Influence of the NaCl or CaCl2 Concentration on the Structure of Heat

Taco Nicolai*. Polyme`res, Colloıdes, Interfaces, UMR CNRS, Université du Maine, 72085 Le Mans Cedex 9, France. Received February 22, 2005; Revised ...
0 downloads 0 Views 235KB Size
Download PDF
Biomacromolecules 2005, 6, 2157-2163

2157

Influence of the NaCl or CaCl2 Concentration on the Structure of Heat-Set Bovine Serum Albumin Gels at pH 7 Laurence Donato, Catherine Garnier, and Jean-Louis Doublier Unite´ de Physicochimie des Macromole´ cules, INRA, Rue de la Ge´ raudie` re, BP 71642, 44316 Nantes Cedex 03, France

Taco Nicolai* Polyme` res, Colloı¨des, Interfaces, UMR CNRS, Universite´ du Maine, 72085 Le Mans Cedex 9, France Received February 22, 2005; Revised Manuscript Received April 19, 2005

The structure of heat-set systems of the globular protein bovine serum albumin (BSA) was investigated at pH 7 in different salt conditions (NaCl or CaCl2) using light scattering. Cross-correlation dynamic light scattering was used to correct for multiple scattering from turbid samples. After heat treatment, aggregates are formed whose size increases as the protein concentration increases. Beyond a critical concentration that decreases with increasing salt concentration, gels are formed. The heterogeneity and the reduced turbidity of the gels were found to increase with increasing salt concentration and to decrease with increasing protein concentration. The structure of the gels is determined by the strength of the repulsive electrostatic interactions between the aggregated proteins. The results obtained in NaCl are similar to those reported in previous studies for other globular proteins. CaCl2 was found to be much more efficient in reducing electrostatic interactions than NaCl at the same ionic strength. Introduction Under heat treatment, globular proteins in aqueous solution undergo structural modification, leading to unfolding of hydrophobic zones and exposition of the cysteine residues. This results in aggregation of the proteins via hydrophobic and/or hydrogen interactions and formation of disulfide bridges. If the protein concentration is high enough, the aggregation can lead to gel formation. The structure of heatset globular protein gels is therefore dependent on the nature and the strength of interactions between proteins, which itself depends on the protein concentration, pH, and the concentration and type of ions present in the medium.1,2 When electrostatic repulsions are strong, i.e., at pH far from the isoelectric point and at low ionic strength, then homogeneous, transparent gels are formed. If the charge of the proteins is decreased (pH near pI) and/or screened by adding salt, the gels become opaque and heterogeneous on the length scale of microns. Recently, the structure of heated systems obtained by two industrially important globular proteins (β-lactoglobulin3 and ovalbumin4) has been described at pH 7 at different NaCl concentrations. Here we complement these studies for another important globular protein, bovine serum albumin (BSA). BSA is a widely studied protein and is the most abundant protein in plasma. BSA consists of 582 amino acid residues and has an average molecular weight of 66 kg/mol. Its isoelectric point is around 5.2.5 At pH 5-7, it contains 17 intrachain disulfide bridges and one sulfhydryle group. BSA is made up of three homologous domains. Serum * Corresponding author. E-mail: [email protected].

albumin was first postulated to be an oblate ellipsoid with dimensions of 141 Å × 41 Å.5 However, X-ray crystallographic data indicated that an oblate ellipsoid structure of albumin was unlikely; rather, a heart-shaped structure was proposed for human serum albumin, and it may be assumed that BSA has the same structure.6 The net negative charge of BSA at pH 7 is -18 and it is irregularly distributed within the three domains of the protein, but the distribution of charges on the ternary structure seems fairly uniform.6 An overview of BSA gelation during heating was published by Clark and Lee-Tuffnell,7 who described the different types of gels that are obtained, depending on the conditions of the medium. In the present paper, we study the influence of adding NaCl or CaCl2 on the structure of heated BSA solutions and gels at pH 7. The results in the presence of NaCl will be compared with those obtained for β-lactoglobulin (β-lg)3 and ovalbumin (OA).4 Adding CaCl2 has a more dramatic effect on globular protein gelation and was not studied before using light scattering. Experimental Section Materials. BSA obtained by cold fractionation to prevent denaturation (98-99 wt % purity) was purchased from ICN Biomedicals (Aurora, OH). The protein powder was defatted by washing with n-pentane in order to eliminate the accompanying fatty acids. Solutions of BSA were prepared by adding BSA powder in water under gentle magnetic stirring at 4 °C overnight. The pH was adjusted at 7 with 1 M NaOH. Sodium azide (3 mM) was added to prevent bacterial growth. Insoluble matter was eliminated by centrifugation (16 000g,

