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Gelation by phase separation in a whey protein system: in-situ kinetics of aggregation
Journal of Biotechnology 79 (2000) 231 – 244 www.elsevier.com/locate/jbiotec
Gelation by phase separation in a whey protein system: in-situ kinetics of aggregation D. Renard a,*, P. Robert a, C. Garnier a, E. Dufour b, J. Lefebvre a a
Unite´ de Physico-Chimie des Macromole´cules, Centre de Recherches INRA, Rue de la Ge´raudie`re, BP 71627 -44316 Nantes Cedex 3, France b De´partement Qualite´ et E´conomie Alimentaires, ENITA Clermont Ferrand-63370 Lempdes, France Received 29 July 1999; received in revised form 22 November 1999; accepted 25 November 1999
Abstract The aggregation and gelation properties of b-lactoglobulin (BLG), a globular protein from milk, was studied in aqueous ethanol solutions at room temperature. The phase state diagrams as a function of pH and ethanol concentration showed that a gel structure appeared after a period ranging from 1 min to 1 week, depending on the physico-chemical conditions. The in-situ kinetics of aggregation were followed by several methods in order to obtain a better understanding of the building of aggregates by the addition of ethanol. It was shown that the aggregation kinetics highly depended upon the pH, the process being fastest at pH 7. Viscoelasticity and infrared measurements indicated that alcohol-induced gelation would proceed via a two-step mechanism: small aggregates loosely connected between them were first built up; a real network took place in a second step. The coarse and irregular structures formed in aqueous ethanol gels revealed by confocal laser scanning microscopy could be analysed in terms of a phase separation. This observation was supported by a syneresis phenomenon visible in the final gel state. BLG in water –ethanol solution would undergo either an inhibition of the demixing by gelation or a binary phase separation accompanied by an irreversible gelation transition. © 2000 Elsevier Science B.V. All rights reserved. Keywords: b-Lactoglobulin; Kinetics; Gelation; Phase separation
1. Introduction In certain physico-chemical conditions, the native structure of a protein may undergo conformational changes such as denaturation and dissociation. Above a critical protein concentra* Corresponding author. Tel.: +33-2-40675052; fax: + 332-40675043. E-mail address: [email protected] (D. Renard)
tion, changes in the structure can lead to aggregation and coagulation/gelation. Numerous authors have investigated the gelation of globular proteins induced by a rise in temperature. The thermal denaturation of b-lactoglobulin (BLG) is characterized by changes in the secondary and tertiary structures exposing hydrophobic residues to the solvent. An aggregation process due to hydrophobic interactions may subsequently take place (Cairoli et al., 1994; Arai et al., 1998; Renard et al., 1998).
0168-1656/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 5 6 ( 0 0 ) 0 0 2 4 0 - 6
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Various methods, including microscopy (Stading et al., 1992), light scattering (LS) (Griffin and Griffin, 1993; Griffin et al., 1993; Gimel et al., 1994) small angle neutron scattering (SANS) (Gimel et al., 1994; Renard et al., 1996a) and infrared spectroscopy (Casal et al., 1988; Boye et al., 1995) have been extensively used to investigate the structure of heat-induced protein aggregates and gels. Depending on the protein charge and the screening of this charge (pH/ionic strength effects), the microstructure of BLG gels may vary from reasonably homogeneous and transparent to turbid and opalescent systems. The corresponding microstructures revealed by electron microscopy and SANS varied from fine-stranded aggregates arranged as ‘string-of beads’ through networks showing only periodic fluctuations in network density to substantially collapsed, or completely phase-separated structures (Stading et al., 1992; Renard, 1994; Clark, 1995; Renard et al., 1996a). Aggregation and gelation of BLG have been studied at the molecular level using infrared spectroscopy. The results obtained indicate that aggregation and gelation are accompanied by the formation of intermolecular hydrogen-bonded anti-parallel b-sheet structures (Casal et al., 1988; Boye et al., 1995). The temperature at which protein denaturation, precipitation, aggregation and gelation occur, as well as the final structure of a gel, are affected by the addition of solute. Elyse´e-Collen and Lencki (1996a,b) investigated the effects of ethanol and ammonium sulfate on the formation of heat-induced gelatin and ovalbumin gels. The phase diagrams showed that the systems displayed different morphologies, depending on the experimental conditions. The addition of a weak protic solvent such as alcohol to proteinaceous solutions changes the bulk dielectric constant of the medium, the solvent – solute interactions, the organization of the hydrophobic moieties in the globulin core and, therefore, has a broad effect on protein conformation (Thomas and Dill, 1993). X-ray crystallography indicates that BLG in its native state is mainly composed of nine anti-parallel b-strands, arranged to form a barrel, and one a-helix (Papiz et al., 1986; Monaco et al., 1987). In solution, various oligomeric states exist de-
pending on pH, ionic strength, temperature and protein concentration (Timasheff and Townend, 1964; Zimmerman et al., 1970; Aymard et al., 1996). BLG is essentially monomeric below pH 3 and at low salt concentration while it is dimeric at neutral pH, irrespective the salt concentration is. A reversible aggregation of BLG resulting in the formation of octamers has been observed at low temperature (4°C) and at pH values ranging from 3.7 to 5.2 (Timasheff and Townend, 1964; Zimmerman et al., 1970). At pH 7.5, the Tanford transition occurs, which is characterized by a change in the tyrosine environment, an increase in the reactivity of a free sulfydryl group and a release of a carboxyl group (Tanford et al., 1959; Qin et al., 1998). The effect of alcohol on structural changes of BLG in dilute solutions (micromolar range) has been extensively studied using spectroscopic methods (Townend et al., 1967; Dufour and Haertle´, 1990; Dufour et al., 1993, 1994a,b; Ragona et al., 1999). Over a wide pH range, the protein predominantly adopts an a-helical conformation if the dielectric constant of the solvent is low (Dufour et al., 1993, 1994a). Indeed, the reversible b-strand/ a-helix transition induced by alcohol involves at least three conformational states: native protein, intermediate state and a-helix shaped BLG (Dufour et al., 1994b). The intermediate state observed in 20% (v/v) ethanol by fluorescence and circular dichroism measurements was identified as being a «molten globule» state (Dufour and Haertle´, 1990; Dufour et al., 1994a). However, there is actually no clear evidence whether it concerns a hierarchical protein folding model, as described above, or a non-hierarchical model, where more than two species could exist at a given ethanol concentration (Shiraki et al., 1995). The b-strand/a-helix transition of BLG at high protein concentration (millimolar range) in water–ethanol solutions has also been studied by infrared spectroscopy (Dufour et al., 1994b). Millimolar solutions of BLG in 50% (v/v) ethanol show a higher a-helix content at pH 8 than at pH 7. In addition, BLG may aggregate and gel at room temperature in aqueous ethanol solutions depending on the pH and the protein concentration (Dufour et al., 1998).
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While the BLG conformational changes induced by various environmental conditions have been extensively characterized at low protein concentration, few studies deal with changes of this protein in the millimolar concentration range. Through the data obtained on BLG gels in water – ethanol solutions at the mesoscopic and macroscopic levels, it was concluded that the spatial arrangements of the aggregates were similar in the final structures whatever the pH (Renard et al., 1999). It therefore seems useful to investigate the detailed mechanisms that control aggregation and gelation of BLG. In the present paper, the in-situ kinetics of BLG aggregation in water – ethanol solution were studied at 25°C by using rheological measurements, confocal laser scanning microscopy (CLSM), light and small angle neutron scattering (SANS) and Fourier transform infrared spectroscopy, in order to get a better understanding of the building of aggregates. The final structure of the gels at mesoscopic and macroscopic scales was observed using SANS and CLSM.
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solubility of the material. The undissolved material was, therefore, removed by centrifugation at 16 000× g for 40 min. The concentration of protein stock solutions was determined from the optical density at 278 nm (corrected for turbidity) using for the specific absorption coefficient A 1% 1 cm= 9.6 (Townend et al., 1960).
2.3. Dynamical rheological measurements
2. Material and methods
Small amplitude oscillatory shear experiments were performed at 25°C on a Carri-Med CS 50 rheometer using the cone-plate geometry (cone diameter 4 cm; angle 3.58°). Frequency sweep of BLG gel at pH 2 (2.56% (w/v)) in 50% (v/v) ethanol solution was recorded between 10 − 2 and 10 Hz under a strain amplitude of 0.03. The viscoelastic response was linear up to a strain amplitude value of 0.08. The aggregation/gelation kinetics of BLG (2.56% (w/v)) in 50% (v/v) ethanol solution were studied for a period of 80 h at pH 7, 8 and 9 using a frequency of 1 Hz and a strain amplitude of 0.01. The strain amplitude was chosen to prevent any disruption of the network build-up. Each experiment was conducted in triplicate.
