheated aggregates in the microdomains is low. This means that their physical association is slow, which allows more time for growth and fusion of the microdomains. Conclusions Mixtures of β-lg aggregates and κ-car phase separate at room temperature leading to the formation of spherical, protein-rich microdomains, which precipitate slowly. The minimum κ-car concentration needed to cause phase separation decreases weakly with increasing concentration and size of the β-lg aggregates. Phase separation leads to fractionation of the polydisperse β-lg aggregates with the larger aggregates in the sedimented microdomains and the smaller aggregates in the supernatant. At room temperature β-lg aggregates inside the microdomains associate via physical bonds. Initially the association is reversible, but with time the microdomains become increasingly stable to dilution. The microdomains tend to stick together, but do not fuse. Acknowledgment. P.C. acknowledges financial support from Unilever Research. LA001674Q (24) Le Bon, C.; Nicolai, T.; Durand, D. Macromolecules 1999, 32, 6120.
