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β-lactoglobulin under high pressure studied by small-angle neutron scattering
Biochimica et Biophysica Acta 1764 (2006) 211 – 216 http://www.elsevier.com/locate/bba
h-lactoglobulin under high pressure studied by small-angle neutron scattering C. Loupiac a,*, M. Bonetti b, S. Pin c, P. Calmettes d a
Equipe d’Inge´nierie Mole´culaire et Sensorielle des Aliments et des Produits de Sante´, ENSBANA, Dijon, France b Service de Physique de l_Etat Condense´, CEA de Saclay, Gif sur Yvette, France c Service de Chimie Mole´culaire, URA 331 CNRS, CEA de Saclay, Gif sur Yvette, France d Laboratoire Le´on Brillouin, UMR 12 CNRS, CEA de Saclay, Gif sur Yvette, France Received 29 July 2005; received in revised form 14 October 2005; accepted 17 October 2005 Available online 7 November 2005
Abstract We used small-angle neutron scattering to study the effects of the high hydrostatic pressure on the structure of h-lactoglobulin. Experiments were carried out at pH 7 on the dimeric form of the protein in a pressure range going from 50 MPa to 300 MPa. These measurements allow the protein size and the interactions between macromolecules to be studied during the application of pressure. Increasing pressure up to 150 MPa leads to a swollen state of the protein that gives rise to an increase of the radius of gyration by about 7%. Within this pressure range, we also show that the interaction between macromolecules weakens although it remains repulsive. The measurements show an aggregation process occurring above 150 MPa. From the spectra analysis, it appears that the aggregation occurs mainly by association of the dimeric units. D 2005 Elsevier B.V. All rights reserved. Keywords: h-lactoglobulin; High pressure; Molten globule; Aggregation; Small-angle neutron scattering
1. Introduction Traditional food-processing methods rely on high temperatures as a way to ensure prolonged shelf-life and food safety. However, the use of such high temperatures is commonly known to cause irreversible damages on the processed products. These undesirable changes modify the nutritional as well as the organoleptic attributes [1,2]. Over the last decade, considerable attention has been given to the impact of high pressures on modification of the foods properties [3– 5]. In foodstuffs, proteins are very often used for their functional properties. Most of the time, their abilities to act as emulsifiant, gelation or foaming agents are related to their structure [6]. Processing foods under high pressure often results at the molecular level in structural changes of the protein [7,8] and/or aggregation [9,10], and possibly at the macroscopic level to a gel formation [11]. The isolation of folding intermediates is crucial to understand protein misfolding and protein aggregation. Experimental and theoretical approaches indicate that one of the underlying mechanism of pressure unfolding is the
* Corresponding author. Tel.: +33 3 80 39 66 84; fax: +33 3 80 39 66 47. E-mail address: [email protected] (C. Loupiac). 1570-9639/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2005.10.012
penetration of water into the protein [12,13]. Several intermediate states of the protein have been shown to exist, with their properties depending on the employed experimental conditions, and the protein might be in the molten globule state [9,14,15]. h-lactoglobulin (BLG) is the main protein constituent of the milk whey from ruminant. This protein is an important functional protein in foods, as it is the major component of many dairy gel and emulsions. Several models for the thermal denaturation and aggregation of h-lactoglobulin have been proposed [16 –18]. Since the early fifties, many available experimental techniques have been used to monitor the effects of various physicochemical parameters (concentrations, ionic strength, pH, temperature) on the protein structure [19 –21]. At pH values between 5 and 8, the dimer is the most stable form at room temperature, although small proportions of bigger aggregates may be present as well. The attracting forces between the single monomers seem to be weak and of hydrophobic nature. During a temperature-induced denaturation, the protein does not unfold completely, and a considerable amount of secondary structure is retained. A molten globule state could occur and two or more intermediate species could exist [12,22]. From a molecular point of view, the role of intermolecular disulfide bonds and the participation of hydro-
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phobic interactions to the thermal aggregation and gelation process have been discussed in earlier studies [20,23]. The S – H/S – S interchange reaction seems to play an important role in the h-lactoglobulin aggregation mechanism. Heating of a high concentrated (>1% w/w) protein solution results in the formation of a gel with fractal like organization [23]. Studies by fluorescence, circular dichroism, electrophoresis, rheology have shown that pressure induces hlactoglobulin denaturation and aggregation/gel formation [11,24 – 26]. Pressure- and thermal-induced gelation mechanisms of this protein differ, probably owing to disruption of hydrophobic and ionic bonds by high pressure, whereas heat-induced denaturation involves breaking down weak bonds (hydrogen bonds and salt bridge), and intermolecular aggregation via the S– H/S – S interchange reaction [27]. Consequently, in contrast to thermal denaturation, the denaturation induced by pressure in a 100- to 400-MPa range might be reversible. A basic challenge of this study was to better understand the mechanism of pressure unfolding, dissociation, and aggregation-gelation of the h-lactoglobulin. We used pressure as a physicochemical perturbation to establish experimental conditions under which a different mechanism of aggregation might occur. From the small-angle neutron scattering (SANS) measurements, the overall conformation of the h-lactoglobulin was studied by measuring its radius of gyration in a medium pressure range going from 50 MPa to 300 MPa. In this pressure range, we can determine whether the dissociation of the dimeric units occurs and if the aggregation mechanism involves the monomeric form of the protein. These experiments were done ‘‘on-line’’ at room temperature by gradually increasing the pressure between each measurement. To determine the pressure effects on the protein interactions and the variation of the value of the actual radius of gyration of the protein, the SANS measurements were performed at different protein concentrations. 