2.1. Material
2.4. Infrared spectroscopy
The BLG sample used was kindly provided by Lactalis (Retiers, France). It was prepared from sweet or mixed whey by a-lactalbumin precipitation at acidic pH, ion-exchange chromatography of the residual whey, ultrafiltration and spray-drying (Rialland and Barbier, 1988). Reversed-phase HPLC indicated that BLG purity was 94% and that a-lactalbumin (3%) and bovine serum albumin (2%) were the main protein contaminants.
The infrared spectra were recorded between 1595 and 1720 cm − 1 at 2 cm − 1 intervals on a Fourier transform spectrometer IFS25 (Brucker). The attenuated total reflection (ATR) cell used in this study was made of a ZnSe crystal allowing six internal reflections (SPECAC). The cell was thermostatted and all experiments were performed at 25°C. A total of 200 scans were collected and averaged for both background and sample measurements. Since water strongly absorbs in the amide I region, the backgrounds were obtained using aqueous ethanol solutions (50% (v/v) ethanol). In this way, autosubtraction of the solvent spectrum was achieved (Dufour et al., 1994b). The aggregation/gelation kinetics performed in 50% (v/v) ethanol solutions were studied at three pH values (7, 8 and 9) for 10 h, the protein concentration being 2.56% (w/v). While the first
2.2. Protein solutions The protein was dissolved in water (stock solution: 10% w/w) and the pH adjusted in the 2 – 9 range with NaOH or HCl solutions (0.1 or 1 M). Sodium azide was added (0.2% (w/v)) to the protein solutions in order to prevent bacterial growth. The solutions were stirred overnight to ensure both complete hydration and maximum
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spectrum was recorded 3 min after the beginning of the kinetics, the following spectra were collected every 5 min for 6.5 h, and every 20 min until 10 h. The experiments were performed in triplicate. A baseline correction of the spectra was achieved using the SPECTRAFILE software (Heyden & Son, GMBH). Second-derivative spectra were assessed by applying the finite-difference method. The second-derivative data multiplied by −1 were thereafter normalized so that the area under each spectrum was constant.
their final state after an equilibrium period of 8 days. The data were normalized for transmission and sample path length and divided by the water spectrum. An absolute intensity scale was obtained using the absolute value of water intensity in units of cross-section (cm − 1). A subtraction of the appropriate incoherent background, taking 80% H/D exchange on the protein into account was realized according to Zaccaı¨ and Jacrot (1983). For globular proteins, the scattering intensity at a given wave-vector q can be written as:
2.5. Light scattering
I(q)= AP(q)S(q) or (Nad 2/(Dr 2))I(q)/C =MP(q)S(q)
Static (SLS) and dynamic (DLS) light scattering measurements were performed using an ALV-5000 multi-bit, multi-tau correlator in combination with a Malvern goniometer and a Spectra-Physics laser emitting vertically polarised light at 514.5 nm. Measurements on BLG (0.2 – 0.9% (w/v)) at pH 8 in 50% (v/v) ethanol solutions were done at u = 90° to minimise the influence of spurious scattering. The correlation functions were analysed using the inverse Laplace transform routine REPES (Stepanek, 1993) in order to obtain the corresponding relaxation time distribution (and hydrodynamic radii distribution). The measurement temperature was fixed at 25°C and controlled within 0.1°C.
P(q) is the «form factor» of the molecule and S(q) the «structure factor» which accounts for intermolecular interactions. The quantity A is a parameter that includes the concentration C, the mass M, the density d, Avogadro’s number Na, and the neutron contrast (Dr)2. The neutron contrast was assessed by taking the scattering length densities of the solvent (50/50% (v/v) D2O/C2D6O solvent) into account. The scattering intensity I(q) was finally expressed in terms of molecular mass, as the density (d=1.332 g cm − 3) and the contrast variation (Dr = 5.55 · 1010 cm − 2) of BLG in a 50/50% (v/v) D2O/C2D6O solution are known.
2.6. Small-angle neutron scattering
2.7. Confocal laser scanning microscopy (CLSM)
Small-angle neutron scattering (SANS) experiments were performed at the ILL (Grenoble, France) using the D22 multidetector instrument. The spectra for BLG solutions at pH 7 (C = 1.79% (w/v)) and pH 8 (C = 2.56% (w/v)) in 50% (v/v) ethanol solutions were recorded every 5 min during 8 h for each sample using a spectrometer configuration l =10 A, (incident wavelength) and d=14 m (distance of the sample to the detector). The range of wave-vector q = 4p/l sin(u/2), u being the scattering angle, thus covered was: 2.35 · 10 − 2 – 4.4 · 10 − 1 nm − 1. The gels formed at pH 2, 7, 8 and 9 in 50% (v/v) ethanol solutions and those formed at pH 7 for various ethanol concentrations were also characterized in
BLG solution at pH 7 C= 3.75% (w/v) in 50% (v/v) ethanol solution was prepared as described above; the protein was stained with Rhodamine IsoThioCyanate (0.25 mg g − 1 protein). Solutions were put inside cavity slides of 0.5 mm diameter. To prevent any evaporation, the slides were covered with glass coverslips and sealed. CSLM observations were performed using a Zeiss LSM 410 Axiovert microscope. The excitation wavelength was adjusted to that of RITC at 543 nm, and the emission above 570 nm was recorded. Pictures were taken every 5 min during 48 h with a water-immersed × 40 objective at a depth of 25 mm beneath the coverslip surface.
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3. Results and discussion
3.1. Static properties of b-lactoglobulin gels in water –ethanol solution Fig. 1 gives an overview of the different morphologies, obtained for b-lactoglobulin (BLG) solutions at pH 8 in 50% (v/v) ethanol concentration, depending on the protein concentration. At low protein concentrations (tube 1: 0.1% w/v; tube 2: 1% w/v), transparent liquids were obtained. While a viscous solution corre-
Fig. 1. b-Lactoglobulin gel morphologies formed at pH 8 in 50/50% (v/v) water – ethanol solutions (observations made after 8 days). 1, liquid (0.1% w/v); 2, liquid (1% w/v); 3, pre-gel state (2% w/v); 4, gel (3% w/v); 5, turbid gel (4% w/v); 6, collapsed gel (after syneresis).
Fig. 2. Frequency sweep at 25°C for b-lactoglobulin gel formed at pH 2 in 50/50% (v/v) water–ethanol solution (C= 2.56%, w/v), g0 = 0.03. , G% (Pa); , G%% (Pa).
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sponding to a pre-gel state was observed at 2% (w/v) concentration (tube 3), a gel really developed at 3% (w/v) concentration (tube 4). The turbidity in gelled samples increased with protein concentration (tube 5, 4% w/v), pH or ethanol concentration. In addition, a syneresis phenomenon is observed at high protein concentration. The tube 6 shows a collapsed gel state after completion of syneresis where the gel diameter is reduced in such a way that there is no more adhesion to the tube walls. The expelled liquid analysed by refractometry, was identified as being a 50/50% (v/v) water–ethanol mixture corresponding to the solvent used. Infra-red spectroscopy revealed that BLG monomers in a-helical form were also present in the expelled liquid. Phase state diagrams of BLG in water–ethanol solutions established as a function of pH and ethanol concentration revealed that the equilibrium gel state was reached in a time scale ranging from 1 min to 1 week, depending on the physico-chemical conditions (Dufour et al., 1998). With decreasing protein concentration, the critical gelation time became longer. Moreover, the critical gelation time, at a given protein concentration, shifted towards higher values as the pH of the solution decreased from 9 to 2 (Renard et al., 1999). The increase of the negative net charge on the protein would favour electrostatic double layer repulsion and slow down the aggregation process of BLG molecules. The critical gelation time at a fixed pH and protein concentration was highest at low ethanol concentration. Moreover, increase of protein concentration resulted in a large increase of turbidity, characteristic of a coarser network structure, whatever the ethanol concentration used. The mechanical properties of the resulting gels were investigated through dynamical rheological measurements. A typical example of a frequency sweep under 3% strain amplitude is shown Fig. 2 for a gel formed at pH 2. Whatever the pH, the signal always corresponds to a viscoelastic material with a slight dependence on the frequency of the storage and loss moduli G% and G%%. This rheological behaviour is characteristic of a weak network structure. However, long-time response by means of a
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Fig. 3. Small angle neutron scattering profiles I (g mol − 1) versus q (nm − 1) on a log-log scale for b-lactoglobulin gels formed in 50/50% (v/v) water–ethanol solution; the slope delimits the fractal regime of the structures. , pH 2, C= 4.45%; D, pH 7, C=2.56%; , pH 8, C= 3.03%; , pH 9, C= 3.02% (w/v).
