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Langmuir 2001, 17, 1896-1904
Structure and Properties of Adsorption Layers of β-Casein Formed from Guanidine Hydrochloride Rich Solutions Adel Aschi,† Abdelhafidh Gharbi,† Lotfi Bitri,‡ Patrick Calmettes,§ Mohamed Daoud,| Ve´ronique Aguie´-Be´ghin,⊥ and Roger Douillard*,⊥ Laboratoire de Physico-Chimie de la Matie` re Condense´ e and Laboratoire de Biochimie et de Technobiologie, Faculte´ des Sciences de Tunis, Campus Universitaire, 2092 El Menzah 9 Tunis, Tunisie, Laboratoire Le´ on Brillouin and Service de Physique de l’Etat Condense´ , C.E. Saclay, 91191 Gif sur Yvette Cedex, France, and E Ä quipe de Biochimie des Macromole´ cules Ve´ ge´ tales, Centre de Recherche Agronomique, 2 Esplanade R. Garros, BP 224, 51686 Reims Cedex 2, France Received August 10, 2000. In Final Form: December 8, 2000 Adsorption layers of β-casein formed at the interface between air and a buffer including various concentrations of guanidine hydrochloride (GuHCl) were studied by neutron reflectivity and by bubble tensiometry. A transition in the structure and in the properties of the adsorption layer seems to occur around a GuHCl concentration of 1.5 M. The data are interpreted assuming that the adsorbed protein molecules behave like multiblock copolymers with alternating hydrophilic and hydrophobic sequences. Below the transition, the hydrophilic coils and the hydrophobic two-dimensional blocks have a fractal dimension larger than that beyond the transition where they have the features of either two-dimensional or three-dimensional excluded volume coils. The effect of temperature on these phenomena indicates that they are not dominated by hydrophobic interactions. Thus, the attractions between amino acids which are broken by GuHCl might be hydrogen bonds which are frequently encountered in the secondary structure of polypeptide chains. These results show that even with the flexible polypeptide chain of β-casein, interactions between amino acids contribute significantly to the structure of the adsorption layer formed from a buffer devoid of denaturing agent.
1. Introduction Protein molecules are amphiphilic, because of the occurrence of polar and nonpolar side chains in the amino acids, forming their polymeric chain. This makes the protein molecules surface active, so that they adsorb at gas-liquid, liquid-liquid, and solid-liquid interfaces.1,2 Such an adsorption allows the stabilization of colloidal dispersions which are thermodynamically unstable and which otherwise phase separate quickly. Thus, proteins play a key role in the stabilization of foams, emulsions, and composite systems in food products and in cosmetics.3-5 In these fields, the use of proteins as stabilizers has been mostly empirical for a long while and is presently being rationalized. Their use is still growing because unlike * Corresponding author: Roger Douillard, E Ä quipe de Biochimie des Macromole´cules Ve´ge´tales, Centre de Recherche Agronomique, 2 Esplanade R. Garros, BP 224, 51686 Reims Cedex 2, France. Tel: 33 3 26 77 35 94. Fax: 33 3 26 77 35 99. E-mail: Roger.Douillard@ reims.inra.fr. † Laboratoire de Physico-Chimie de la Matie ` re Condense´e, Campus Universitaire. ‡ Laboratoire de Biochimie et de Technobiologie, Campus Universitaire. § Laboratoire Le ´ on Brillouin, C.E. Saclay. | Service de Physique de l’Etat Condense ´ , C.E. Saclay. ⊥ E Ä quipe de Biochimie des Macromole´cules Ve´ge´tales, Centre de Recherche Agronomique. (1) Proteins at liquid interfaces; Mo¨bius, D., Miller, R., Eds.; Elsevier: Amsterdam, 1998; p 498. (2) Brash, J. L.; Wojciechowski, P. W. Interfacial phenomena and bioproducts; Marcel Dekker: New York, 1996; p 510. (3) Mangino, M. E. Protein interactions in emulsions: protein-lipid interactions. In Protein functionality in food systems; Hettiarachchy, N. S., Ziegler, G. R., Eds.; Marcel Dekker: New York, 1994; pp 147179. (4) German, J. B.; Phillips, L. Protein interactions in foams: proteingas-phase interactions. In Protein functionality in food systems; Hettiarachchy, N. S., Ziegler, G. R., Eds.; Marcel Dekker: New York, 1994; pp 181-208.