10.1021/bm050132q CCC: $30.25 © 2005 American Chemical Society Published on Web 05/26/2005

2158

Biomacromolecules, Vol. 6, No. 4, 2005

Donato et al.

30 min) and the solutions were extensively dialyzed against salt-free Milli-Q water at pH 7 with 3 mM sodium azide added. After dialysis, salt solutions were added to obtain desired salt concentrations ranging from 0.01 to 0.2 M NaCl and 0.1 to 3 mM CaCl2. All samples were filtered through 0.45 µm pore size Anotope filters. The protein concentration was measured by UV absorption at 278 nm using an extinction coefficient of 0.677 g.L-1.cm -1. Light Scattering. Light scattering measurements were done at 20 °C using a commercial version of the 3D crosscorrelation instrument described in ref 8 (LS Instruments, Fribourg, Switzerland). The light source was a diode laser with wavelength λ ) 685 nm. Photon correlation was done with a digital correlator (ALV-5000E; ALV, Langen, Germany). The relative excess scattering intensity (Ir) was determined as the total intensity minus the solvent scattering divided by the scattering intensity of toluene at 20 °C. Ir was corrected for multiple scattering and the transmission as described in refs 9 and 10. The structure factor [S(q)] of the system can be derived from the dependence of Ir on the scattering wave vector [q ) 4πn/λ sin(θ/2), where n is the refractive index of the solution and θ is the scattering angle]:11,12 Ir ) KCMaS(q), where K is an optical constant, Ma is an apparent molar mass, and C is the concentration. In dilute solution, when interactions are negligible, Ma represents the weight-average molar mass (Mw) while the z-average radius of gyration (Rgz) can be calculated from the initial q-dependence of S(q): S(q) ) [1 + (qRgz)2/3]-1. If interactions cannot be ignored, the initial q-dependence can sometimes be used to calculate an apparent radius of gyration (Ra): S(q) ) [1 + (qRa)2/3]-1

(1)

Turbidity Measurements. Turbidity measurements were done using a UV spectrometer (Perkin-Elmer Lambda 2) equipped with a thermostated stage (PTP-6 Peltier System), which allowed temperature control as a function of time. The turbidity (τ) was determined at 685 nm from the ratio of the light transmitted through the sample (Is) and that transmitted through the solvent (I0) using the relation Is/I0 ) exp(-τl), where l is the path length of the light through the samples (between 0.1 and 1 cm). BSA solutions were placed in quartz spectrometer cells at 20 °C. The temperature was raised rapidly from 20 to 80 °C and kept at 80 °C for 48 h or longer. Determination of Critical Concentration of Gelation. BSA solutions in glass tubes that were hermetically sealed with Parafilm were heated for 12 or 48 h in a thermostated bath at 80 °C and subsequently rapidly cooled to 20 °C. After staying for 12 h at 20 °C, the solutions were checked for gelation, which we operationally defined by the absence of flow when tilting the tubes. Confocal Laser Scanning Microscopy (CLSM). CLSM was used in the fluorescence mode. Observations were made with a Carl Zeiss LSM 410 Axiovert (Le Pecq, France) using a water-immersed ×40 objective. The proteins were labeled by adding rhodamine B isothiocyanate (RITC) (2.5 mg of RITC/g BSA) to the BSA solution and stirring during 1 h at

Figure 1. Critical gel concentrations of BSA solutions at pH 7 heated for 48 h at 80 °C as a function of the NaCl concentration (a) or the CaCl2 concentration (b). The lower and upper error bars indicate, respectively, the highest protein concentration tested where no gel was observed and the lowest concentration where a gel was observed. The arrow in Figure 1a shows Cg in the absence of added NaCl. The dashed line in Figure 1b separates the domain where less than one calcium ion is available per protein (top left) from the area where more than one calcium ion is available (bottom right).