2. Materials and methods 2.1. Protein sample preparation h-lactoglobulin has been purified from raw milk following the method described by Fox et al. [28]. h-lactoglobulin is isolated from other milk proteins because among the whey protein, it is the most resistant to precipitation by trichloroacetic acid (TCA). After a first centrifugation to eliminate the milk fat content, caseins were precipitated by addition of acetate buffer at pH 4.6. The precipitated casein was removed by a first filtration through filter papers and through a second filtration on cellulose nitrate membrane (5 Am pore size, 47 mm diameter). Around 800 ml of acid whey proteins were recovered from 1 l of milk. 31 g of TCA per liter of whey proteins were added to precipitate all the proteins except the h-lactoglobulin. The precipitated proteins were eliminated by centrifugation at 17,000g for 30 min at 4 -C. Pure h-lactoglobulin is recovered in supernatant at a concentration around 1.5 mg cm3. The solution is dialyzed against water and then buffered to eliminate all the salt added during the purification process. To obtain a sufficient scattered intensity within a reasonable amount of time, the protein was further concentrated at the end of the purification. We used Centriplus filters from Millipore (Amicon, cut off of 3500 Da) to obtain a solution at a concentration around 100 mg cm3. To clear off the protein solution from any aggregate that would generate an undesirable forward-
scattered intensity in the SANS spectra, a gel permeation chromatography (size exclusion sephadex S200 column) was performed. At the end of this step, the protein concentration is around 15 mg cm3. For the high pressure experiments, this stock is dialyzed 3 times against heavy water-buffer (BisTris, 100 mM) until almost all accessible hydrogen were exchanged. After purification, the pD value of the sample is around 6.6 so the pH is 7 (pH = pD + 0.4). Consequently, proteins in solution are mostly in the dimeric form. Sodium azide at 0.02% was added to the solution to avoid any bacterial contamination. All samples were centrifuged at 20,000g during 5 min prior to the experiments. The concentration of the protein stock at the end of the sample preparation was very close to the BLG concentration in the milk (our upper concentration limit: 15 mg cm3). For the SANS measurements made as a function of the protein concentration, we had to make dilution to minimize multiple scattering and to decrease the effect of proteins interaction. However, to have an acceptable scattering intensity in a reasonable time of acquisition (typically 12 h), we kept the protein concentration above 5 mg cm3. Thus the range of studied concentration will be limited by a factor 3, going from 15 to 5 mg cm3.
2.2. High-pressure cell A sapphire-anvil cell specially designed to perform SANS measurements has been used [29]. The sample is pressurized by squeezing between two opposed 20 mm thick sapphire anvils a stainless steel gasket with a central hole that contains the protein solution. The gasket hole is 10 mm in diameter and 2 mm thick. The sample volume is about 160 mm3. The hole is filled with the protein solution at atmospheric pressure by means of a syringe. Cleaning the faces of the anvils, changing the squeezed gasket, and filling with a new protein sample are simple operations that do not require the removal of the cell from the SANS spectrometer. In the studied pressure range, pressure calibration was performed by monitoring the fluorescence emission wavelength shift of a Sm2+ :SrFCl crystals [30] that has a large pressure coefficient (dk/ dP) = (1.11 T 0.11) nm GPa 1. With the present gasket dimensions and because of the low yield strength of the stainless steel the largest working pressure is around 360 MPa. Above this pressure, the gasket plastically flows and any further squeezing of the gasket does not allow a higher pressure to be achieved [29].
2.3. SANS measurements The SANS measurements were performed at the Laboratoire Le´on Brillouin (CEA-CNRS, Saclay, France) using the PACE spectrometer. This spectrometer is equipped with 30 concentric annular rings. The incident neutron wavelength was k = 1.1 nm. The corresponding neutron beam transmission of the empty cell is å0.72. The range of the wave-number transfer q = (4p/k) sin(h/2) is between 0.07 and 0.74 nm1, with h, the scattering angle. Each raw spectrum from the protein solution was measured at room temperature (T = 20 -C) and divided by the corresponding transmission. The scattered intensity from the buffer was measured at a single pressure, P = 50 MPa, as it is pressure independent. It was then subtracted from the spectrum of each protein sample. Finally, to account for the non-uniform detector efficiency, the result was normalized to the incoherent scattering spectrum of a 2-mm thick 50% H2O – 50% D2O water mixture, from which the spectrum of the empty anvil-cell was previously subtracted.
3. Results Fig. 1 shows the SANS spectra measured at different hlactoglobulin concentrations c (P = 0.1 MPa) = 7.1, 10.7 and 14.3 mg cm3 for increasing pressures P = 50, 150 and 300 MPa (Fig. 1a, b, c, respectively). Several spectra of one or two h were recorded successively at each pressure. For pressure below 150 MPa, the scattered intensity does not change with time and indicates that the protein is stable.