numerous synthetic amphiphiles they are edible and biodegradable. The mechanism of adsorption of proteins at interfaces has been extensively studied at the air-buffer interface. In the case of a usual soluble globular protein such as bovine serum albumin, it is believed to proceed through a diffusion step6 and partial unfolding of the molecule at the interface.7-13 This mechanism has also been often studied with β-casein whose structure in solution is rather flexible.14 Thus, it looks more or less like a “naturally unfolded protein”. In fact, it seems that an important number of proline units in its polypeptide chain imposes such an open structure instead of a globular or moltenglobule-like structure.15 Its molecular mass is 23 980 for 209 amino acids, and its sequence consists of three irregular alternating hydrophilic-hydrophobic diblocks.16 A salient feature of its sequence is the occurrence of a 50 amino acid long hydrophilic block at its N-terminal end. (5) Phillips, L. G.; Whitehead, D. M.; Kinsella, J. Structure-function properties of food proteins; Academic Press: San Diego, CA, 1994; p 271. (6) Ybert, C.; di Meglio, J. M. Langmuir 1998, 14, 471-475. (7) Birdi, K. S. Lipid and biopolymer at liquid interfaces; Plenum Press: New York, 1989. (8) Damodaran, S. Adv. Nutr. Res. 1990, 34, 1. (9) Sadana, A. Chem. Rev. 1992, 92, 1799. (10) Ball, A.; Jones, R. A. L. Langmuir 1995, 11, 3542. (11) Claesson, P. M.; Blomberg, E.; Fro¨berg, J. C.; Nylander, T.; Arnebrant, T. Adv. Colloid Interface Sci. 1995, 57, 161. (12) Stahmann, K. P.; Bo¨ddecker, T.; Sahm, H. Eur. J. Biochem. 1997, 224, 220. (13) Izmaı¨lova, V. N.; Yampolskaya, G. P. In Protein at liquid interfaces; Mo¨bius, D., Miller, R., Eds.; Elsevier: Amsterdam, 1998; p 103. (14) Swaisgood, H. E. In Development in Dairy Chemistry; Fox, P. F., Ed.; Elsevier Applied Science Publishers: London, 1982; Vol. 1, p 1-58. (15) Fink, A. L. In Methods in Molecular Biology: Theory and Practice, Vol. 40; Humana Press Inc.: Totowa, NJ, 1995; p 343. (16) Dalgleish, D. G.; Leaver, J. J. Colloid Interface Sci. 1991, 141, 288-294.
10.1021/la001159s CCC: $20.00 © 2001 American Chemical Society Published on Web 02/15/2001
Adsorption Layers of β-Casein
It is believed that when it adsorbs at an interface the unfolding (or conformation change) is much reduced as compared to the case of a globular protein. Its surface properties have been studied for more than 20 years.16-26 Surface concentration, surface pressure, dilational modulus, and concentration profile of its adsorption layers are now well documented. A polymer model has been developed recently to describe its surface properties as those of a multiblock copolymer, that is, a polymer made of alternating hydrophilic and hydrophobic sequences.27 This approach might be valid if the adsorbed molecule behaves in fact as a polymer: swollen three-dimensional coils for the hydrophilic blocks in solution and two-dimensional excluded volume coils for the hydrophobic blocks at the air-water interface (it is difficult to define the nature of the solvent of a block lying flat at the interface; nevertheless, its structure defined as a self-avoiding walk implies that it has a fractal structure, just as a three-dimensional coil swollen by a solvent). However, polypeptide chains are known to interact heavily with themselves through hydrogen bonding of the peptide bonds, in helices and β-sheets, for instance,28 or through hydrophobic interactions.29 Thus, to check if some residual interactions of the polypeptide chain with itself are significant features of the adsorbed molecule, an investigation was undertaken to compare the properties of β-casein adsorption layers formed from a buffered solution with or without a strong denaturant of proteins, guanidine hydrochloride (GuHCl). For a large enough concentration in the bulk, this compound is known to unfold most proteins completely and to make them behave as polymers freely swollen by the solvent.30-36 2. Material and Methods Materials. β-Casein was obtained from the skimmed milk of a single cow homozygous for the three major caseins (Rs1B, βB, and κB), purified according to the method of Mercier et al. (1968),37 (17) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1978a, 70, 403-414. (18) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1978b, 70, 415-426. (19) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1978c, 70, 427-439. (20) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1980a, 76, 227-239. (21) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1980b, 76, 240-250. (22) Damodaran, S.; Song, K. Colloids Surf. 1990, 50, 75-86 (23) Dickinson, E.; Horne, D. S.; Phipps, J. S.; Richardson, R. M. Langmuir 1993, 9, 242-248. (24) Nylander, T.; Wahlgren, N. M. Langmuir 1997, 13, 6219-6225. (25) Mellema, M.; Clark, D. C.; Husband, F. A.; Mackie, A. R. Langmuir 1998, 14, 1753-1758. (26) Harzallah, B.; Aguie´-Be´ghin, V.; Douillard, R.; Bosio, L. Int. J. Biol. Macromol. 