creep-recovery test displayed a liquid-like type viscoelastic behaviour typical of a transient network (results not shown). The liquid-like behaviour of these gels means that the connections between the structural elements of the network are not permanent at the time scale of the experiments. This result agrees with observations made on both heat-set BLG and bovine serum albumin gels where, in all cases, a small but definite irrecoverable strain at the end of the recovery test was observed (Renard et al., 1996b; Lefebvre et al., 1998). The structure of BLG aggregates in the gels was investigated at a mesoscopic scale through small angle neutron scattering (SANS) experiments. This technique allows the determination of shape (1 – 100 nm scale) in self-assembly structures and to precise the interactions between the structures, even in turbid samples. The scattering intensity as a function of the wave-vector q is displayed in Fig. 3 for BLG gels formed at four pH values. The curves are quite identical at pH 7, 8 and 9
and reveal similarities with those obtained at the same pH values for heat-set BLG gels formed at high ionic strength (Renard et al., 1999). The similar intensity values observed at q higher than 1 nm − 1 suggest that the elementary subunit constitutive of the aggregates is the same. The shape in the intermediate q range indicates that compact structures are formed when aggregation takes place in water–ethanol solvent. On the contrary, the structure of the aggregates formed at pH 2 is completely different. A fractal regime at low q values seems to appear with an apparent fractal dimensionality value of about 1.3 characteristic of more or less linear aggregates with slight branching. Moreover, a correlation peak with a maximum in intensity at q* appears in the intermediate q range giving a mean inter-aggregate distance of 23 nm (2p/q*). These observations indicate that stiff charged aggregates are formed and that preferential distances are maintained between these structures giving rise to a correlation peak at the SANS scale.
Fig. 4. Small angle neutron scattering profiles I (g mol − 1) versus q (nm − 1) on a log – log scale for b-lactoglobulin gels at pH 7. , 70/30% (v/v) ethanol – water, C =4.63%; , 60/40% (v/v) ethanol – water, C =3.53%; D, 50/50% (v/v) ethanol/water, C =2.56% (w/v).
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Fig. 5. Second-derivative infrared spectra of b-lactoglobulin pH 8 C =2.56% (w/v) in 50/50% (v/v) water – ethanol solution as a function of time. – – , 3 min; ---, 28 min; …, 298 min. The second derivative data are multiplied by −1.
Fig. 4 displays the scattering intensities obtained for BLG gels at pH 7 as a function of ethanol percentage. The curves are very similar for ethanol concentrations between 50 and 70% (v/v). The shape of the curves confirms the formation of large and compact structures, the compactness being maximal in 50% (v/v) ethanol concentration. Considering the results obtained at different observation scales as reported above, we decided to investigate the build-up of these structures leading to a network as a function of time, in order to clarify the mechanisms that control aggregation and gelation in these systems.
3.2. In-situ kinetics of aggregation of b-lactoglobulin in water– ethanol solution Aggregation of BLG (2.56% (w/v)) at pH 8 in 50% (v/v) ethanol concentration was first studied at the molecular level using Fourier transformed infrared spectroscopy (FTIR). The second derivative spectra recorded at various times are given in Fig. 5. After 3 min, the BLG spectrum exhibits a peak at about 1650 cm − 1 and a shoulder at
around 1620 cm − 1. The band at 1650 cm − 1 is characteristic of a-helices appearing with the addition of ethanol. A time dependent decrease of the band at 1650 cm − 1 accompanied by an increase of the peak at 1620 cm − 1 is observed. The development of the band at 1620 cm − 1 may be assigned to b-strands exposed to the solvent or to the formation of intermolecular H-bonded bsheets. In addition, it was observed that the absorption band assigned to a-helix structures shifts from 1650 to 1660 cm − 1 during aggregation. Novskaya and Chirgadze (1976) reported that the frequency of a-helices steadily rises when the number of amino-acid residues involved in a-helical structures decreases. The in-situ kinetics of aggregation of BLG in water–ethanol solvent were assessed by measuring the integrated intensity of the normalized second derivative absorption band at 1620 cm − 1. Fig. 6 displays different kinetics obtained for three BLG concentrations in 50% (v/v) ethanol concentration. The intermolecular b-sheet structures quickly increased and reached a plateau at about 200 min. The initial rate seems to increase
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Fig. 6. Integrated intensity at 1620 cm − 1 as a function of time for b-lactoglobulin pH 8 in 50/50% (v/v) water–ethanol solution. , C =1%; , C=1.5%; , C= 2.56% (w/v).
with BLG concentration. Moreover, one can observe an increase in the final formation of the aggregates by increasing BLG concentration. At the macroscopic level, the viscoelasticity of the networks formed at various pH values by
adding ethanol was followed by measuring the storage and loss moduli G% and G%% at a fixed frequency of 1 Hz under 1% of strain amplitude (Fig. 7). The progress curves were obtained over a period of 80 h. The gelation kinetics greatly depended on the pH value of the solution. An increase in the net charge on the protein would tend to slow down the kinetic process as it will increase repulsive barriers between charged molecules. Consequently, one expect that moduli recorded at pH 9 would be lower than those observed at pH 7 and 8. Our results are not in agreement with this hypothesis, presumably because a slow denaturation that is known to occur at pH 9 promotes the aggregation process. Nevertheless, G%/G%% ratio increased with increasing pH of the solution. Equilibrium values for both G% and G%% moduli were reached after 80 h except in the case where syneresis after gelation occurred. The protein concentration dependence on the moduli G% and G%% was considerably high. In particular, the viscoelastic parameters diverged near the ‘isolated aggregates – connected aggregates’ transition (threshold located around the critical concentration for gelation). This result
Fig. 7. Gelation kinetics of b-lactoglobulin (C= 2.56% (w/v)) in 50/50% (v/v) water – ethanol solution; evolution of the storage modulus G% (filled symbols) and the loss modulus G%% (empty symbols) as a function of time (h) at 25°C; f= 1 Hz, g0 =0.01. , pH 7; , pH 8; , pH 9.
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Fig. 8. Changes of the secondary structure (A1620/A1650 ratio) (symbol) of b-lactoglobulin C= 2.25% (w/v) at pH 7 in 50/ 50% (v/v) water – ethanol solution as a function of time between 0 and 600 min. Comparison with the evolution of the storage modulus G% (Pa) (line) during the kinetic carried out at the same pH value.
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was previously observed on heat-set BLG gels where two regimes took place: one regime with no concentration dependence of the elastic modulus G% (isolated aggregates), a second regime characterized by a concentration dependence of G% (connected aggregates leading to a network) (Renard et al., 1995). In the particular case of BLG in diluted regime (2.25% (w/v)), Fig. 8 shows the evolution time in the first stages of aggregation of the storage modulus G% and of the absorbance ratio A1620/A1650 cm − 1 for alcohol gelation of BLG at pH 7. At this concentration, most of the secondary conformational changes were observed when the storage modulus remained close to zero. Consequently, it is suggested that at room temperature BLG monomers first connected through intermolecular b-sheets leading to small aggregates that did not develop a measurable viscoelastic response. The protein network was built up in a second step after 10 h.
Fig. 9. In-situ kinetics of aggregation followed by static and dynamic light scattering (T= 25°C) of b-lactoglobulin solutions at pH 8 in 50/50% (v/v) water –ethanol solution as a function of protein concentration. (a) Scattering intensity (expressed as the ratio of the scattering intensity of the sample over the scattering intensity of toluene) recorded at a fixed angle u= 90° and T =25°C as a function of time. , C =0.2%; , C= 0.3%; , C= 0.5%; , C = 0.7%; ", C = 0.8%; +, C =0.9% (w/v). (b) Apparent hydrodynamic radii Rh (nm) recorded at a fixed angle u=90° and T= 25°C as a function of time. , C = 0.2%; , C = 0.5% (w/v).
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Fig. 10. In-situ kinetics of gelation followed by small angle neutron scattering of b-lactoglobulin solutions in 50/50% (v/v) water– ethanol solution as a function of pH. The slopes delimit the fractal regime of the structures. (a) Scattering intensity I (g mol − 1) versus q (nm − 1) on a log-log scale for b-lactoglobulin at pH 7, C=1.79% (w/v) recorded as a function of time. , 5 min; , 25 min; , 85 min; , 8 h; ", 192 h. (b) Scattering intensity I (g mol − 1) versus q (nm − 1) on a log-log scale for b-lactoglobulin at pH 8 C =2.56% (w/v) recorded as a function of time. , 5 min; , 25 min; , 50 min; , 80 min; ("), 8 h; ( ), 192 h.