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Guinier approximation [31]: Iðq; P; cÞ ffi Ið0; P; cÞexp½ q2 R2g ðP; cÞ=3
ð1Þ
where R g(P, c) is the apparent radius of gyration of the protein and I(0, P, c), the forward-scattered intensity. For almost spherical proteins, Eq. (1) is valid to within 1% for q R g(P, c) 1.3, that corresponds, in the present case, to a maximum wave-number transfer q max å 0.5 nm1. Fig. 1 shows the natural logarithm of the scattered intensity I(0, P, c) as a function of q 2. In a q-domain where the Guinier approximation is valid, the slope of the linear regression is related to the value of the apparent radius of gyration R g (P, c). Its value increases with increasing pressure P. The estimated error of the apparent radius of gyration obtained from the regression analysis varies from T 1% to T 2% according to the protein concentration and pressure. The value of the actual radius of gyration R g(P, 0) was inferred from the apparent radius of gyration R g(P, c) using the following relation: 2 R2 g ðP; cÞ ¼ Rg ðP; c ¼ 0Þ½1 þ 2B2 ðPÞMp cðPÞ þ N
ð2Þ
where B 2(P) has a similar meaning as the second virial coefficient A 2(P) in Eq. (3) describing the interaction between macromolecules. Fig. 3 shows the square of the inverse of R g (P, c) as a function of the protein concentration, c, for the two studied pressure (P = 50 and 150 MPa) at which the protein is stable. The protein concentration c(P) is calculated using the tabulated values of the H2O density as a function of pressure P given in the NBS Steam Tables [32]. According to Eq. (2), the intercept at c = 0 of the linear regression of R g 2(P, c) versus c(P) allows the actual radius of gyration R g(P, c = 0) to be determined. The values at P = 50 and 150 MPa are R g = (2.06 T 0.05) nm and (2.21 T 0.05) nm, respectively, and are shown in Fig. 4. The error of the actual radius of gyration R g(P, c = 0) is given by the regression analysis and varies between T 1.5 and T 2%. The Fig. 1. Logarithm of the scattered intensity, I(q, P, c), as a function of the square of the wave-number transfer, q. h-lactoglobulin concentration, c(P = 0.1 MPa): (a) 7.1 mg cm3, (b) 10.7 mg cm3, and (c) 14.3 mg cm3. Pressure, P: (o) 50 MPa, (g) 150 MPa, and (N) 300 MPa. The full lines are a fit with Eq. (1). Temperature is 20 -C.
Therefore, the spectra at 50 and 150 MPa are averaged over several spectra. At a pressure around 300 MPa, the scattered intensity at low q increases with time as shown in Fig. 2. This indicates that a pressure-induced aggregation of the macromolecules occurs in the sample. To confirm the irreversible mechanism of the protein aggregation, the pressure was then lowered from 300 MPa to 150 MPa: the scattered intensity does not decrease to the value initially measured at 150 MPa, but rather remains constant (Fig. 2). The data reduction of the scattered spectra is based upon the analysis as thoroughly discussed by Loupiac et al. [15]. The scattered intensity from a protein can be described by the
Fig. 2. Time variation of the scattered intensity I(q, P, c) as a function of the wave-number transfer, q. h-lactoglobulin concentration c(P = 0.1 MPa) = 7.1 mg cm3, P = 300 MPa. (N), (‚) and (Q): Spectra recorded during 2 h successively. The irreversibility of the aggregation mechanism is evidenced when the pressure is lowered from 300 MPa to 150 MPa, ( ).
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Fig. 4. Value of the actual radius of gyration, R g(c = 0), as a function of pressure, P.
Fig. 5 shows the second virial coefficient A 2 as a function of pressure P. The positive sign of A 2 at 50 and 150 MPa shows that the interaction between two dimers is repulsive. It appears that this repulsive interaction becomes weaker by increasing the pressure from 50 to 150 MPa. A large increase of A 2 is observed at P = 300 MPa but this value is very approximate if one considers the amount of aggregates that are formed at this pressure (see Fig. 2). Fig. 3. Inverse of the square of apparent radius of gyration, R g(P, c), as a function of the BLG concentration, c(P). (a) P = 50 MPa; (b) P = 150 MPa. The solid line is a linear regression to determine the value of the actual radius of gyration at vanishing protein concentration.
value at P = 50 MPa (2.06) nm is very close to the one obtained either by SAXS (2.10 nm) [26] or by SANS (between 2.00 and 2.26 nm) (without pressure) [23,33] measurements (see Table 1). Applying pressure from 50 to 150 MPa gives a 7% increase of R g. In Fig. 4, the value of R g at P = 300 MPa is also shown. However, the growth of the forward-scattered intensity (see Fig. 1) suggests a pressure-induced aggregation mechanism. Thereby the value of R g at P = 300 MPa which shows a large increment around 30% in comparison to the one at 50 MPa is biased by the aggregation process. The inverse of the forward-scattered intensity I(q = 0, P, c) from an hydrated protein of molecular weight M p and concentration c(P) has the following virial expansion: cðPÞ 1 NA ¼ 1 þ 2A2 ð PÞMp cð PÞ þ N 2 M Ið0; P; cÞ P K ð PÞ
4. Discussion One of the questions addressed in this work is to know whether or not the aggregation process induced by applying pressure involves the dimeric unit or monomeric unit of the protein. This has been answered by the analysis of the evolution of the radius of gyration as a function of applied pressure. The behavior of the forward-scattered intensity allows the critical pressure at which the aggregation process takes place to be determined and the change of the protein interactions induced by pressure to be studied by means of the second virial coefficient. The measured value of the actual radius of gyration of the protein, R g (P = 50 MPa) = 2.06 nm, is very close to the one obtained in other studies for h-lactoglobulin dimeric forms [23,26]. To analyze correctly the pressure effects on the value
ð3Þ
where K(P) is the pressure dependent specific contrast of the protein molecule with respect to the buffer, N A is the Avogadro number, and A 2(P) is the second virial coefficient. Eq. (3) is correct as far as the solute concentration is low, meaning that higher order terms in c(P) are negligible. The coefficient A 2(P) describes the interaction between two proteins: For repulsive interactions it is positive, whereas for attractive proteins it is negative [34]. According to Eq. (3), the slope of c(P)[K(P)]2/ I(0,P,c) vs. c(P) allows the second virial coefficient, A 2(P), to be determined.