1998, 23, 73-84. (27) Aguie´-Be´ghin, V.; Leclerc, E.; Daoud, M.; Douillard, R. J. Colloid Interface Sci. 1999, 214, 143. (28) Richardson, J. S.; Richardson, D. C. In Prediction of protein structure and the principles of protein conformation; Fasman, G. D., Ed.; Plenum Press: New York, 1989; pp 1-98. (29) Poupon, A.; Mornon, J. P. Proteins 1998, 33, 329-342. (30) Tanford, C. Adv. Protein Chem. 1968, 23, 121-282. (31) Shirley, B. A. In Protein stability and folding: theory and practice; Shirley, B. A., Ed.; Humana Press Inc.: Totowa, NJ, 1985; pp 177-190. (32) Calmettes, P.; Durand, D.; Smith, J. C.; Desmadril, D.; Minard, P.; Douillard, R. J. Phys. IV 1993, 3, 253-256. (33) Calmettes, P.; Roux, B.; Durand, D.; Desmadril, D.; Smith, J. C. J. Mol. Biol. 1993, 231, 840-848. (34) Calmettes, P.; Durand, D.; Desmadril, D.; Minard, P.; Douillard, R. Physica B 1995, 213/214, 754-756. (35) Gupta, R.; Yadav, S.; Ahmad, F. Biochemistry 1996, 35, 1192511930. (36) Zhou, J. M.; Fan, Y. X.; Kihara, H.; Kimura, K.; Amemiya, Y. FEBS Lett. 1997, 415, 183-185. (37) Mercier, J. C.; Maubois, J. L.; Poznanski, S.; Ribadeau-Dumas, B. Bull. Soc. Chim. Biol. 1968, 50, 521.
Langmuir, Vol. 17, No. 6, 2001 1897 and freeze-dried. Acid-precipitated casein was fractionated by ion exchange chromatography on a DEAE column (5 PW, Waters) using a NaCl gradient in a 20 mM imidazole buffer at pH 7 including 3.3 M urea and 1 mM dithiothreitol (DTT). The fraction corresponding to β-casein was rechromatographed in the same conditions, and its purity was checked by polyacrylamide gel electrophoresis. The extinction coefficient used to determine the volume concentration of this protein is E1cm1% ) 4.6 at 278 nm. The neutron coherent scattering length density of β-casein (the Nb parameter of the neutron reflectivity experiments) in a fully deuterated solvent was calculated assuming total exchange of the mobile protons and found to be Nb ) 2.945 10-6 Å-2, assuming a partial specific volume of 0.742 cm3 g-1.14 Before use for neutron reflectivity, the solutions of β-casein were dialyzed against the bulk buffer which was deposited in the trough in order to match exactly the scattering length densities of the two solutions. Guanidine hydrochloride (Pierce, Sequanal Grade) was found to be contaminated by surface active molecules, as evaluated from a significant drift of the surface tension of a 1 M solution after 12 h. It was further purified by crystallization in water until no surface tension drift was noticed. Its protons, which are all mobile, were exchanged with deuterons as described previously.38 The Nb of the fully deuterated compound was found to be Nb ) 7.184 10-6 Å-2. The molar concentration C of guanidine hydrochloride was obtained from the measurement of the index of refraction of the sample using the relation
C (M) ) 57.141 ∆n + 3.68(∆n)2 - 91(∆n)3
(1)
where ∆n is the difference between the index of refraction of the buffer with and without GuHCl.39 Surface Tension Measurements. For measurement of static or dynamic surface tension, a bubble (or drop) tensiometer (IT Concept, Longessaigne, France) was used.40 The surface tension was measured through shape analysis of an air bubble formed at the tip of a stainless steel needle dipped in the solution. In this way, protein depletion in the solution during the adsorption process is always negligible.41 The needle is attached to a syringe whose plunger is precisely controlled by a micrometer screw driven by an electric motor. The bubble is illuminated with a beam of parallel light. The image is recorded by a CCD camera and is digitized to allow the analysis of its shape. The interfacial tension measurements are based on the axisymmetric bubble shape analysis method in which the experimental profile of the bubble is analyzed according to the Laplace equation:
1 d(x sin θ) 2 ∆F ) -g z x dx b γ
(2)