To test this assumption about the intermolecular b-sheet structures developing with time, in-situ light scattering experiments were performed at a fixed angle. Scattered light intensity and hydrodynamic radii were recorded as a function of time during the first stages of aggregation (Fig. 9a, b). On Fig. 9a, the sample to toluene intensities ratio at a fixed protein concentration increased rapidly as a function of time meaning that an increase in the size and/or the number of scattering elements occured. Aggregation went faster as protein concentration increased. On Fig. 9b, the size of the scattering elements (apparent hydrodynamic radii) were measured through dynamic light scattering. Two different populations of scattering elements were observed whatever the BLG concentration (0.2%, 0.5% (w/v)). The two populations exhibit a rapid increase in the first 15 min, followed by a slow and progressive increase. While the size values of the scattering elements after 60 min of aggregation time were 4 and 45 nm for 0.2% (w/v) BLG concentration, they were 8 and 65 nm in the case of 0.5% (w/v) BLG concentration.
The estimated mass ratio between large and small aggregates assessed after 60 min were found to be 5.4 and 1.2 for 0.2 and 0.5% (w/v) protein concentrations, respectively. This mass ratio changed with time and was higher at the beginning of the aggregation process (6.8 and 2.1 for 0.2 and 0.5% (w/v), respectively). In addition, the results indicate that both aggregates molecular mass and size are not proportional to the protein concentration. The internal structure of the aggregates formed at higher protein concentration in water–ethanol solutions was investigated by means of SANS experiments. The in-situ kinetics of aggregation of BLG at pH 7 and 8 in 50% (v/v) ethanol concentration were followed by recording the scattered intensities as a function of the wave-vector q (Fig. 10a,b). The scattered intensities for BLG aggregates at pH 7 (Fig. 10a) displayed a linear dependence with the wave-vector q. The apparent negative slope values assessed for each aggregation time ranged from 1 to 2.8. Similar trends were observed at pH 8 (Fig. 10b), with just a slow
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Fig. 11. In-situ kinetics of gelation followed by confocal laser scanning microscopy (lexc =543 nm) of b-lactoglobulin labelled with RITC at pH 7 C= 3.75% (w/v) in 50/50% (v/v) water–ethanol solution. (a) After 5 min; (b) after 44 h.
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down of the aggregation process, despite the higher protein concentration. The slope values are in agreement with the formation of fractal linear aggregates at the beginning of the process. Branching would develop with time giving higher apparent fractal dimensions. The final structures would be made of highly compact clusters. The intensity dependence of the compact clusters had an apparent slope value much higher than those corresponding to the diffusion or reaction-limited cluster – cluster aggregation models which values are of 1.75 and 2.1, respectively (Kolb et al., 1983; Meakin, 1983; Kolb and Jullien, 1984; Brown and Ball, 1985). From the above observations, a question arose: did we have such a large polydispersity in the heterogeneous networks that slope values would not reflect a fractal regime in the aggregates structure? To answer the question, in-situ kinetics of BLG gelation in 50% (v/v) ethanol concentration were studied using confocal laser scanning microscopy (CLSM). Fig. 11a and b show micrographs of BLG gels (3.75% (w/v)) formed at pH 7 after 5 min and 44 h. Fluorescent proteinrich domains appear light on the picture while protein-poor phases are dark (Fig. 11a). While initial structures corresponding to dense and polydisperse clusters appeared in the first minutes, with many very small aggregates and few very large ones, an overall connected morphology of the protein-rich phase seems to have formed in a second step (Fig. 11b). The coexistence of dense clusters included in a network in the final state would suggest that competition between gelation and phase separation would occur leading to a non-equilibrium gel state. Demixing would be caused by less favorable interactions between the solvent and BLG in its denatured form (a-helical form). Bansil et al. (1992) studying gelatin gelation in water–methanol solution by optical microscopy revealed similar structures and concluded to the existence of a phase separation process due to spinodal decomposition. In addition, they found that the kinetics of phase separation depended very strongly on temperature: for deep
quenches, where the rates of phase separation and gelation were comparable, the phase-separation process did not go to completion. San Biagio et al. (1999) recently reported that bovine serum albumin coagulation proceeds through three simultaneous processes: conformational change of the protein, mesoscopic demixing of the solution and protein cross-linking. Several parameters such as temperature (T= 30–70°C for 10 min) or dilution (1:1 ratio) on BLG aggregated diluted systems were tested to elucidate the reversible character of the BLG aggregation in water–ethanol solution. No reversible transition of the aggregation was observed even after 24 h, meaning that high energy levels were implied in the inter-aggregate bonds.
4. Conclusion From the molten globule and helix-shaped BLG forms induced in water–ethanol solvent, aggregation may take place below a critical protein concentration leading to linear fractal aggregates during the first stages of the process. These small aggregates that appear loosely connected to each other further develop with time and lead more or less rapidly to a phase-separated network. From the coarse and irregular structures formed, it is postulated that BLG in water–ethanol solution would undergo a frustration of demixion by gelation or would undergo a simultaneous binary phase separation and irreversible gelation transition. As in general, the system does not fully separated into two phases, the early stage characteristic of the spinodal decomposition is ‘pinned’ owing to the onset of gelation. Recent work on heat-set globular protein gelation resulted in similar conclusions (San Biagio et al., 1996; Tobitani and RossMurphy, 1997). If this process of aggregation/ demixing would be adjustable through variation of gelling conditions, potentially it would lead to almost infinite possibilities for BLG gel structure and biopolymeric networks in general.
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References Arai, M., Ikura, T., Semisotnov, G.V., Kihara, H., Amemiya, Y., Kuwajima, K., 1998. Kinetic refolding of b-lactoglobulin. Studies by synchrotron X-ray scattering, and circular dichroism, absorption and fluorescence spectroscopy. J. Mol. Biol. 275, 149 – 162. Aymard, P., Durand, D., Nicolai, T., 1996. The effect of temperature and ionic strength on the dimerisation of b-lactoglobulin. Int. J. Biol. Macromol. 19, 213–221. Bansil, R., Lal, J., Carvalho, B.L., 1992. Effects of gelation on spinodal decomposition kinetics in gelatin. Polymer 33, 2961 – 2969. Boye, J.I., Alli, I., Ismail, A.A., Gibbs, B.F., Konishi, Y., 1995. Factors affecting molecular characteristics of whey protein gelation. Int. Dairy J. 5, 337–353. Brown, W., Ball, R., 1985. Computer simulation of chemically limited aggregation. J. Phys. A 18, L517. Cairoli, S., Iametti, S., Bonomi, F., 1994. Reversible and irreversible modifications of b-lactoglobulin upon exposure to heat. J. Protein Chem. 13, 347–354. Casal, H.L., Kohler, U., Mantsch, H.H., 1988. Structural and conformational changes of b-lactoglobulin B: an infrared spectroscopy study of the effect of pH and temperature. Biochim. Biophys. Acta 957, 11–20. Clark, A.H., 1995. Kinetics of demixing. In: Harding, S.E., Hill, S.E., Mitchell, J.R. (Eds.), Biopolymer Mixtures. Nottingham University Press, Nottingham, UK, pp. 37– 64. Dufour, E., Haertle´, T., 1990. Alcohol-induced changes of b-lactoglobulin– retinol-binding stoichiometry. Protein Eng. 4, 185 – 190. Dufour, E., Bertrand – Harb, C., Haertle´, T., 1993. Reversible effects of medium dielectric constant on structural transformation of b-lactoglobulin and its retinol binding. Biopolymers 33, 589 – 598. Dufour, E., Genot, C., Haertle´, T., 1994a. b-Lactoglobulin binding properties during its folding changes studied by fluorescence spectroscopy. Biochim. Biophys. Acta 1205, 105 – 112. Dufour, E., Robert, P., Bertrand, D., Haertle´, T., 1994b. Conformation changes of b-lactoglobulin: an ATR infrared spectroscopic study of the effect of pH and ethanol. J. Protein Chem. 13, 143–149. Dufour, E., Robert, P., Renard, D., Llamas, G., 1998. Investigation of b-lactoglobulin gelation in water–ethanol solutions. Int. Dairy J. 8, 87–93. Elyse´e-Collen, B., Lencki, R.W., 1996a. Protein ternary phase diagrams. 1. Effect of ethanol, ammonium sulfate, and temperature on the phase behavior of type B gelatin. J. Agric. Food Chem. 44, 1651–1657. Elyse´e-Collen, B., Lencki, R.W., 1996b. Protein ternary phase diagrams. 2. Effect of ethanol, ammonium sulfate, and temperature on the phase behavior of (S)-ovalbumin. J. Agric. Food Chem. 44, 1658–1663. Gimel, J.-C., Durand, D., Nicolai, T., 1994. Structure and distribution of aggregates formed after heat-induced denaturation of globular proteins. Macromolecules 27, 583–589.