Fig. 5. Second virial coefficient, A 2(P), as a function of pressure, P.
C. Loupiac et al. / Biochimica et Biophysica Acta 1764 (2006) 211 – 216 Table 1 Values of the apparent radius of gyration R g obtained in previous studies by different scattering methods and in various experimental conditions Methods
R g (nm)
Experimental conditions
Reference
SANS SAXS SANS SAXS
2.26 2.10 2.00 1.29
pH pH pH pH
[33] [26] [23] [35]
6.9, 0.1 M NaCl 7, 10 mM Tris 7, 0.1 M ammonium acetate 2, 0.1 M NaCl
of the radius of gyration, the measurements were made at different protein concentrations and the results extrapolated at vanishing protein concentration. Our analysis shows that the size of the h-lactoglobulin increases with increasing pressure. A change of 7% of the actual radius of gyration is observed between 50 MPa and 150 MPa and an increase of 31% is observed when applying a pressure of 300 MPa. At this highest pressure, the measured value is approximate because of the beginning of an aggregation process. In a study concerning the structure and the distribution of aggregates formed after a heat process, Gimel at al., [23] give the geometrical parameters of the native dimeric units of h-lactoglobulin determined by neutron and light scattering measurements. To support our discussion, we listed in Table 1 the values of the radius of gyration obtained in previous studies. The dimeric protein is represented by two connected spheres with radius 1.7 nm and a distance from center to center around 3.3 nm with a 2 nm radius of gyration very close to our measurement. Pessen at al. [35] have determined the radius of gyration of the monomeric form of protein at pH 2 by SAXS at atmospheric pressure and gave a value around 1.29 nm. Therefore, we can affirm that no dissociation of the dimer occurs in the 50 – 150 MPa pressure range as our measured radius of gyration is far away from the monomeric one. It is possible that the protein at 150 MPa could be in a swollen state. Different parameters could lead to this swollen state of the protein: hindrance of water inside the protein matrix and/or changes in the hydrogen bonds network, and/ or breaking down the electrostatic bonds and some of the protein hydrophobic interactions [13,27]. This description is in good agreement with previous measurements made by fluorescence [25] which show that the tryptophan (Trp) residues of the protein after applying pressure becomes part of a more hydrophilic surrounding. This change of exposure can be interpreted as an opening of the structure leading the Trp residues to be more solvent exposed or by the new proximity of water molecule inside the protein matrix. Applying pressure gives rise to a reorganization of the protein water interaction. These changes of water organization imply local structural reorganization of amino acids. Inside the h-lactoglobulin exists a large hydrophobic cavity which should be destabilized by the water entrance in the matrix. One way to keep the protein structure intact could be to swell to decrease the antagonist interactions between hydrophobic amino acids and water. This reorganization could initiate the unfolding of the protein. Then, the thiolate of the hlactoglobulin, initially buried inside the protein, becomes
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very exposed to the solvent. This group is then much more reactive and in these pH conditions (pH = 7) can form intermolecular disulfide bridge, leading to the final irreversible aggregate state. The implication of the free thiolate of the protein in the aggregation after high pressure treatments has been shown in previous studies [24,25]. In the same pH conditions and similar protein concentrations, Funterberger et al., [24] have shown by gel electrophoresis that high pressure promotes BLG aggregation through SH/S –S interchanges reactions. The pressure induced formation of S –S intermolecular bonded oligomers. This mechanism is in good agreement with the change of values we observed for the second virial coefficient (A 2) of the protein between 50 MPa and 150 MPa going respectively from 8.7 104 cm3 mol g2 to 5 104 cm3 mol g2. Positive values of A 2 indicate that the interactions between two protein molecules in the solution are repulsive, and the decrease with pressure shows that this repulsive interaction becomes weaker by increasing the pressure from 50 to 150 MPa. This change of interaction could be linked to the change of hydration organization suggested as hypothesis before to justify the increase of size of the protein. Breaking down hydrophobic interactions, salt bridges and electrostatic bonds of the protein leads to a change of the overall charge repartition of the protein. The macromolecules in this swollen state, as there are less repulsive, can interact together and initiate the aggregation process. Also, the aggregation process is clearly observed in our spectra for pressure around 300 MPa whatever the protein concentration studied. We therefore confirm by the present measurements that relatively high pressures lead to an aggregation process. After applying 300 MPa pressure, we have released the pressure to 150 MPa. The results showed that this process is irreversible. It can be stated in these experimental conditions (protein concentration, pH, salt) that a pressure value around 150 MPa leads to a swollen state of the h-lactoglobulin and that at a pressure around 300 MPa the protein begins to form irreversible aggregates. This aggregation occurs between dimeric units of the protein. In the future it will be interesting to see the repercussion of this aggregation between dimeric units on the gel properties. Small-angle neutron scattering measurements allows the range of the wave-number transfer to be easily modified in order to probe either the macromolecule scale (this study) or the gel organization. References [1] D. Knorr, Novel approaches in food-processing technology: new technologies for preserving foods and modifying function, Curr. Opin. Biotechnol. 10 (1999) 485 – 491. [2] B. Krebbers, A.M. Matser, M. Koets, P.V. Bartels, R.W. Van den Berg, High pressure – temperature processing as an alternative for preserving basil, High Press. Res. 22 (2002) 711 – 714. [3] J.C. Cheftel, Review: high pressure, microbial inactivation and food preservation, Food Sci. Technol. Int. 1 (1995) 75 – 90. [4] W. Messens, J. Van Camp, A. Huyghebaert, The use of high pressure to modify the functionality of food proteins, Trends Food Sci. Technol. 8 (1997) 107 – 112.