where x and z are the Cartesian coordinates at any point of the bubble profile, b is the curvature radius at the apex of the bubble (x ) z ) 0), θ is the angle of the tangent to the bubble profile formed with the x axis, g is the acceleration of gravity, and ∆F is the difference of volumetric mass between the two fluids. The surface pressure is, as usual, the difference between the surface tension of the pure solvent γ0 and that of the solution with surface active molecules γ:
π ) γ0 - γ
(3)
The surface dilational modulus, , is defined as the ratio between the variation of surface tension, dγ, and the relative change in surface area, dA/A ) d ln(A):42 (38) Calmettes, P.; Durand, D.; Desmadril, D.; Minard, P.; Receveur, V.; Smith, J. C. Biophys. Chem. 1994, 53, 105-114. (39) Nozaki, Y. Methods Enzymol. 1970, 26, 43-50. (40) Labourdenne, S.; Gaudry-Rolland, N.; Letellier, S.; Lin, M.; Cagna, A.; Esposito, G.; Verger, R.; Rivie`re, C. Chem. Phys. Lipids 1994, 71, 163. (41) Miller, R.; Fainerman, V. B.; Wu¨stneck, R.; Kra¨gel, J.; Trukhin, D. V. Colloids Surf., A 1998, 131, 225-230. (42) Lucassen-Reynders, E. H. In Anionic Surfactants; Lucassen, E. H., Ed.; Dekker: New York, 1981; p 173.
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dγ d ln(A)
Aschi et al. (4)
It was determined during sine wave deformation of the area of the bubble performed by moving periodically the plunger of the syringe. The area and the surface tension were calculated several times a second. A Fourier transform of the data was performed, and only the first harmonic was retained. It was verified that the calculated dilational modulus was independent of the amplitude when this quantity was less than 20% of the area. The experimental setup allows measurements of at frequencies smaller than 0.2 Hz (2 surface tension and area measurements per second, 10 points during one period). At low frequencies, the adsorption of protein is significant during one period at the beginning of the adsorption and is not calculated in quasiequilibrium conditions. Thus, we have chosen a standard frequency of 0.1 Hz where the surface tension does not change significantly during one period, the bulk β-casein concentration being 0.1 g/L. No change of the y slope (see results and discussion section) was noticed between 0.1 and 0.2 Hz. In these conditions, the surface pressure was 17-22 mN/m after 20 min (1200 s). Neutron Reflectivity. Specular reflection was measured at the reflectometer DESIR in the Orphe´e reactor (Laboratoire Le´on Brillouin, Saclay). By analysis of the ratio R of reflected to incident intensity as a function of the wave-vector, q ) (4π/λ) sin(θ), information about the profile of the refractive index in the sample near the interface can be obtained. Variation in q is achieved by variation of the wavelength (polychromatic source). The reflectivity of an interface can be calculated from its refractive index profile n(z). The n(z) profile of the interfacial layer is divided into a number of uniform layers. Then, the Fresnel reflection and transmission coefficients are calculated for each interface and combined to give the total reflectivity from the adsorption layer. The refractive index is simply related to the neutron coherent scattering length density, Nb(z) by43
n(z) ) 1 -
λ2Nb(z) 2π
(5)
For a single homogeneous layer at the depth z, Nb(z) can be expressed in terms of the volume fraction, φ(z), of the molecule at a distance z from the interface:
Nb(z) ) φ(z) Nbm + (1 - φ(z))Nbs
(6)
where Nbm is the coherent scattering length density of the molecule and Nbs is that of the solvent. At the interface between air and a medium 2 of index n, Descarte’s law can be written as
cos(θ1) ) n cos(θ2)
(7)
where θ1 is the grazing-incidence angle and θ2 is the refraction angle. Total reflection occurs if θ1 is lower than θC, where θC is defined when θ2 ) 0, which means that
cos(θC) ) n
sin(θC) ) λC
x
λ ) (h/mL)t
(11)
where h is Planck’s constant and m is the neutron mass. The accessed range of the neutron wavelength was 3 e λ e 17.4 Å. All the reflectivity spectra were determined 8 h after pouring the solution in the trough so that the adsorption reaches a quasiequilibrium state. A background noise was determined for each spectrum between 2 and 3 Å where the intensity of the beam is practically zero. It was assumed to be constant throughout the whole spectrum and was subtracted from the measured spectra.