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b-lactoglobulin revealed by intrinsic fluorescence studies. Int. J. Biol. Macromol. 22, 41–49. Renard, D., Lefebvre, J., Robert, P., Llamas, G., Dufour, E., 1999. Structural investigation of b-lactoglobulin gelation in water – ethanol solutions. Int. J. Biol. Macromol. 26, 35–44. Rialland, J.P., Barbier, J.P., 1988. Process for selectively separating the alpha-lactalbumin from the proteins of whey. US Patent 4, 782, 138. San Biagio, P.L., Bulone, D., Emanuele, A., Palma, M.U., 1996. Self-assembly of biopolymeric structures below the threshold of random cross-link percolation. Biophys. J. 70, 494 – 499. San Biagio, P.L., Martorana, V., Emanuele, A., et al., 1999. Interacting processes in protein coagulation. Proteins: Struct. Funct. Genet. 37, 116–120. Shiraki, K., Nishikawa, K., Goto, Y., 1995. Trifluoroethanolinduced stabilization of the a-helical structure of b-lactoglobulin: implication for non-hierarchical protein folding. J. Mol. Biol. 245, 180–194. Stading, M., Langton, M., Hermansson, A.M., 1992. Inhomogeneous fine-stranded b-lactoglobulin gels. Food Hydrocolloids 5, 455 – 470. Stepanek, P., 1993. Data analysis in dynamic light scattering. In: Brown, W. (Ed.), Dynamic Light Scattering. The Methods and Some Applications. Clarendon Press, Oxford, UK, pp. 177 – 241.
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Tanford, C., Bunville, L.G., Nozaki, Y., 1959. The reversible transformation of b-lactoglobulin at pH 7.5. J. Am. Chem. Soc. 81, 4032 – 4036. Thomas, P.D., Dill, K.A., 1993. Local and nonlocal interactions in globular proteins and mechanisms of alcohol denaturation. Protein Sci. 2, 2050 – 2065. Timasheff, S.N., Townend, R., 1964. Structure of the b-lactoglobulin tetramer. Nature 203, 517 – 519. Tobitani, A., Ross-Murphy, S.B., 1997. Heat-induced gelation of globular proteins. 1. Model for the effects of time and temperature on the gelation time of BSA gels. Macromolecules 30, 4845 – 4854. Townend, R., Winterbottom, R.J., Timasheff, S.N., 1960. Molecular interactions in b-lactoglobulin. II. Ultracentrifugal and electrophoretic studies of the association of b-lactoglobulin below its isoelectric point. J. Am. Chem. Soc. 82, 3161 – 3168. Townend, R., Kumosinski, T.F., Timasheff, S.N., 1967. The circular dichroism of variants of b-lactoglobulin. J. Biol. Chem. 242, 4538 – 4545. Zaccaı¨, G., Jacrot, B., 1983. Small angle neutron scattering. Ann. Rev. Biophys. Bioeng. 12, 139 – 157. Zimmerman, J.K., Barlow, G.H., Klotz, I.M., 1970. Dissociation of b-lactoglobulin near neutral pH. Arch. Biochem. Biophys. 138, 101 – 109.
Gelation by phase separation in a whey protein system: in-situ kinetics of aggregation D. Renard a,*, P. Robert a, C. Garnier a, E. Dufour b, J. Lefebvre a a
Unite´ de Physico-Chimie des Macromole´cules, Centre de Recherches INRA, Rue de la Ge´raudie`re, BP 71627 -44316 Nantes Cedex 3, France b De´partement Qualite´ et E´conomie Alimentaires, ENITA Clermont Ferrand-63370 Lempdes, France Received 29 July 1999; received in revised form 22 November 1999; accepted 25 November 1999
Abstract The aggregation and gelation properties of b-lactoglobulin (BLG), a globular protein from milk, was studied in aqueous ethanol solutions at room temperature. The phase state diagrams as a function of pH and ethanol concentration showed that a gel structure appeared after a period ranging from 1 min to 1 week, depending on the physico-chemical conditions. The in-situ kinetics of aggregation were followed by several methods in order to obtain a better understanding of the building of aggregates by the addition of ethanol. It was shown that the aggregation kinetics highly depended upon the pH, the process being fastest at pH 7. Viscoelasticity and infrared measurements indicated that alcohol-induced gelation would proceed via a two-step mechanism: small aggregates loosely connected between them were first built up; a real network took place in a second step. The coarse and irregular structures formed in aqueous ethanol gels revealed by confocal laser scanning microscopy could be analysed in terms of a phase separation. This observation was supported by a syneresis phenomenon visible in the final gel state. BLG in water –ethanol solution would undergo either an inhibition of the demixing by gelation or a binary phase separation accompanied by an irreversible gelation transition. © 2000 Elsevier Science B.V. All rights reserved. Keywords: b-Lactoglobulin; Kinetics; Gelation; Phase separation
1. Introduction In certain physico-chemical conditions, the native structure of a protein may undergo conformational changes such as denaturation and dissociation. Above a critical protein concentra* Corresponding author. Tel.: +33-2-40675052; fax: + 332-40675043. E-mail address: [email protected] (D. Renard)
tion, changes in the structure can lead to aggregation and coagulation/gelation. Numerous authors have investigated the gelation of globular proteins induced by a rise in temperature. The thermal denaturation of b-lactoglobulin (BLG) is characterized by changes in the secondary and tertiary structures exposing hydrophobic residues to the solvent. An aggregation process due to hydrophobic interactions may subsequently take place (Cairoli et al., 1994; Arai et al., 1998; Renard et al., 1998).
0168-1656/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 5 6 ( 0 0 ) 0 0 2 4 0 - 6
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Various methods, including microscopy (Stading et al., 1992), light scattering (LS) (Griffin and Griffin, 1993; Griffin et al., 1993; Gimel et al., 1994) small angle neutron scattering (SANS) (Gimel et al., 1994; Renard et al., 1996a) and infrared spectroscopy (Casal et al., 1988; Boye et al., 1995) have been extensively used to investigate the structure of heat-induced protein aggregates and gels. Depending on the protein charge and the screening of this charge (pH/ionic strength effects), the microstructure of BLG gels may vary from reasonably homogeneous and transparent to turbid and opalescent systems. The corresponding microstructures revealed by electron microscopy and SANS varied from fine-stranded aggregates arranged as ‘string-of beads’ through networks showing only periodic fluctuations in network density to substantially collapsed, or completely phase-separated structures (Stading et al., 1992; Renard, 1994; Clark, 1995; Renard et al., 1996a). Aggregation and gelation of BLG have been studied at the molecular level using infrared spectroscopy. The results obtained indicate that aggregation and gelation are accompanied by the formation of intermolecular hydrogen-bonded anti-parallel b-sheet structures (Casal et al., 1988; Boye et al., 1995). The temperature at which protein denaturation, precipitation, aggregation and gelation occur, as well as the final structure of a gel, are affected by the addition of solute. Elyse´e-Collen and Lencki (1996a,b) investigated the effects of ethanol and ammonium sulfate on the formation of heat-induced gelatin and ovalbumin gels. The phase diagrams showed that the systems displayed different morphologies, depending on the experimental conditions. The addition of a weak protic solvent such as alcohol to proteinaceous solutions changes the bulk dielectric constant of the medium, the solvent – solute interactions, the organization of the hydrophobic moieties in the globulin core and, therefore, has a broad effect on protein conformation (Thomas and Dill, 1993). X-ray crystallography indicates that BLG in its native state is mainly composed of nine anti-parallel b-strands, arranged to form a barrel, and one a-helix (Papiz et al., 1986; Monaco et al., 1987). In solution, various oligomeric states exist de-
pending on pH, ionic strength, temperature and protein concentration (Timasheff and Townend, 1964; Zimmerman et al., 1970; Aymard et al., 1996). BLG is essentially monomeric below pH 3 and at low salt concentration while it is dimeric at neutral pH, irrespective the salt concentration is. A reversible aggregation of BLG resulting in the formation of octamers has been observed at low temperature (4°C) and at pH values ranging from 3.7 to 5.2 (Timasheff and Townend, 1964; Zimmerman et al., 1970). At pH 7.5, the Tanford transition occurs, which is characterized by a change in the tyrosine environment, an increase in the reactivity of a free sulfydryl group and a release of a carboxyl group (Tanford et al., 1959; Qin et al., 1998). The effect of alcohol on structural changes of BLG in dilute solutions (micromolar range) has been extensively studied using spectroscopic methods (Townend et al., 1967; Dufour and Haertle´, 1990; Dufour et al., 1993, 1994a,b; Ragona et al., 1999). Over a wide pH range, the protein predominantly adopts an a-helical conformation if the dielectric constant of the solvent is low (Dufour et al., 1993, 1994a). Indeed, the reversible b-strand/ a-helix transition induced by alcohol involves at least three conformational states: native protein, intermediate state and a-helix shaped BLG (Dufour et al., 1994b). The intermediate state observed in 20% (v/v) ethanol by fluorescence and circular dichroism measurements was identified as being a «molten globule» state (Dufour and Haertle´, 1990; Dufour et al., 1994a). However, there is actually no clear evidence whether it concerns a hierarchical protein folding model, as described above, or a non-hierarchical model, where more than two species could exist at a given ethanol concentration (Shiraki et al., 1995). The b-strand/a-helix transition of BLG at high protein concentration (millimolar range) in water–ethanol solutions has also been studied by infrared spectroscopy (Dufour et al., 1994b). Millimolar solutions of BLG in 50% (v/v) ethanol show a higher a-helix content at pH 8 than at pH 7. In addition, BLG may aggregate and gel at room temperature in aqueous ethanol solutions depending on the pH and the protein concentration (Dufour et al., 1998).