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h-lactoglobulin under high pressure studied by small-angle neutron scattering C. Loupiac a,*, M. Bonetti b, S. Pin c, P. Calmettes d a
Equipe d’Inge´nierie Mole´culaire et Sensorielle des Aliments et des Produits de Sante´, ENSBANA, Dijon, France b Service de Physique de l_Etat Condense´, CEA de Saclay, Gif sur Yvette, France c Service de Chimie Mole´culaire, URA 331 CNRS, CEA de Saclay, Gif sur Yvette, France d Laboratoire Le´on Brillouin, UMR 12 CNRS, CEA de Saclay, Gif sur Yvette, France Received 29 July 2005; received in revised form 14 October 2005; accepted 17 October 2005 Available online 7 November 2005
Abstract We used small-angle neutron scattering to study the effects of the high hydrostatic pressure on the structure of h-lactoglobulin. Experiments were carried out at pH 7 on the dimeric form of the protein in a pressure range going from 50 MPa to 300 MPa. These measurements allow the protein size and the interactions between macromolecules to be studied during the application of pressure. Increasing pressure up to 150 MPa leads to a swollen state of the protein that gives rise to an increase of the radius of gyration by about 7%. Within this pressure range, we also show that the interaction between macromolecules weakens although it remains repulsive. The measurements show an aggregation process occurring above 150 MPa. From the spectra analysis, it appears that the aggregation occurs mainly by association of the dimeric units. D 2005 Elsevier B.V. All rights reserved. Keywords: h-lactoglobulin; High pressure; Molten globule; Aggregation; Small-angle neutron scattering
1. Introduction Traditional food-processing methods rely on high temperatures as a way to ensure prolonged shelf-life and food safety. However, the use of such high temperatures is commonly known to cause irreversible damages on the processed products. These undesirable changes modify the nutritional as well as the organoleptic attributes [1,2]. Over the last decade, considerable attention has been given to the impact of high pressures on modification of the foods properties [3– 5]. In foodstuffs, proteins are very often used for their functional properties. Most of the time, their abilities to act as emulsifiant, gelation or foaming agents are related to their structure [6]. Processing foods under high pressure often results at the molecular level in structural changes of the protein [7,8] and/or aggregation [9,10], and possibly at the macroscopic level to a gel formation [11]. The isolation of folding intermediates is crucial to understand protein misfolding and protein aggregation. Experimental and theoretical approaches indicate that one of the underlying mechanism of pressure unfolding is the
* Corresponding author. Tel.: +33 3 80 39 66 84; fax: +33 3 80 39 66 47. E-mail address: [email protected] (C. Loupiac). 1570-9639/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2005.10.012
penetration of water into the protein [12,13]. Several intermediate states of the protein have been shown to exist, with their properties depending on the employed experimental conditions, and the protein might be in the molten globule state [9,14,15]. h-lactoglobulin (BLG) is the main protein constituent of the milk whey from ruminant. This protein is an important functional protein in foods, as it is the major component of many dairy gel and emulsions. Several models for the thermal denaturation and aggregation of h-lactoglobulin have been proposed [16 –18]. Since the early fifties, many available experimental techniques have been used to monitor the effects of various physicochemical parameters (concentrations, ionic strength, pH, temperature) on the protein structure [19 –21]. At pH values between 5 and 8, the dimer is the most stable form at room temperature, although small proportions of bigger aggregates may be present as well. The attracting forces between the single monomers seem to be weak and of hydrophobic nature. During a temperature-induced denaturation, the protein does not unfold completely, and a considerable amount of secondary structure is retained. A molten globule state could occur and two or more intermediate species could exist [12,22]. From a molecular point of view, the role of intermolecular disulfide bonds and the participation of hydro-
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phobic interactions to the thermal aggregation and gelation process have been discussed in earlier studies [20,23]. The S – H/S – S interchange reaction seems to play an important role in the h-lactoglobulin aggregation mechanism. Heating of a high concentrated (>1% w/w) protein solution results in the formation of a gel with fractal like organization [23]. Studies by fluorescence, circular dichroism, electrophoresis, rheology have shown that pressure induces hlactoglobulin denaturation and aggregation/gel formation [11,24 – 26]. Pressure- and thermal-induced gelation mechanisms of this protein differ, probably owing to disruption of hydrophobic and ionic bonds by high pressure, whereas heat-induced denaturation involves breaking down weak bonds (hydrogen bonds and salt bridge), and intermolecular aggregation via the S– H/S – S interchange reaction [27]. Consequently, in contrast to thermal denaturation, the denaturation induced by pressure in a 100- to 400-MPa range might be reversible. A basic challenge of this study was to better understand the mechanism of pressure unfolding, dissociation, and aggregation-gelation of the h-lactoglobulin. We used pressure as a physicochemical perturbation to establish experimental conditions under which a different mechanism of aggregation might occur. From the small-angle neutron scattering (SANS) measurements, the overall conformation of the h-lactoglobulin was studied by measuring its radius of gyration in a medium pressure range going from 50 MPa to 300 MPa. In this pressure range, we can determine whether the dissociation of the dimeric units occurs and if the aggregation mechanism involves the monomeric form of the protein. These experiments were done ‘‘on-line’’ at room temperature by gradually increasing the pressure between each measurement. To determine the pressure effects on the protein interactions and the variation of the value of the actual radius of gyration of the protein, the SANS measurements were performed at different protein concentrations. 