3. Theoretical Background The adsorption of β-casein at the air-buffer interface may be modeled as that of a multiblock copolymer exhibiting alternating blocks of hydrophobic and hydrophilic monomers. This kind of structure has been clearly evidenced by Dalgleish and Leaver16 for β-casein with three hydrophilic and three hydrophobic blocks. However, the most prominent block is the hydrophilic one at the N-terminal end, which accounts for roughly 50 amino acids. Thus, the question is that of the actual nature of the rest of the molecule: is it a hydrophobic C-terminal block or is it composed of five alternating hydrophobic and hydrophilic blocks? In other words, is β-casein composed of two blocks or of six blocks (three hydrophilichydrophobic diblocks)? However, the number of diblocks is small and this protein, once adsorbed, should behave more like a diblock than like a long polymer. The theory of adsorption of long polymers and of diblocks at the gas/ liquid interface has been developed recently27 and is briefly summarized hereafter. In the theory mentioned above, the polymer is composed of N diblocks each consisting of a hydrophobic block with ZA monomers and of a hydrophilic block with ZB monomers. The junctions between the hydrophilic and the hydrophobic blocks are supposed to stick strictly to the interface plane, and the blocks do not cross the interface. Once adsorbed, the polymer adopts a conformation where the hydrophobic sequences form two-dimensional pancakes in contact with the gas and with the liquid and where the hydrophilic sequences form three-dimensional coils in the liquid phase. The conformations of these pancakes and coils are defined as random self-avoiding walks and are characterized by a radius in the three-dimensional case or by a radius and a thickness in the two-dimensional case. Such a definition implies that each block has a fractal structure where the fractal dimension (D) is the reverse of the Flory exponent, ν:
(8)
Equations 5 and 8 lead to
Nb(0) π
technique enables separation of neutrons of various wavelength of a white beam pulse. The de Broglie relation gives the time t for a neutron with the associated wavelength λ to travel a distance L:
(9)
R = aZν
(12)
D ) 1/ν
(13)
The reflectivity spectra, R(q), were obtained at a fixed angle for wavelengths shorter than λC. The value of each wavelength is determined by the time-of-flight technique (TOF).44 This
where R is the radius of the block, a is the size of monomers (statistical units), and Z is their number in the block. The sign = means that all numerical coefficients are purposely ignored in the relation. When the surface concentration increases, the polymer molecules and the blocks overlap. The interface enters in successive semidilute regimes whose nature is determined partly by the ratio R ) ZA/ZB and partly by the surface concentration of adsorbed multiblocks. The various regimes and their properties have been calculated using
(43) Penfold, J.; Thomas, R. K. J. Phys.: Condens. Matter 1990, 2, 1369.
(44) Lee, L. T.; Mann, E. K.; Langevin, D.; Farnoux, B. Langmuir 1991, 7, 3076.
or, because θC is small,
θC ≈ λC
x
Nb(0) π
(10)
Adsorption Layers of β-Casein
Langmuir, Vol. 17, No. 6, 2001 1899
When the two-dimensional blocks impose their behavior to the interfacial layer, y g 3. When the properties of the interface are dominated by three-dimensional coils forced to form quasi-brushes (they are forced to have an extension in a direction perpendicular to the interface), y ) 1, whatever the fractal dimension of the coil in the dilute regime. Equation 14 holds for the surface pressure whether the surface concentration Γ is expressed as mass or molecules per unit surface. Taking into account the expressions of the dilational modulus (eq 4) and of the surface pressure (eq 3) and assuming that adsorption and desorption are not significant during one oscillation (m is constant in eq 15),
) Figure 1. Phase diagram of the regimes occurring on both sides of the gas-liquid interface as a function of the surface concentration and of the ratio R of hydrophobic to hydrophilic sequences. The unit of the vertical axis is the reduced surface concentration G ) Γ/Γ/pol where Γ is the surface concentration and Γ/pol is the overlap surface concentration between the dilute (gaseous) regime and the first semidilute regime of the interface. / G/pol is the overlap concentration of the polymer chains; Gblock , / is the overlap concentration of the B block sequences; Gpan is the overlap concentration of the A block sequences; G// pan is the overlap concentration of the monomers of the A block sequences; Gmelt is the overlap surface concentration at which the A block sequences begin to stretch (from ref 27). Abbreviations: D, dilute regime. The other regimes are defined by the properties of the polymer or of the two different blocks on each side of the interface. For the A block sequences on the gas side of the interface, the local regimes are as follows. p, isolated pancakes; SDp, two-dimensional semidilute regime of the polymer chain (the statistical units are the A block sequences); SDb, twodimensional semidilute regime of the block sequences (the statistical units are the monomers); M, quasi-melt; Mqb, quasibrushes are formed in the melt. In the case of the B blocks on the liquid side of the interface, the regimes listed next may happen. m, isolated mushrooms; SDp, two-dimensional semidilute regime of the polymer chain (the statistical units are the B block sequences); B, quasi-brush regime in the liquid phase.