ordinary temperature. It was verified that labeling of the protein affected neither the denaturation temperature measured by DSC nor the rheological behavior of BSA solutions upon heat treatment. Labeled solutions were poured between a concave slide and a flat coverslip that was hermetically sealed. A heat treatment of 30 min at 80 °C was applied using a thermostated stage (Linkam PE 60, London, UK). Observation of the systems was made by excitation of RITC at 543 nm, the emission being recorded between 575 and 640 nm. For each system, images were taken after 1 h at 20 °C at the end of the heat treatment. Results Figure 1 displays the sol-gel state diagram obtained after 48 h of heating at 80 °C as a function of salt concentration (NaCl, Figure 1a; CaCl2, Figure 1b). The error bars indicate the lowest concentration tested where the system gelled and the highest concentration tested where the system remained liquid. Gels are obtained above a critical protein concentration (Cg) that decreases as the ionic strength increases. Gels formed at low protein concentrations (C < 4 g/L), i.e., at high salt concentrations, are very weak and collapse under

Structure of Heat-Set Systems of BSA

Figure 2. Variations of the reduced turbidity (τ/C) as a function of time during heating at 80 °C for different BSA concentrations at pH 7 at two NaCl concentrations: 100 mM (a) and 50 mM (b).

their own weight, resulting in precipitation. Protein concentrations higher than about 5 g/L are needed to form gels that do not flow when the vials are tilted. Curves similar to those shown in Figure 1a have been obtained for other globular proteins in NaCl.4 In CaCl2 the critical concentrations are much lower than in NaCl at the same ionic strength, suggesting that Ca2+ has a specific affinity for BSA Visual observations of the BSA solutions heated for 48 h at 80 °C showed that transparent systems were obtained at NaCl concentrations up till 10 mM and CaCl2 concentrations up till 0.5 mM. In NaCl, the solutions became turbid or opaque from 25 mM upward. In CaCl2, the systems became turbid at low concentrations starting from 0.75 mM CaCl2, but remained transparent up till 1.5 mM at high protein concentrations. Turbidity. The turbidity was measured in-situ while heating at 80 °C for different protein concentrations at different NaCl or CaCl2 concentrations. The range of protein concentrations that could be probed at higher salt concentrations was limited on the low concentration side by the formation of a precipitate. Figures 2 and 3 show the variations of the reduced turbidity (τ/C) as a function of time at two NaCl concentrations (50 and 100 mM) and two CaCl2 concentrations (0.75 and 1.5 mM), respectively. We normalized the turbidity by C in order to remove the trivial concentration dependence and observe more clearly the effect of structural changes.

Biomacromolecules, Vol. 6, No. 4, 2005 2159

Figure 3. Variations of the reduced turbidity (τ/C) as a function of time during heating at 80 °C for different BSA concentrations at pH 7 at two CaCl2 concentrations: 1.5 mM (a) and 0.75 mM (b).

Both in 100 and 50 mM NaCl (Figure 2a,b), the turbidity increased rapidly while the temperature increased to 80 °C. At higher protein concentrations (C > 20 g/L) this initial increase was followed by a distinct plateau, after which τ increased again to reach a second plateau. The reduced turbidity at the first plateau was approximately the same for the intermediate concentrations between 40 and 70 g/L. At 90 g/L, however, the first plateau value was significantly higher. The second increase took a period of at least 10 h to complete and appeared to occur somewhat sooner at higher protein concentrations. The terminal reduced turbidity was again similar between 40 and 70 g/L, but it was significantly higher at higher protein concentrations in 100 and 200 mM NaCl (not shown). At 200 mM NaCl, the turbidity became too high to be measured for C > 70 g/L. At concentrations of 20 g/L and lower, the two-step increase was less obvious and it took increasingly longer heating to reach a steady value of the turbidity. The concentration dependence of the reduced turbidity after 48 h heating is shown in Figure 4. The reduced turbidity after 48 h in NaCl (Figure 4a) decreased as the protein concentration increased but leveled off at high concentrations and even seemed to increase again at the highest concentration at 100 mM NaCl. We note that the values of τ/C up till 10 g/L are lower estimates of the terminal turbidity, because they still increase after 48 h of heating. As in NaCl, a two-step increase of the turbidity was seen in CaCl2 (see Figure 3), except at 5 g/L in 0.75 mM, where