2. Materials and methods 2.1. Protein sample preparation h-lactoglobulin has been purified from raw milk following the method described by Fox et al. [28]. h-lactoglobulin is isolated from other milk proteins because among the whey protein, it is the most resistant to precipitation by trichloroacetic acid (TCA). After a first centrifugation to eliminate the milk fat content, caseins were precipitated by addition of acetate buffer at pH 4.6. The precipitated casein was removed by a first filtration through filter papers and through a second filtration on cellulose nitrate membrane (5 Am pore size, 47 mm diameter). Around 800 ml of acid whey proteins were recovered from 1 l of milk. 31 g of TCA per liter of whey proteins were added to precipitate all the proteins except the h-lactoglobulin. The precipitated proteins were eliminated by centrifugation at 17,000g for 30 min at 4 -C. Pure h-lactoglobulin is recovered in supernatant at a concentration around 1.5 mg cm3. The solution is dialyzed against water and then buffered to eliminate all the salt added during the purification process. To obtain a sufficient scattered intensity within a reasonable amount of time, the protein was further concentrated at the end of the purification. We used Centriplus filters from Millipore (Amicon, cut off of 3500 Da) to obtain a solution at a concentration around 100 mg cm3. To clear off the protein solution from any aggregate that would generate an undesirable forward-
scattered intensity in the SANS spectra, a gel permeation chromatography (size exclusion sephadex S200 column) was performed. At the end of this step, the protein concentration is around 15 mg cm3. For the high pressure experiments, this stock is dialyzed 3 times against heavy water-buffer (BisTris, 100 mM) until almost all accessible hydrogen were exchanged. After purification, the pD value of the sample is around 6.6 so the pH is 7 (pH = pD + 0.4). Consequently, proteins in solution are mostly in the dimeric form. Sodium azide at 0.02% was added to the solution to avoid any bacterial contamination. All samples were centrifuged at 20,000g during 5 min prior to the experiments. The concentration of the protein stock at the end of the sample preparation was very close to the BLG concentration in the milk (our upper concentration limit: 15 mg cm3). For the SANS measurements made as a function of the protein concentration, we had to make dilution to minimize multiple scattering and to decrease the effect of proteins interaction. However, to have an acceptable scattering intensity in a reasonable time of acquisition (typically 12 h), we kept the protein concentration above 5 mg cm3. Thus the range of studied concentration will be limited by a factor 3, going from 15 to 5 mg cm3.
2.2. High-pressure cell A sapphire-anvil cell specially designed to perform SANS measurements has been used [29]. The sample is pressurized by squeezing between two opposed 20 mm thick sapphire anvils a stainless steel gasket with a central hole that contains the protein solution. The gasket hole is 10 mm in diameter and 2 mm thick. The sample volume is about 160 mm3. The hole is filled with the protein solution at atmospheric pressure by means of a syringe. Cleaning the faces of the anvils, changing the squeezed gasket, and filling with a new protein sample are simple operations that do not require the removal of the cell from the SANS spectrometer. In the studied pressure range, pressure calibration was performed by monitoring the fluorescence emission wavelength shift of a Sm2+ :SrFCl crystals [30] that has a large pressure coefficient (dk/ dP) = (1.11 T 0.11) nm GPa 1. With the present gasket dimensions and because of the low yield strength of the stainless steel the largest working pressure is around 360 MPa. Above this pressure, the gasket plastically flows and any further squeezing of the gasket does not allow a higher pressure to be achieved [29].
2.3. SANS measurements The SANS measurements were performed at the Laboratoire Le´on Brillouin (CEA-CNRS, Saclay, France) using the PACE spectrometer. This spectrometer is equipped with 30 concentric annular rings. The incident neutron wavelength was k = 1.1 nm. The corresponding neutron beam transmission of the empty cell is å0.72. The range of the wave-number transfer q = (4p/k) sin(h/2) is between 0.07 and 0.74 nm1, with h, the scattering angle. Each raw spectrum from the protein solution was measured at room temperature (T = 20 -C) and divided by the corresponding transmission. The scattered intensity from the buffer was measured at a single pressure, P = 50 MPa, as it is pressure independent. It was then subtracted from the spectrum of each protein sample. Finally, to account for the non-uniform detector efficiency, the result was normalized to the incoherent scattering spectrum of a 2-mm thick 50% H2O – 50% D2O water mixture, from which the spectrum of the empty anvil-cell was previously subtracted.
3. Results Fig. 1 shows the SANS spectra measured at different hlactoglobulin concentrations c (P = 0.1 MPa) = 7.1, 10.7 and 14.3 mg cm3 for increasing pressures P = 50, 150 and 300 MPa (Fig. 1a, b, c, respectively). Several spectra of one or two h were recorded successively at each pressure. For pressure below 150 MPa, the scattered intensity does not change with time and indicates that the protein is stable.