scaling law arguments (Figure 1). Increasing the surface concentration, at a fixed value of R, yields a succession of regimes. It was surprising to realize that this succession of regimes may happen only in three distinct manners according to the value of R. At small R values, the phase diagram is largely influenced by the properties of the semidilute regime in the liquid medium. At large values of R, the phase diagram reflects in priority the properties of the hydrophobic blocks. At intermediate values of R, the phase diagram is influenced by the properties of both sides of the interface. It is then possible to calculate the surface pressure π as a function of the surface concentration Γ in these three situations. In the semidilute regimes, using a scaling law approach, the surface pressure was found to be45
π = kBTΓy
(14)
where kB is the Boltzmann constant, T is the absolute temperature, y is an exponent characteristic of the regime of the interface and of the fractal dimension of the block in the dilute regime, and Γ is the surface concentration defined as
Γ ) m/A
(15)
where m is the mass of polymer (protein) adsorbed at the interface of area A.
dπ d ln(Γ)
(16)
Combining eqs 14 and 16 gives
) yπ
(17)
Thus, representing as a function of π is just a mathematical transformation of the state equation 14 because in eq 16 is a function of only π and Γ. From an experimental standpoint, it is more convenient to measure than Γ, and thus one may use eq 17 rather than eq 14 to check how experiment complies with theory (Figure 2). 4. Results and Discussion All experiments were performed using a 0.1 M phosphate buffer at pH 7 containing 0.1 M NaCl to allow comparison of our results with those previously obtained in the same conditions on micelle formation by β-casein in solution.46-48 Neutron reflectivity is expected to give information on the structure of the interfacial layer and thus to give some insight on the effect of GuHCl on the interactions of the blocks with the solvent. This approach should be completed by micelle structure analysis in the bulk.48 Surface tensiometry and especially dilational modulus measurements should permit the evaluation of the regime of the interface and in some instances of the fractal dimension of the hydrophobic blocks lying flat on the interface. Neutron Reflectivity. A first set of spectra was obtained with various GuHCl concentrations at 10 and 20 °C for adsorption layers formed from a 100 mg/L β-casein solution. It was analyzed using a multilayer model. In all cases, the adjustment was satisfactory with a two-layer model. An additional layer never improved the quality of the fits. When no denaturing agent is present, the total amount of β-casein adsorbed is 2.9 mg m-2, a value very close to 2.8 mg m-2 which was previously determined by neutron reflectivity from a 100 mg/L β-casein solution49 using a two-layer model and not too far from 2.22 mg m-2 determined by X-ray reflectivity with a power law model.26 However, the structure of the adsorption layer seems quite different because its total thickness is 13.8 nm and was previously found to be 6.9 nm.49 These differences are more important in the first layer (the layer closer to the interface) which is 5.2 nm wide with a volume fraction of 0.32 whereas these quantities were previously found to be 3.1 nm and 0.52. The data seem to indicate that the (45) Douillard, R. Colloids Surf., B 1993, 1, 333. (46) Leclerc, E.; Calmettes, P. Phys. Rev. Lett. 1997, 78, 150. (47) Leclerc, E.; Calmettes, P. Physica B 1998, 241, 1141-1143. (48) Aschi, A.; Gharbi, A.; Bitri, L.; Aguie´-Be´ghin, V.; Douillard, R.; Daoud, M.; Calmettes, P. