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Interactions in micellar solutions of β-casein
ELSEVIER
Physica B 234 236 (1997) 207-209
Interactions in micellar solutions of j3-casein E. Leclerc, P. Calmettes* Laboratoire Ldon Brillouin (CEA-CNRS), CEA-Saelay, 91191 Gif-sur- Yvette, Cedex, France
Abstract
[3-casein is a protein which forms micelles in aqueous solvents. The magnitude and the range of the weight-average interactions between the diverse solute particles are inferred from small-angle neutron scattering measurements made on various [3-casein solutions. Well above the critical micelle concentration (CMC), these interactions are repulsive. They weaken with decreasing protein concentration, and finally become strongly attractive near the CMC. Although indispensable for micelle formation this fact has never been reported so far.
Keywords: Micelles; Proteins; Biological structures; Small-angle neutron scattering
Little attention has been paid to the interactions existing between the different structural species present in very dilute micellar solutions. Most of the experimental works concerns large-micelle systems where the isolated molecules contribute only slightly to the solution properties [1, 2]. Due to both the smallness of usual amphiphilic molecules and the very low values of the critical micelle concentration (CMC) small-micelle solutions are very difficult to study experimentally. Nevertheless, the presence of specific interactions between the various solute particles has been recognized. However, their theoretical description remains challenging, especially when experimental information is lacking [-3, 4]. The aim of this paper is to provide such information. To this end, small-angle neutron scattering (SANS) was used to study the structure of [3-casein solutions near the CMC. [3-casein is an amphiphilic milk protein of 24 kg/mol. Starting from the N-terminus, many of the first 50 amino acids are hydrophilic at pH
* Corresponding author.
7 whereas the remaining 159 residues are mainly hydrophobic. Consequently, [3-casein can form micelles in aqueous solvents [5]. This protein is almost devoid of secondary and tertiary structure and may behave as a random coil [6]. [3-casein was purified from the skimmed milk of a single cow, according to the method of Mercier et al. [7]. It was exhaustively dialyzed against D / O buffered with 0.1 M Na phosphates and 0.1 M NaC1 at pH 7. SANS experiments were carried out at protein concentrations, c, ranging from 0.64 to 10 mg/cm a and at various temperatures T, between 4.5 and 70°C. Scattering spectra, I(q), were recorded f o r 7 x l 0 - 3 ~ < q ~ < 7 x l 0 - 2 A- 1,whereqis the wave number transfer. The data were processed as usual. The small q regions of the solute spectra may be described by the Guinier approximation
I(q;c)~-I(O;c) exp [ - q2R'g2(c)/3],
(1)
where I(0;c) is the forward scattered intensity and R'g(c) the apparent radius of gyration of the scatterers. The forward intensity can be written I(O;c) =
0921-4526/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved PII $ 0 9 2 1 - 4 5 2 6 ( 9 6 ) 0 0 9 1 3- 1
KcMw(c) / [1 + B.(c) Mw(c) c],
(2)
208
E. Leclerc, P. Calmettes / Physica B 234-236 (1997) 207-209
where K ~ 10- 3 mol c m 2 / g 2 sr is an experimental constant. Mw(c) is the weight average of the solute molecular weight and Bw(c) is a mean interaction parameter related to the virial coefficients of the osmotic pressure. Guinier plots of some SANS spectra are shown in Fig. 1. Except for particular values of c and T, I(0) and Rg are found to depend on the range of q used to fit Eq. (1) to the spectra. Since the validity of Eq. (1) is limited to small values of qR'g, a theoretical model is required to describe the micelle structure at larger q values. A relatively dense spherical core surrounded by a spherical corona of lower density was found to describe quite well all the spectra up to q = 0.6 × 10- 2 /~- 1. This is consistent with the usual picture of micelle structure I-8]. In the range of c and T investigated the core density varies between 0.4 and 0.9 g / c m 3 whereas the density of a globular protein is about 1.35g/cm 3. The density of the outer shell is much lower, between 0.025 and 0.14 g / c m 3, respectively. The previous model was used to infer from each spectrum the value of the forward scattered intensity would have in absence of interaction. The actual value of the forward scattered intensity I(0), was deduced from the small q region of the spectra
(q ~< 1.4 x 10 -2 A - l ) by means of the Guinier approximation. Using Eq. (2), these two values allow the quantity Bw(c) M,~(c) to be estimated. The results are shown in Fig. 2. Close to the CMC ( ~ 1 mg/ml) the mean interactions are attractive and concentration dependent. Furthermore, they spread over about 100 A. When either the protein concentration or the temperature is high enough the mean interactions become weakly repulsive. Their range agrees well with what is expected from the micelle morphology for usual streric repulsions. Its value is indeed always larger than the diameter of the dense core and smaller than the one of the whole micelle. To conclude, it must be stressed that interactions are an important feature of micellar systems. Near the CMC the mean interactions are strongly attractive. This result is satisfactory because it explains well why micelles can form. It reflects the existence of large fluctuating aggregates of few molecules whose structure is much looser than that of the full-grown micelles formed well beyond the CMC. Full-grown micelles interact as soft spheres. This is in fair agreement with their internal structure consisting of a relatively dense hydrophobic core surrounded by a penetrable hydrophilic shell of lower density.