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While the BLG conformational changes induced by various environmental conditions have been extensively characterized at low protein concentration, few studies deal with changes of this protein in the millimolar concentration range. Through the data obtained on BLG gels in water – ethanol solutions at the mesoscopic and macroscopic levels, it was concluded that the spatial arrangements of the aggregates were similar in the final structures whatever the pH (Renard et al., 1999). It therefore seems useful to investigate the detailed mechanisms that control aggregation and gelation of BLG. In the present paper, the in-situ kinetics of BLG aggregation in water – ethanol solution were studied at 25°C by using rheological measurements, confocal laser scanning microscopy (CLSM), light and small angle neutron scattering (SANS) and Fourier transform infrared spectroscopy, in order to get a better understanding of the building of aggregates. The final structure of the gels at mesoscopic and macroscopic scales was observed using SANS and CLSM.
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solubility of the material. The undissolved material was, therefore, removed by centrifugation at 16 000× g for 40 min. The concentration of protein stock solutions was determined from the optical density at 278 nm (corrected for turbidity) using for the specific absorption coefficient A 1% 1 cm= 9.6 (Townend et al., 1960).
2.3. Dynamical rheological measurements
2. Material and methods
Small amplitude oscillatory shear experiments were performed at 25°C on a Carri-Med CS 50 rheometer using the cone-plate geometry (cone diameter 4 cm; angle 3.58°). Frequency sweep of BLG gel at pH 2 (2.56% (w/v)) in 50% (v/v) ethanol solution was recorded between 10 − 2 and 10 Hz under a strain amplitude of 0.03. The viscoelastic response was linear up to a strain amplitude value of 0.08. The aggregation/gelation kinetics of BLG (2.56% (w/v)) in 50% (v/v) ethanol solution were studied for a period of 80 h at pH 7, 8 and 9 using a frequency of 1 Hz and a strain amplitude of 0.01. The strain amplitude was chosen to prevent any disruption of the network build-up. Each experiment was conducted in triplicate.
2.1. Material
2.4. Infrared spectroscopy
The BLG sample used was kindly provided by Lactalis (Retiers, France). It was prepared from sweet or mixed whey by a-lactalbumin precipitation at acidic pH, ion-exchange chromatography of the residual whey, ultrafiltration and spray-drying (Rialland and Barbier, 1988). Reversed-phase HPLC indicated that BLG purity was 94% and that a-lactalbumin (3%) and bovine serum albumin (2%) were the main protein contaminants.
The infrared spectra were recorded between 1595 and 1720 cm − 1 at 2 cm − 1 intervals on a Fourier transform spectrometer IFS25 (Brucker). The attenuated total reflection (ATR) cell used in this study was made of a ZnSe crystal allowing six internal reflections (SPECAC). The cell was thermostatted and all experiments were performed at 25°C. A total of 200 scans were collected and averaged for both background and sample measurements. Since water strongly absorbs in the amide I region, the backgrounds were obtained using aqueous ethanol solutions (50% (v/v) ethanol). In this way, autosubtraction of the solvent spectrum was achieved (Dufour et al., 1994b). The aggregation/gelation kinetics performed in 50% (v/v) ethanol solutions were studied at three pH values (7, 8 and 9) for 10 h, the protein concentration being 2.56% (w/v). While the first
2.2. Protein solutions The protein was dissolved in water (stock solution: 10% w/w) and the pH adjusted in the 2 – 9 range with NaOH or HCl solutions (0.1 or 1 M). Sodium azide was added (0.2% (w/v)) to the protein solutions in order to prevent bacterial growth. The solutions were stirred overnight to ensure both complete hydration and maximum
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spectrum was recorded 3 min after the beginning of the kinetics, the following spectra were collected every 5 min for 6.5 h, and every 20 min until 10 h. The experiments were performed in triplicate. A baseline correction of the spectra was achieved using the SPECTRAFILE software (Heyden & Son, GMBH). Second-derivative spectra were assessed by applying the finite-difference method. The second-derivative data multiplied by −1 were thereafter normalized so that the area under each spectrum was constant.
their final state after an equilibrium period of 8 days. The data were normalized for transmission and sample path length and divided by the water spectrum. An absolute intensity scale was obtained using the absolute value of water intensity in units of cross-section (cm − 1). A subtraction of the appropriate incoherent background, taking 80% H/D exchange on the protein into account was realized according to Zaccaı¨ and Jacrot (1983). For globular proteins, the scattering intensity at a given wave-vector q can be written as:
2.5. Light scattering
I(q)= AP(q)S(q) or (Nad 2/(Dr 2))I(q)/C =MP(q)S(q)
Static (SLS) and dynamic (DLS) light scattering measurements were performed using an ALV-5000 multi-bit, multi-tau correlator in combination with a Malvern goniometer and a Spectra-Physics laser emitting vertically polarised light at 514.5 nm. Measurements on BLG (0.2 – 0.9% (w/v)) at pH 8 in 50% (v/v) ethanol solutions were done at u = 90° to minimise the influence of spurious scattering. The correlation functions were analysed using the inverse Laplace transform routine REPES (Stepanek, 1993) in order to obtain the corresponding relaxation time distribution (and hydrodynamic radii distribution). The measurement temperature was fixed at 25°C and controlled within 0.1°C.
P(q) is the «form factor» of the molecule and S(q) the «structure factor» which accounts for intermolecular interactions. The quantity A is a parameter that includes the concentration C, the mass M, the density d, Avogadro’s number Na, and the neutron contrast (Dr)2. The neutron contrast was assessed by taking the scattering length densities of the solvent (50/50% (v/v) D2O/C2D6O solvent) into account. The scattering intensity I(q) was finally expressed in terms of molecular mass, as the density (d=1.332 g cm − 3) and the contrast variation (Dr = 5.55 · 1010 cm − 2) of BLG in a 50/50% (v/v) D2O/C2D6O solution are known.
2.6. Small-angle neutron scattering
2.7. Confocal laser scanning microscopy (CLSM)
Small-angle neutron scattering (SANS) experiments were performed at the ILL (Grenoble, France) using the D22 multidetector instrument. The spectra for BLG solutions at pH 7 (C = 1.79% (w/v)) and pH 8 (C = 2.56% (w/v)) in 50% (v/v) ethanol solutions were recorded every 5 min during 8 h for each sample using a spectrometer configuration l =10 A, (incident wavelength) and d=14 m (distance of the sample to the detector). The range of wave-vector q = 4p/l sin(u/2), u being the scattering angle, thus covered was: 2.35 · 10 − 2 – 4.4 · 10 − 1 nm − 1. The gels formed at pH 2, 7, 8 and 9 in 50% (v/v) ethanol solutions and those formed at pH 7 for various ethanol concentrations were also characterized in
BLG solution at pH 7 C= 3.75% (w/v) in 50% (v/v) ethanol solution was prepared as described above; the protein was stained with Rhodamine IsoThioCyanate (0.25 mg g − 1 protein). Solutions were put inside cavity slides of 0.5 mm diameter. To prevent any evaporation, the slides were covered with glass coverslips and sealed. CSLM observations were performed using a Zeiss LSM 410 Axiovert microscope. The excitation wavelength was adjusted to that of RITC at 543 nm, and the emission above 570 nm was recorded. Pictures were taken every 5 min during 48 h with a water-immersed × 40 objective at a depth of 25 mm beneath the coverslip surface.