2160

Biomacromolecules, Vol. 6, No. 4, 2005

Figure 4. BSA concentration dependence of the reduced turbidity obtained after 48 h of heating at 80 °C at different NaCl concentrations (a) and different CaCl2 concentrations (b).

the increase was monotonic. The second increase appeared sooner with increasing protein concentration in 1.5 mM CaCl2 as it did in NaCl, but it appeared later as the protein concentration increased in 0.75 mM CaCl2. In CaCl2, the most striking effect was the strong decrease of the final value of τ/C as the protein concentration increased (see Figure 4b), reflecting the formation of more homogeneous gels; see below. Light Scattering. The scattered light intensity was measured as a function of the scattering wave vector for a series of systems heated for 48 h at 80 °C at different protein and salt concentrations. The structure factors [S(q))] as a function of qRa obtained at 25, 50, and 100 mM NaCl are shown in Figure 5a and those obtained at 0.5, 0.75, and 1 mM CaCl2 are shown in Figure 5b. The structure factors are constructed by superimposition of results obtained on aggregates formed at several different protein concentrations below Cg, as explained in refs 3, 13, and 14. The solid lines in Figure 5 represent eq 1. The structure factors of the heated systems formed at different protein and salt concentrations are similar and deviate somewhat from eq 1 at large values of qRa. Nevertheless, the apparent molar mass (Ma) and radius of gyration (Ra) can be determined from the initial q-dependence using eq 1. Figure 6 shows the variations of Ma as a function of Ra for all systems where they could be reliably determined. Ra represents the so-called correlation length beyond which the system becomes homogeneous. For self-similar structures, Ma increases with Radf, where df is the so-called fractal

Donato et al.

Figure 5. Structure factors of BSA solutions after heating for 48 h at 80 °C at different NaCl (a) and CaCl2 (b) concentrations. The symbols for each salt concentration represent results obtained at several protein concentrations between 0.2 and 100 g/L. The solid line is described by eq 1.

Figure 6. Apparent molar mass Ma as a function of the apparent radius of gyration for BSA solutions after heating for 48 h at 80 °C at different NaCl and CaCl2 concentrations. The straight line (slope 2) is displayed to show the tendency.

dimension. We do not have enough data to derive an accurate relationship between Ma and Ra at each salt concentration, but the overall trend is compatible with a power law relation with df close to two or perhaps slightly higher. This value for df is consistent with the q-dependence of S(q) at large q, which for self-similar structures is given by: S(q) ∝ q-df. The prefactor of the power law relationship between Ma and Ra appears to be larger as the salt concentration increases, indicating a denser local structure. But the data are too sparse and contain too much noise to draw firm conclusions. An

Structure of Heat-Set Systems of BSA

Biomacromolecules, Vol. 6, No. 4, 2005 2161

Figure 8. Confocal microscopy images of BSA solutions heat treated for 30 min at 80 °C at 200 mM NaCl (top) or 3 mM CaCl2 (bottom). The observations are made after 1 h at 20 °C (scale bar ) 25 µm)..

Figure 7. BSA concentration dependence of the apparent molar mass of samples after heating for 48 h at 80 °C at different NaCl (a) and CaCl2 (b) concentrations.