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Guinier approximation [31]: Iðq; P; cÞ ffi Ið0; P; cÞexp½ q2 R2g ðP; cÞ=3
ð1Þ
where R g(P, c) is the apparent radius of gyration of the protein and I(0, P, c), the forward-scattered intensity. For almost spherical proteins, Eq. (1) is valid to within 1% for q R g(P, c) 1.3, that corresponds, in the present case, to a maximum wave-number transfer q max å 0.5 nm1. Fig. 1 shows the natural logarithm of the scattered intensity I(0, P, c) as a function of q 2. In a q-domain where the Guinier approximation is valid, the slope of the linear regression is related to the value of the apparent radius of gyration R g (P, c). Its value increases with increasing pressure P. The estimated error of the apparent radius of gyration obtained from the regression analysis varies from T 1% to T 2% according to the protein concentration and pressure. The value of the actual radius of gyration R g(P, 0) was inferred from the apparent radius of gyration R g(P, c) using the following relation: 2 R2 g ðP; cÞ ¼ Rg ðP; c ¼ 0Þ½1 þ 2B2 ðPÞMp cðPÞ þ N
ð2Þ
where B 2(P) has a similar meaning as the second virial coefficient A 2(P) in Eq. (3) describing the interaction between macromolecules. Fig. 3 shows the square of the inverse of R g (P, c) as a function of the protein concentration, c, for the two studied pressure (P = 50 and 150 MPa) at which the protein is stable. The protein concentration c(P) is calculated using the tabulated values of the H2O density as a function of pressure P given in the NBS Steam Tables [32]. According to Eq. (2), the intercept at c = 0 of the linear regression of R g 2(P, c) versus c(P) allows the actual radius of gyration R g(P, c = 0) to be determined. The values at P = 50 and 150 MPa are R g = (2.06 T 0.05) nm and (2.21 T 0.05) nm, respectively, and are shown in Fig. 4. The error of the actual radius of gyration R g(P, c = 0) is given by the regression analysis and varies between T 1.5 and T 2%. The Fig. 1. Logarithm of the scattered intensity, I(q, P, c), as a function of the square of the wave-number transfer, q. h-lactoglobulin concentration, c(P = 0.1 MPa): (a) 7.1 mg cm3, (b) 10.7 mg cm3, and (c) 14.3 mg cm3. Pressure, P: (o) 50 MPa, (g) 150 MPa, and (N) 300 MPa. The full lines are a fit with Eq. (1). Temperature is 20 -C.
Therefore, the spectra at 50 and 150 MPa are averaged over several spectra. At a pressure around 300 MPa, the scattered intensity at low q increases with time as shown in Fig. 2. This indicates that a pressure-induced aggregation of the macromolecules occurs in the sample. To confirm the irreversible mechanism of the protein aggregation, the pressure was then lowered from 300 MPa to 150 MPa: the scattered intensity does not decrease to the value initially measured at 150 MPa, but rather remains constant (Fig. 2). The data reduction of the scattered spectra is based upon the analysis as thoroughly discussed by Loupiac et al. [15]. The scattered intensity from a protein can be described by the
Fig. 2. Time variation of the scattered intensity I(q, P, c) as a function of the wave-number transfer, q. h-lactoglobulin concentration c(P = 0.1 MPa) = 7.1 mg cm3, P = 300 MPa. (N), (‚) and (Q): Spectra recorded during 2 h successively. The irreversibility of the aggregation mechanism is evidenced when the pressure is lowered from 300 MPa to 150 MPa, ( ).
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Fig. 4. Value of the actual radius of gyration, R g(c = 0), as a function of pressure, P.
Fig. 5 shows the second virial coefficient A 2 as a function of pressure P. The positive sign of A 2 at 50 and 150 MPa shows that the interaction between two dimers is repulsive. It appears that this repulsive interaction becomes weaker by increasing the pressure from 50 to 150 MPa. A large increase of A 2 is observed at P = 300 MPa but this value is very approximate if one considers the amount of aggregates that are formed at this pressure (see Fig. 2). Fig. 3. Inverse of the square of apparent radius of gyration, R g(P, c), as a function of the BLG concentration, c(P). (a) P = 50 MPa; (b) P = 150 MPa. The solid line is a linear regression to determine the value of the actual radius of gyration at vanishing protein concentration.
value at P = 50 MPa (2.06) nm is very close to the one obtained either by SAXS (2.10 nm) [26] or by SANS (between 2.00 and 2.26 nm) (without pressure) [23,33] measurements (see Table 1). Applying pressure from 50 to 150 MPa gives a 7% increase of R g. In Fig. 4, the value of R g at P = 300 MPa is also shown. However, the growth of the forward-scattered intensity (see Fig. 1) suggests a pressure-induced aggregation mechanism. Thereby the value of R g at P = 300 MPa which shows a large increment around 30% in comparison to the one at 50 MPa is biased by the aggregation process. The inverse of the forward-scattered intensity I(q = 0, P, c) from an hydrated protein of molecular weight M p and concentration c(P) has the following virial expansion: cðPÞ 1 NA ¼ 1 þ 2A2 ð PÞMp cð PÞ þ N 2 M Ið0; P; cÞ P K ð PÞ
4. Discussion One of the questions addressed in this work is to know whether or not the aggregation process induced by applying pressure involves the dimeric unit or monomeric unit of the protein. This has been answered by the analysis of the evolution of the radius of gyration as a function of applied pressure. The behavior of the forward-scattered intensity allows the critical pressure at which the aggregation process takes place to be determined and the change of the protein interactions induced by pressure to be studied by means of the second virial coefficient. The measured value of the actual radius of gyration of the protein, R g (P = 50 MPa) = 2.06 nm, is very close to the one obtained in other studies for h-lactoglobulin dimeric forms [23,26]. To analyze correctly the pressure effects on the value
ð3Þ
where K(P) is the pressure dependent specific contrast of the protein molecule with respect to the buffer, N A is the Avogadro number, and A 2(P) is the second virial coefficient. Eq. (3) is correct as far as the solute concentration is low, meaning that higher order terms in c(P) are negligible. The coefficient A 2(P) describes the interaction between two proteins: For repulsive interactions it is positive, whereas for attractive proteins it is negative [34]. According to Eq. (3), the slope of c(P)[K(P)]2/ I(0,P,c) vs. c(P) allows the second virial coefficient, A 2(P), to be determined.
Fig. 5. Second virial coefficient, A 2(P), as a function of pressure, P.