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3. Results and discussion
3.1. Static properties of b-lactoglobulin gels in water –ethanol solution Fig. 1 gives an overview of the different morphologies, obtained for b-lactoglobulin (BLG) solutions at pH 8 in 50% (v/v) ethanol concentration, depending on the protein concentration. At low protein concentrations (tube 1: 0.1% w/v; tube 2: 1% w/v), transparent liquids were obtained. While a viscous solution corre-
Fig. 1. b-Lactoglobulin gel morphologies formed at pH 8 in 50/50% (v/v) water – ethanol solutions (observations made after 8 days). 1, liquid (0.1% w/v); 2, liquid (1% w/v); 3, pre-gel state (2% w/v); 4, gel (3% w/v); 5, turbid gel (4% w/v); 6, collapsed gel (after syneresis).
Fig. 2. Frequency sweep at 25°C for b-lactoglobulin gel formed at pH 2 in 50/50% (v/v) water–ethanol solution (C= 2.56%, w/v), g0 = 0.03. , G% (Pa); , G%% (Pa).
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sponding to a pre-gel state was observed at 2% (w/v) concentration (tube 3), a gel really developed at 3% (w/v) concentration (tube 4). The turbidity in gelled samples increased with protein concentration (tube 5, 4% w/v), pH or ethanol concentration. In addition, a syneresis phenomenon is observed at high protein concentration. The tube 6 shows a collapsed gel state after completion of syneresis where the gel diameter is reduced in such a way that there is no more adhesion to the tube walls. The expelled liquid analysed by refractometry, was identified as being a 50/50% (v/v) water–ethanol mixture corresponding to the solvent used. Infra-red spectroscopy revealed that BLG monomers in a-helical form were also present in the expelled liquid. Phase state diagrams of BLG in water–ethanol solutions established as a function of pH and ethanol concentration revealed that the equilibrium gel state was reached in a time scale ranging from 1 min to 1 week, depending on the physico-chemical conditions (Dufour et al., 1998). With decreasing protein concentration, the critical gelation time became longer. Moreover, the critical gelation time, at a given protein concentration, shifted towards higher values as the pH of the solution decreased from 9 to 2 (Renard et al., 1999). The increase of the negative net charge on the protein would favour electrostatic double layer repulsion and slow down the aggregation process of BLG molecules. The critical gelation time at a fixed pH and protein concentration was highest at low ethanol concentration. Moreover, increase of protein concentration resulted in a large increase of turbidity, characteristic of a coarser network structure, whatever the ethanol concentration used. The mechanical properties of the resulting gels were investigated through dynamical rheological measurements. A typical example of a frequency sweep under 3% strain amplitude is shown Fig. 2 for a gel formed at pH 2. Whatever the pH, the signal always corresponds to a viscoelastic material with a slight dependence on the frequency of the storage and loss moduli G% and G%%. This rheological behaviour is characteristic of a weak network structure. However, long-time response by means of a
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Fig. 3. Small angle neutron scattering profiles I (g mol − 1) versus q (nm − 1) on a log-log scale for b-lactoglobulin gels formed in 50/50% (v/v) water–ethanol solution; the slope delimits the fractal regime of the structures. , pH 2, C= 4.45%; D, pH 7, C=2.56%; , pH 8, C= 3.03%; , pH 9, C= 3.02% (w/v).
creep-recovery test displayed a liquid-like type viscoelastic behaviour typical of a transient network (results not shown). The liquid-like behaviour of these gels means that the connections between the structural elements of the network are not permanent at the time scale of the experiments. This result agrees with observations made on both heat-set BLG and bovine serum albumin gels where, in all cases, a small but definite irrecoverable strain at the end of the recovery test was observed (Renard et al., 1996b; Lefebvre et al., 1998). The structure of BLG aggregates in the gels was investigated at a mesoscopic scale through small angle neutron scattering (SANS) experiments. This technique allows the determination of shape (1 – 100 nm scale) in self-assembly structures and to precise the interactions between the structures, even in turbid samples. The scattering intensity as a function of the wave-vector q is displayed in Fig. 3 for BLG gels formed at four pH values. The curves are quite identical at pH 7, 8 and 9
and reveal similarities with those obtained at the same pH values for heat-set BLG gels formed at high ionic strength (Renard et al., 1999). The similar intensity values observed at q higher than 1 nm − 1 suggest that the elementary subunit constitutive of the aggregates is the same. The shape in the intermediate q range indicates that compact structures are formed when aggregation takes place in water–ethanol solvent. On the contrary, the structure of the aggregates formed at pH 2 is completely different. A fractal regime at low q values seems to appear with an apparent fractal dimensionality value of about 1.3 characteristic of more or less linear aggregates with slight branching. Moreover, a correlation peak with a maximum in intensity at q* appears in the intermediate q range giving a mean inter-aggregate distance of 23 nm (2p/q*). These observations indicate that stiff charged aggregates are formed and that preferential distances are maintained between these structures giving rise to a correlation peak at the SANS scale.
Fig. 4. Small angle neutron scattering profiles I (g mol − 1) versus q (nm − 1) on a log – log scale for b-lactoglobulin gels at pH 7. , 70/30% (v/v) ethanol – water, C =4.63%; , 60/40% (v/v) ethanol – water, C =3.53%; D, 50/50% (v/v) ethanol/water, C =2.56% (w/v).
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Fig. 5. Second-derivative infrared spectra of b-lactoglobulin pH 8 C =2.56% (w/v) in 50/50% (v/v) water – ethanol solution as a function of time. – – , 3 min; ---, 28 min; …, 298 min. The second derivative data are multiplied by −1.
Fig. 4 displays the scattering intensities obtained for BLG gels at pH 7 as a function of ethanol percentage. The curves are very similar for ethanol concentrations between 50 and 70% (v/v). The shape of the curves confirms the formation of large and compact structures, the compactness being maximal in 50% (v/v) ethanol concentration. Considering the results obtained at different observation scales as reported above, we decided to investigate the build-up of these structures leading to a network as a function of time, in order to clarify the mechanisms that control aggregation and gelation in these systems.
3.2. In-situ kinetics of aggregation of b-lactoglobulin in water– ethanol solution Aggregation of BLG (2.56% (w/v)) at pH 8 in 50% (v/v) ethanol concentration was first studied at the molecular level using Fourier transformed infrared spectroscopy (FTIR). The second derivative spectra recorded at various times are given in Fig. 5. After 3 min, the BLG spectrum exhibits a peak at about 1650 cm − 1 and a shoulder at
around 1620 cm − 1. The band at 1650 cm − 1 is characteristic of a-helices appearing with the addition of ethanol. A time dependent decrease of the band at 1650 cm − 1 accompanied by an increase of the peak at 1620 cm − 1 is observed. The development of the band at 1620 cm − 1 may be assigned to b-strands exposed to the solvent or to the formation of intermolecular H-bonded bsheets. In addition, it was observed that the absorption band assigned to a-helix structures shifts from 1650 to 1660 cm − 1 during aggregation. Novskaya and Chirgadze (1976) reported that the frequency of a-helices steadily rises when the number of amino-acid residues involved in a-helical structures decreases. The in-situ kinetics of aggregation of BLG in water–ethanol solvent were assessed by measuring the integrated intensity of the normalized second derivative absorption band at 1620 cm − 1. Fig. 6 displays different kinetics obtained for three BLG concentrations in 50% (v/v) ethanol concentration. The intermolecular b-sheet structures quickly increased and reached a plateau at about 200 min. The initial rate seems to increase
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Fig. 6. Integrated intensity at 1620 cm − 1 as a function of time for b-lactoglobulin pH 8 in 50/50% (v/v) water–ethanol solution. , C =1%; , C=1.5%; , C= 2.56% (w/v).
with BLG concentration. Moreover, one can observe an increase in the final formation of the aggregates by increasing BLG concentration. At the macroscopic level, the viscoelasticity of the networks formed at various pH values by
adding ethanol was followed by measuring the storage and loss moduli G% and G%% at a fixed frequency of 1 Hz under 1% of strain amplitude (Fig. 7). The progress curves were obtained over a period of 80 h. The gelation kinetics greatly depended on the pH value of the solution. An increase in the net charge on the protein would tend to slow down the kinetic process as it will increase repulsive barriers between charged molecules. Consequently, one expect that moduli recorded at pH 9 would be lower than those observed at pH 7 and 8. Our results are not in agreement with this hypothesis, presumably because a slow denaturation that is known to occur at pH 9 promotes the aggregation process. Nevertheless, G%/G%% ratio increased with increasing pH of the solution. Equilibrium values for both G% and G%% moduli were reached after 80 h except in the case where syneresis after gelation occurred. The protein concentration dependence on the moduli G% and G%% was considerably high. In particular, the viscoelastic parameters diverged near the ‘isolated aggregates – connected aggregates’ transition (threshold located around the critical concentration for gelation). This result
Fig. 7. Gelation kinetics of b-lactoglobulin (C= 2.56% (w/v)) in 50/50% (v/v) water – ethanol solution; evolution of the storage modulus G% (filled symbols) and the loss modulus G%% (empty symbols) as a function of time (h) at 25°C; f= 1 Hz, g0 =0.01. , pH 7; , pH 8; , pH 9.