important observation is, however, that there is not a large difference between the structures formed in NaCl and in CaCl2. The concentration dependence of Ma at different NaCl or CaCl2 contents is shown in Figure 7, parts a and b, respectively. Without added salt and at low NaCl (10 mM) or CaCl2 (0.1 mM) concentrations, Ma remains small over the whole range of protein concentrations. On the basis of the results obtained on other globular proteins,13,14 we do not expect much aggregation at very low protein concentrations (at least in NaCl), so Ma tends to the molar mass of monomeric BSA (6.6 × 104g/mol) as C goes to zero. Unfortunately, it is difficult to measure Ma at very low concentrations where the signal-to-noise level is low. With increasing protein concentration Ma increases because aggregates are formed. However, at higher concentrations, Ma decreases again with increasing protein concentration, even though gels are formed. This decrease is caused by the increasing electrostatic and excluded volume interactions between the aggregated proteins, which leads to increased order and thus increased destructive interference of the scattered light. The increase of Ma caused by aggregation and the subsequent decrease caused by repulsive interactions are much better seen at higher salt concentrations, especially at 25 mM NaCl and 0.5 mM CaCl2. The maximum value of Ma under these salt conditions was situated at a concentration close to Cg. At 50 mM NaCl the q-dependence of the scattered light intensity showed a power law dependence at C ) 70 g/L over almost the whole accessible range,

indicating that Ra > 1µm and Ma > 5 × 109 g/mol. At even higher salt concentrations, measurements could not be done over a range of protein concentrations around Cg for which the heated systems precipitated or became too turbid. However, even at high salt concentrations, Ma decreased strongly with increasing protein concentration, especially in CaCl2. Figure 8 shows confocal microscopy images of heated samples in 200 mM NaCl (top a) and 3 mM CaCl2 (bottom b). For practical reasons, the samples have been heated at 80 °C during only 30 min, and since the systems evolve with heating time, they cannot be directly compared with the other results obtained after 48 h of heating. Images of the samples at lower salt concentrations showed no features after 30 min of heating. Figure 8b shows that at 3 mM CaCl2 the heterogeneity of heated samples increases with decreasing protein concentration and are heterogeneous on length scales of tens of micrometers at low protein concentrations. The systems obtained at 200 mM NaCl (Figure 8a) were more homogeneous than at 3 mM CaCl2 for all protein concentrations. When comparing different concentrations at 200 mM NaCl, the sample at 10 g/L appeared to be more heterogeneous than those at 5, 20, or 40 g/L, as might be expected from the light-scattering results presented above (see Figure 7a). However, at 80 g/L the sample appeared more heterogeneous than at 40 g/L. This is consistent with the observation that the reduced turbidity leveled off at high protein concentrations and even increased again at 100 mM NaCl. Although at 200 mM NaCl the turbidity became too high to be measured for C > 70 g/L, Figure 8a shows that the heterogeneity increased again at the highest protein concentrations. Discussion Heat-denatured BSA at neutral pH has been shown to aggregate in the form of linear strands of still globular proteins.7 In water electrostatic repulsion inhibits crosslinking of the strands and gels are formed only at relatively high protein concentrations. With increasing salt concentration the aggregates become more densely cross-linked and form a gel at lower protein concentrations. The aggregates have a self-similar overall structure characterized by a fractal dimension in dilute solution of 2.1 at a ionic strength of 100 mM.15 These results are similar to those obtained for β-lg and OA under the same conditions.16,13 The fractal dimension of the aggregates is somewhat smaller if they are formed at lower ionic strength.14,17