t
~
l
i
I
i
i
i
I
I
t
i
i
I
-1
5
-2
-3
i
-4 0
i
5 x 10 "4
10 "I
q 2 (A-2)
Fig. 1. Guinier plots of the neutron scattering spectra I(q), of 13-casein at 4.5 °C. The protein concentrations are 1.25 (0), 2.5 (©), 5.0 (11) and 10 mg/cm 3 (D). Full lines are the results of the fit to the model described in the text.
E. Leclerc, P. Calmettes / Physica B 234-236 (1997) 207 209
209
50
A
E
-50
-100
-150
-200 2
4
6
8
10
12
C (mg/cm 3)
Fig. 2. Variation of the interaction coefficient Bw(c) M~(c) with the protein concentration c. Bw(c) M~(c) is defined by Eq. (2). The temperatures are 4.5 (11), 9.6 (CO), 15 (e) and 23 °C (O). The solid lines are guide for the eye.
References [1] K.L. Mittal and B. Lindman, eds., Surfactants in Solution, Vol. l-3 (Plenum, New York, 1984). [2] Proc. Int. School of Physics Enrico Fermi, Physics of Amphiphiles : Micelles, Vesicles and Microemulsions, eds. V. Degiorgio and M. Corti (North-Holland, Amsterdam, 1985). [3] W.M. Gelbart, in: Micelles, Membranes, Microemulsions and Monolayers, eds. W.M. Gelbart, A. Ben-Shaul and D. Roux (Springer, New York, 1994) pp. 1-104.
[4] D. Blankschtein, G.M. Thurston and G.B. Benedek, J. Chem. Phys. 85 (1986) 7268. [5] D.G. Schmidt and T.A.J. Payens, J. Colloid Interface Sci. 39 (1972) 655. [6] C. Tanford, Advan. Protein Chem. 23 (1968) 121. [7] J.C. Mercier, J.L. Maubois, S. Poznanski and B. RibadeauDumas, Bull. Soc. Chim. Biol. 50 (1968) 521. [8] J.B. Hayter in Ref. [2] pp. 59-93.
Physica B 234 236 (1997) 207-209
Interactions in micellar solutions of j3-casein E. Leclerc, P. Calmettes* Laboratoire Ldon Brillouin (CEA-CNRS), CEA-Saelay, 91191 Gif-sur- Yvette, Cedex, France
Abstract
[3-casein is a protein which forms micelles in aqueous solvents. The magnitude and the range of the weight-average interactions between the diverse solute particles are inferred from small-angle neutron scattering measurements made on various [3-casein solutions. Well above the critical micelle concentration (CMC), these interactions are repulsive. They weaken with decreasing protein concentration, and finally become strongly attractive near the CMC. Although indispensable for micelle formation this fact has never been reported so far.
Keywords: Micelles; Proteins; Biological structures; Small-angle neutron scattering
Little attention has been paid to the interactions existing between the different structural species present in very dilute micellar solutions. Most of the experimental works concerns large-micelle systems where the isolated molecules contribute only slightly to the solution properties [1, 2]. Due to both the smallness of usual amphiphilic molecules and the very low values of the critical micelle concentration (CMC) small-micelle solutions are very difficult to study experimentally. Nevertheless, the presence of specific interactions between the various solute particles has been recognized. However, their theoretical description remains challenging, especially when experimental information is lacking [-3, 4]. The aim of this paper is to provide such information. To this end, small-angle neutron scattering (SANS) was used to study the structure of [3-casein solutions near the CMC. [3-casein is an amphiphilic milk protein of 24 kg/mol. Starting from the N-terminus, many of the first 50 amino acids are hydrophilic at pH
* Corresponding author.