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Fig. 8. Changes of the secondary structure (A1620/A1650 ratio) (symbol) of b-lactoglobulin C= 2.25% (w/v) at pH 7 in 50/ 50% (v/v) water – ethanol solution as a function of time between 0 and 600 min. Comparison with the evolution of the storage modulus G% (Pa) (line) during the kinetic carried out at the same pH value.
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was previously observed on heat-set BLG gels where two regimes took place: one regime with no concentration dependence of the elastic modulus G% (isolated aggregates), a second regime characterized by a concentration dependence of G% (connected aggregates leading to a network) (Renard et al., 1995). In the particular case of BLG in diluted regime (2.25% (w/v)), Fig. 8 shows the evolution time in the first stages of aggregation of the storage modulus G% and of the absorbance ratio A1620/A1650 cm − 1 for alcohol gelation of BLG at pH 7. At this concentration, most of the secondary conformational changes were observed when the storage modulus remained close to zero. Consequently, it is suggested that at room temperature BLG monomers first connected through intermolecular b-sheets leading to small aggregates that did not develop a measurable viscoelastic response. The protein network was built up in a second step after 10 h.
Fig. 9. In-situ kinetics of aggregation followed by static and dynamic light scattering (T= 25°C) of b-lactoglobulin solutions at pH 8 in 50/50% (v/v) water –ethanol solution as a function of protein concentration. (a) Scattering intensity (expressed as the ratio of the scattering intensity of the sample over the scattering intensity of toluene) recorded at a fixed angle u= 90° and T =25°C as a function of time. , C =0.2%; , C= 0.3%; , C= 0.5%; , C = 0.7%; ", C = 0.8%; +, C =0.9% (w/v). (b) Apparent hydrodynamic radii Rh (nm) recorded at a fixed angle u=90° and T= 25°C as a function of time. , C = 0.2%; , C = 0.5% (w/v).
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Fig. 10. In-situ kinetics of gelation followed by small angle neutron scattering of b-lactoglobulin solutions in 50/50% (v/v) water– ethanol solution as a function of pH. The slopes delimit the fractal regime of the structures. (a) Scattering intensity I (g mol − 1) versus q (nm − 1) on a log-log scale for b-lactoglobulin at pH 7, C=1.79% (w/v) recorded as a function of time. , 5 min; , 25 min; , 85 min; , 8 h; ", 192 h. (b) Scattering intensity I (g mol − 1) versus q (nm − 1) on a log-log scale for b-lactoglobulin at pH 8 C =2.56% (w/v) recorded as a function of time. , 5 min; , 25 min; , 50 min; , 80 min; ("), 8 h; ( ), 192 h.
To test this assumption about the intermolecular b-sheet structures developing with time, in-situ light scattering experiments were performed at a fixed angle. Scattered light intensity and hydrodynamic radii were recorded as a function of time during the first stages of aggregation (Fig. 9a, b). On Fig. 9a, the sample to toluene intensities ratio at a fixed protein concentration increased rapidly as a function of time meaning that an increase in the size and/or the number of scattering elements occured. Aggregation went faster as protein concentration increased. On Fig. 9b, the size of the scattering elements (apparent hydrodynamic radii) were measured through dynamic light scattering. Two different populations of scattering elements were observed whatever the BLG concentration (0.2%, 0.5% (w/v)). The two populations exhibit a rapid increase in the first 15 min, followed by a slow and progressive increase. While the size values of the scattering elements after 60 min of aggregation time were 4 and 45 nm for 0.2% (w/v) BLG concentration, they were 8 and 65 nm in the case of 0.5% (w/v) BLG concentration.
The estimated mass ratio between large and small aggregates assessed after 60 min were found to be 5.4 and 1.2 for 0.2 and 0.5% (w/v) protein concentrations, respectively. This mass ratio changed with time and was higher at the beginning of the aggregation process (6.8 and 2.1 for 0.2 and 0.5% (w/v), respectively). In addition, the results indicate that both aggregates molecular mass and size are not proportional to the protein concentration. The internal structure of the aggregates formed at higher protein concentration in water–ethanol solutions was investigated by means of SANS experiments. The in-situ kinetics of aggregation of BLG at pH 7 and 8 in 50% (v/v) ethanol concentration were followed by recording the scattered intensities as a function of the wave-vector q (Fig. 10a,b). The scattered intensities for BLG aggregates at pH 7 (Fig. 10a) displayed a linear dependence with the wave-vector q. The apparent negative slope values assessed for each aggregation time ranged from 1 to 2.8. Similar trends were observed at pH 8 (Fig. 10b), with just a slow
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Fig. 11. In-situ kinetics of gelation followed by confocal laser scanning microscopy (lexc =543 nm) of b-lactoglobulin labelled with RITC at pH 7 C= 3.75% (w/v) in 50/50% (v/v) water–ethanol solution. (a) After 5 min; (b) after 44 h.
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down of the aggregation process, despite the higher protein concentration. The slope values are in agreement with the formation of fractal linear aggregates at the beginning of the process. Branching would develop with time giving higher apparent fractal dimensions. The final structures would be made of highly compact clusters. The intensity dependence of the compact clusters had an apparent slope value much higher than those corresponding to the diffusion or reaction-limited cluster – cluster aggregation models which values are of 1.75 and 2.1, respectively (Kolb et al., 1983; Meakin, 1983; Kolb and Jullien, 1984; Brown and Ball, 1985). From the above observations, a question arose: did we have such a large polydispersity in the heterogeneous networks that slope values would not reflect a fractal regime in the aggregates structure? To answer the question, in-situ kinetics of BLG gelation in 50% (v/v) ethanol concentration were studied using confocal laser scanning microscopy (CLSM). Fig. 11a and b show micrographs of BLG gels (3.75% (w/v)) formed at pH 7 after 5 min and 44 h. Fluorescent proteinrich domains appear light on the picture while protein-poor phases are dark (Fig. 11a). While initial structures corresponding to dense and polydisperse clusters appeared in the first minutes, with many very small aggregates and few very large ones, an overall connected morphology of the protein-rich phase seems to have formed in a second step (Fig. 11b). The coexistence of dense clusters included in a network in the final state would suggest that competition between gelation and phase separation would occur leading to a non-equilibrium gel state. Demixing would be caused by less favorable interactions between the solvent and BLG in its denatured form (a-helical form). Bansil et al. (1992) studying gelatin gelation in water–methanol solution by optical microscopy revealed similar structures and concluded to the existence of a phase separation process due to spinodal decomposition. In addition, they found that the kinetics of phase separation depended very strongly on temperature: for deep
quenches, where the rates of phase separation and gelation were comparable, the phase-separation process did not go to completion. San Biagio et al. (1999) recently reported that bovine serum albumin coagulation proceeds through three simultaneous processes: conformational change of the protein, mesoscopic demixing of the solution and protein cross-linking. Several parameters such as temperature (T= 30–70°C for 10 min) or dilution (1:1 ratio) on BLG aggregated diluted systems were tested to elucidate the reversible character of the BLG aggregation in water–ethanol solution. No reversible transition of the aggregation was observed even after 24 h, meaning that high energy levels were implied in the inter-aggregate bonds.
4. Conclusion From the molten globule and helix-shaped BLG forms induced in water–ethanol solvent, aggregation may take place below a critical protein concentration leading to linear fractal aggregates during the first stages of the process. These small aggregates that appear loosely connected to each other further develop with time and lead more or less rapidly to a phase-separated network. From the coarse and irregular structures formed, it is postulated that BLG in water–ethanol solution would undergo a frustration of demixion by gelation or would undergo a simultaneous binary phase separation and irreversible gelation transition. As in general, the system does not fully separated into two phases, the early stage characteristic of the spinodal decomposition is ‘pinned’ owing to the onset of gelation. Recent work on heat-set globular protein gelation resulted in similar conclusions (San Biagio et al., 1996; Tobitani and RossMurphy, 1997). If this process of aggregation/ demixing would be adjustable through variation of gelling conditions, potentially it would lead to almost infinite possibilities for BLG gel structure and biopolymeric networks in general.
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