2162

Biomacromolecules, Vol. 6, No. 4, 2005

Clark and Lee-Tuffnell determined the small-scale structure of BSA gels in water and 125 mM NaCl at neutral pH using small-angle X-ray scattering (SAXS).18 In water, they observed a maximum in the q-dependence of Ir, which points to strong repulsive interactions that keep the strands at a preferential distance. In the presence of NaCl, the interaction peak disappeared and instead a strong increase of the scattering intensity at low q-values was observed. Similar observations were reported for β-lg3,19 and OA.20 It has not been possible to study the large-scale structure of protein gels formed in the presence of salt using light scattering, because multiple scattering perturbs the results. However, this problem can be overcome by using the recently developed technique of cross-correlation DLS. This technique has been used to study the large-scale structure of β-lg and OA gels formed at pH 7 and different NaCl concentrations.3,4 The results obtained here for BSA are similar to those previously obtained for β-lg3 and for OA.4 In all cases, gels are obtained above a critical concentration that decreases with increasing salt concentration. Transparent systems are obtained at NaCl concentrations below roughly 50 mM and increasingly turbid systems are obtained at higher salt concentrations, although the borderline between transparent and turbid systems depends somewhat on the protein concentration and the type of protein. Gelation occurs by the growth of aggregates until they overlap and connect to form a system-spanning structure. If there is no subsequent restructuring of the gels at length scales below Ra, then the local structure of the gels is the same as that of diluted aggregates formed below the gel point. The equivalence of the structure of the gels and the aggregates was clearly shown for β-lg3 and OA.4 For BSA, the correspondence is not yet fully established, but the fractal dimension of the gels is compatible with that observed for diluted aggregates formed under the same conditions.13 For all three types of proteins, the heterogeneity of the heated systems, as reflected by Ma and Ra, increased first with increasing concentration to a maximum beyond which it decreased. The increase at low concentrations is caused by the increase of the aggregate size with increasing protein concentration. However, the strength of the repulsive interactions also increases with increasing concentration, which renders the systems more homogeneous. The second effect dominates at high concentrations and causes the maximum. A surprising observation for OA gels is that the heterogeneity increased again at very high concentrations. The onset of this increase occurred at lower protein concentrations when the NaCl concentration was higher. An increase of the heterogeneity at high protein concentrations has not been observed for β-lg, but we observed a similar increase of the heterogeneity at high protein concentrations for BSA at 100 and 200 mM NaCl, albeit less marked than for OA. We have as yet no explanation for this phenomenon. The effect of Ca2+ ions on the structure of heated protein systems has not yet been investigated by light scattering for β-lg and OA. Comparing the results obtained on BSA in NaCl and CaCl2, it is obvious that the difference cannot simply be explained by the difference in valence. At the same ionic strength, CaCl2 is much more efficient in facilitating

Donato et al.

aggregation than NaCl. Even a CaCl2 concentration as low as 0.1 mM influences the aggregation process at low protein concentrations. The effect of Ca2+ is not simple screening of electrostatic interactions between the proteins, but probably involves specific interaction with BSA. Therefore, one might expect the molar ratio R ) [Ca2+]/[BSA] to be important and that the influence of Ca2+ on aggregation becomes important as soon as R is significantly larger than unity. The protein concentration where R equals 1 is indicated by the dashed line in Figure 1b. For [CaCl2] < 0.5 mM, gels are formed only at BSA concentrations where R < 1, which might explain why Cg is no longer a strong function of [CaCl2]. In fact, the gels formed at such low CaCl2 concentrations are similar to those formed in the absence of salt. The importance of R rather than the absolute salt concentration also explains the much stronger decrease of Ma with increasing protein concentration in CaCl2 than in NaCl, because with increasing protein concentration R decreases and fewer charges per protein are specifically screened. Therefore, the repulsive interactions do not only increase because more proteins are present, but also because each protein carries a higher effective charge. Finally, we need to discuss the time dependence of the aggregation and gelation process of BSA. Apparently, the increase of the turbidity occurs in two steps, at least at higher protein concentrations. This two-step increase is particular for BSA, because for β-lg and OA only a single monotonic increase of the turbidity to a plateau is observed. The initial increase of the turbidity is in all cases caused by aggregation of the proteins, which leads to gelation. This step occurs at higher protein concentrations during the increase of the sample temperature to 80 °C. The aggregation rate decreases with decreasing concentration below 10 g/L and is slower at 50 mM than at 100 mM NaCl, as might be expected from the decreased screening of the electrostatic repulsion. In CaCl2 the initial aggregation is fast also at low protein concentrations. The second increase is observed only for BSA and occurs when the system has already gelled, which implies that the gel is not a rigid system of irreversibly attached proteins. The gel slowly reorganizes until the final structure is obtained. The restructuring of the gels is probably allowed by the flexibility of the systems and not by detachment and reattachment of individual proteins, because in no case could the gels be dissolved in water. Conclusions Heat-denatured BSA forms aggregates in aqueous solution and gels above a critical concentration that decreases with increased concentration of added salt. Solutions and gels formed after intensive heat treatment have structures that are characterized by a self-similar structure on length scales smaller than Ra and are homogeneous on larger length scales. Ra depends strongly on the salt and protein concentrations. The stronger the repulsive interactions between the aggregated proteins are, the more homogeneous the heated systems are and the smaller Ra is. Repulsive interactions increase with increasing protein concentration and decreasing