7 whereas the remaining 159 residues are mainly hydrophobic. Consequently, [3-casein can form micelles in aqueous solvents [5]. This protein is almost devoid of secondary and tertiary structure and may behave as a random coil [6]. [3-casein was purified from the skimmed milk of a single cow, according to the method of Mercier et al. [7]. It was exhaustively dialyzed against D / O buffered with 0.1 M Na phosphates and 0.1 M NaC1 at pH 7. SANS experiments were carried out at protein concentrations, c, ranging from 0.64 to 10 mg/cm a and at various temperatures T, between 4.5 and 70°C. Scattering spectra, I(q), were recorded f o r 7 x l 0 - 3 ~ < q ~ < 7 x l 0 - 2 A- 1,whereqis the wave number transfer. The data were processed as usual. The small q regions of the solute spectra may be described by the Guinier approximation
I(q;c)~-I(O;c) exp [ - q2R'g2(c)/3],
(1)
where I(0;c) is the forward scattered intensity and R'g(c) the apparent radius of gyration of the scatterers. The forward intensity can be written I(O;c) =
0921-4526/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved PII $ 0 9 2 1 - 4 5 2 6 ( 9 6 ) 0 0 9 1 3- 1
KcMw(c) / [1 + B.(c) Mw(c) c],
(2)
208
E. Leclerc, P. Calmettes / Physica B 234-236 (1997) 207-209
where K ~ 10- 3 mol c m 2 / g 2 sr is an experimental constant. Mw(c) is the weight average of the solute molecular weight and Bw(c) is a mean interaction parameter related to the virial coefficients of the osmotic pressure. Guinier plots of some SANS spectra are shown in Fig. 1. Except for particular values of c and T, I(0) and Rg are found to depend on the range of q used to fit Eq. (1) to the spectra. Since the validity of Eq. (1) is limited to small values of qR'g, a theoretical model is required to describe the micelle structure at larger q values. A relatively dense spherical core surrounded by a spherical corona of lower density was found to describe quite well all the spectra up to q = 0.6 × 10- 2 /~- 1. This is consistent with the usual picture of micelle structure I-8]. In the range of c and T investigated the core density varies between 0.4 and 0.9 g / c m 3 whereas the density of a globular protein is about 1.35g/cm 3. The density of the outer shell is much lower, between 0.025 and 0.14 g / c m 3, respectively. The previous model was used to infer from each spectrum the value of the forward scattered intensity would have in absence of interaction. The actual value of the forward scattered intensity I(0), was deduced from the small q region of the spectra
(q ~< 1.4 x 10 -2 A - l ) by means of the Guinier approximation. Using Eq. (2), these two values allow the quantity Bw(c) M,~(c) to be estimated. The results are shown in Fig. 2. Close to the CMC ( ~ 1 mg/ml) the mean interactions are attractive and concentration dependent. Furthermore, they spread over about 100 A. When either the protein concentration or the temperature is high enough the mean interactions become weakly repulsive. Their range agrees well with what is expected from the micelle morphology for usual streric repulsions. Its value is indeed always larger than the diameter of the dense core and smaller than the one of the whole micelle. To conclude, it must be stressed that interactions are an important feature of micellar systems. Near the CMC the mean interactions are strongly attractive. This result is satisfactory because it explains well why micelles can form. It reflects the existence of large fluctuating aggregates of few molecules whose structure is much looser than that of the full-grown micelles formed well beyond the CMC. Full-grown micelles interact as soft spheres. This is in fair agreement with their internal structure consisting of a relatively dense hydrophobic core surrounded by a penetrable hydrophilic shell of lower density.
t
~
l
i
I
i
i
i
I
I
t
i
i
I
-1
5
-2
-3
i
-4 0
i
5 x 10 "4
10 "I
q 2 (A-2)
Fig. 1. Guinier plots of the neutron scattering spectra I(q), of 13-casein at 4.5 °C. The protein concentrations are 1.25 (0), 2.5 (©), 5.0 (11) and 10 mg/cm 3 (D). Full lines are the results of the fit to the model described in the text.
E. Leclerc, P. Calmettes / Physica B 234-236 (1997) 207 209
209
50
A
E
-50
-100
-150
-200 2
4
6
8
10
12
C (mg/cm 3)
Fig. 2. Variation of the interaction coefficient Bw(c) M~(c) with the protein concentration c. Bw(c) M~(c) is defined by Eq. (2). The temperatures are 4.5 (11), 9.6 (CO), 15 (e) and 23 °C (O). The solid lines are guide for the eye.
References [1] K.L. Mittal and B. Lindman, eds., Surfactants in Solution, Vol. l-3 (Plenum, New York, 1984). [2] Proc. Int. School of Physics Enrico Fermi, Physics of Amphiphiles : Micelles, Vesicles and Microemulsions, eds. V. Degiorgio and M. Corti (North-Holland, Amsterdam, 1985). [3] W.M. Gelbart, in: Micelles, Membranes, Microemulsions and Monolayers, eds. W.M. Gelbart, A. Ben-Shaul and D. Roux (Springer, New York, 1994) pp. 1-104.
[4] D. Blankschtein, G.M. Thurston and G.B. Benedek, J. Chem. Phys. 85 (1986) 7268. [5] D.G. Schmidt and T.A.J. Payens, J. Colloid Interface Sci. 39 (1972) 655. [6] C. Tanford, Advan. Protein Chem. 23 (1968) 121. [7] J.C. Mercier, J.L. Maubois, S. Poznanski and B. RibadeauDumas, Bull. Soc. Chim. Biol. 50 (1968) 521. [8] J.B. Hayter in Ref. [2] pp. 59-93.