C. Loupiac et al. / Biochimica et Biophysica Acta 1764 (2006) 211 – 216 Table 1 Values of the apparent radius of gyration R g obtained in previous studies by different scattering methods and in various experimental conditions Methods
R g (nm)
Experimental conditions
Reference
SANS SAXS SANS SAXS
2.26 2.10 2.00 1.29
pH pH pH pH
[33] [26] [23] [35]
6.9, 0.1 M NaCl 7, 10 mM Tris 7, 0.1 M ammonium acetate 2, 0.1 M NaCl
of the radius of gyration, the measurements were made at different protein concentrations and the results extrapolated at vanishing protein concentration. Our analysis shows that the size of the h-lactoglobulin increases with increasing pressure. A change of 7% of the actual radius of gyration is observed between 50 MPa and 150 MPa and an increase of 31% is observed when applying a pressure of 300 MPa. At this highest pressure, the measured value is approximate because of the beginning of an aggregation process. In a study concerning the structure and the distribution of aggregates formed after a heat process, Gimel at al., [23] give the geometrical parameters of the native dimeric units of h-lactoglobulin determined by neutron and light scattering measurements. To support our discussion, we listed in Table 1 the values of the radius of gyration obtained in previous studies. The dimeric protein is represented by two connected spheres with radius 1.7 nm and a distance from center to center around 3.3 nm with a 2 nm radius of gyration very close to our measurement. Pessen at al. [35] have determined the radius of gyration of the monomeric form of protein at pH 2 by SAXS at atmospheric pressure and gave a value around 1.29 nm. Therefore, we can affirm that no dissociation of the dimer occurs in the 50 – 150 MPa pressure range as our measured radius of gyration is far away from the monomeric one. It is possible that the protein at 150 MPa could be in a swollen state. Different parameters could lead to this swollen state of the protein: hindrance of water inside the protein matrix and/or changes in the hydrogen bonds network, and/ or breaking down the electrostatic bonds and some of the protein hydrophobic interactions [13,27]. This description is in good agreement with previous measurements made by fluorescence [25] which show that the tryptophan (Trp) residues of the protein after applying pressure becomes part of a more hydrophilic surrounding. This change of exposure can be interpreted as an opening of the structure leading the Trp residues to be more solvent exposed or by the new proximity of water molecule inside the protein matrix. Applying pressure gives rise to a reorganization of the protein water interaction. These changes of water organization imply local structural reorganization of amino acids. Inside the h-lactoglobulin exists a large hydrophobic cavity which should be destabilized by the water entrance in the matrix. One way to keep the protein structure intact could be to swell to decrease the antagonist interactions between hydrophobic amino acids and water. This reorganization could initiate the unfolding of the protein. Then, the thiolate of the hlactoglobulin, initially buried inside the protein, becomes
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very exposed to the solvent. This group is then much more reactive and in these pH conditions (pH = 7) can form intermolecular disulfide bridge, leading to the final irreversible aggregate state. The implication of the free thiolate of the protein in the aggregation after high pressure treatments has been shown in previous studies [24,25]. In the same pH conditions and similar protein concentrations, Funterberger et al., [24] have shown by gel electrophoresis that high pressure promotes BLG aggregation through SH/S –S interchanges reactions. The pressure induced formation of S –S intermolecular bonded oligomers. This mechanism is in good agreement with the change of values we observed for the second virial coefficient (A 2) of the protein between 50 MPa and 150 MPa going respectively from 8.7 104 cm3 mol g2 to 5 104 cm3 mol g2. Positive values of A 2 indicate that the interactions between two protein molecules in the solution are repulsive, and the decrease with pressure shows that this repulsive interaction becomes weaker by increasing the pressure from 50 to 150 MPa. This change of interaction could be linked to the change of hydration organization suggested as hypothesis before to justify the increase of size of the protein. Breaking down hydrophobic interactions, salt bridges and electrostatic bonds of the protein leads to a change of the overall charge repartition of the protein. The macromolecules in this swollen state, as there are less repulsive, can interact together and initiate the aggregation process. Also, the aggregation process is clearly observed in our spectra for pressure around 300 MPa whatever the protein concentration studied. We therefore confirm by the present measurements that relatively high pressures lead to an aggregation process. After applying 300 MPa pressure, we have released the pressure to 150 MPa. The results showed that this process is irreversible. It can be stated in these experimental conditions (protein concentration, pH, salt) that a pressure value around 150 MPa leads to a swollen state of the h-lactoglobulin and that at a pressure around 300 MPa the protein begins to form irreversible aggregates. This aggregation occurs between dimeric units of the protein. In the future it will be interesting to see the repercussion of this aggregation between dimeric units on the gel properties. Small-angle neutron scattering measurements allows the range of the wave-number transfer to be easily modified in order to probe either the macromolecule scale (this study) or the gel organization. References [1] D. Knorr, Novel approaches in food-processing technology: new technologies for preserving foods and modifying function, Curr. Opin. Biotechnol. 10 (1999) 485 – 491. [2] B. Krebbers, A.M. Matser, M. Koets, P.V. Bartels, R.W. Van den Berg, High pressure – temperature processing as an alternative for preserving basil, High Press. Res. 22 (2002) 711 – 714. [3] J.C. Cheftel, Review: high pressure, microbial inactivation and food preservation, Food Sci. Technol. Int. 1 (1995) 75 – 90. [4] W. Messens, J. Van Camp, A. Huyghebaert, The use of high pressure to modify the functionality of food proteins, Trends Food Sci. Technol. 8 (1997) 107 – 112.
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