Structure of Heat-Set Systems of BSA

salt concentration. Below Cg, aggregates are formed that increase in size with increasing protein concentration. The combined effect of increasing aggregate size and increasing interactions leads to a maximum in Ra and Ma at a given protein concentration. The results obtained for BSA in NaCl are very similar to those obtained for β-lg5 and OA.6 Ca2+ ions seem to have a specific affinity to BSA, which influences the heat-induced aggregation process already at very low CaCl2 concentrations. At low CaCl2 concentrations the important factor is the molar ratio between Ca2+ and BSA monomers. This is consistent with rheological observations previously reported,21,22 which showed a significant increase of the rigidity modulus of the gels in the presence of a low amount of Ca2+. Acknowledgment. We thank Dominique Durand for critically reading the manuscript and Matthieu Pouzot for his help with the cross-correlation experiments. References and Notes (1) Clark, A. H. In Functional properties of food macromolecules, 2nd ed; Hill, S. E., Ledward, D. A., Mitchell, J. R., Eds.; Aspen Publishers: Gaithersburg, 1998; p77. (2) Gosal, W.; Murphy, S. B. Curr. Opin. Colloid Interface Sci. 2000, 5, 188. (3) Pouzot, M.; Durand, D.; Nicolai, T. Macromolecules 2004, 37, 8703. (4) Weijers, M.; Visschers, R. W.; Nicolai, T. Macromolecules 2004, 37, 8709. (5) Peters, T., Jr. In AdVances in Protein Chemistry; Anfinsen, C. B., Edsall, J. T., Richards, F. N., Eds.; Academic Press Inc.: New York, 1985; p 161.

Biomacromolecules, Vol. 6, No. 4, 2005 2163 (6) Carter, D. C.; Ho, J. X. In AdVances in Protein Chemistry; Schumaker, V. N., Ed.; Academic Press Inc.: New York, 1994; p 153. (7) Clark, A. H.; Lee-Tuffnell, C. D. In Functional Properties of Food Macromolecules; Mitchell, J. R., Ed.; Elsevier Applied Science: London, 1986; p 203. (8) Urban, C.; Schurtenberger, P. J. Colloid Interface Sci. 1998, 207, 150. (9) Nicolai, T.; Urban, C.; Schurtenberger, P. J. Colloid Int. Sci. 2001, 240, 419. (10) Pouzot, M.; Nicolai, T.; Durand, D.; Benyahia, L. Macromolecules 2004, 37, 614. (11) Higgins, J. S.; Benoit, H. C. Polymers and Neutron Scattering; Clarendon Press: Oxford, 1994. (12) Brown W., Ed. Light Scattering. Principles and DeVelopments; Clarendon Press: Oxford, 1996. (13) Weijers, M.; Visschers, R. W.; Nicolai, T. Macromolecules 2002, 35, 4753. (14) Baussay K.; Le Bon C.; Durand, D.; Nicolai, T. Int. J. Biol. Macromol. 2004, 34, 21. (15) Hagiwara, T.; Kumagai, H.; Matsunaga, T.; Nakamura, K. Biosci. Biotechnol. Biochem. 1997, 61, 1663. (16) Gimel, J. C.; Durand, D.; Nicolai, T. Macromolecules 1994, 27, 583. (17) Pouzot M.; Nicolai T.; Weijers, M.; Visschers, R. W. Food Hydrocolloids 2005, 19, 231 . (18) Clark, A. H.; Lee-Tuffnell, C. D. Int. J. Pept. Res. 1980, 16, 339. (19) Renard, D.; Axelos, M.; Lefebvre, J. In Food Macromolecules and Colloids; Dickinson, E., Lorient, D., Eds.; Royal Society of Chemistry: Cambridge, 1995; p 390. (20) Weijers, M.; Visschers, R. W.; Cohen Stuart, M. A.; Barneveld, P. A. Colloids Surf. A, in press. (21) Donato, L.; Garnier, C.; Novales, B.; Doublier, J. L. Food Hydrocolloids 2005, 19, 549. (22) Donato, L.; Garnier, C.; Novales, B.; Durand, S.; Doublier, J. L. Biomacromolecules 2005, 6, 374.

BM050132Q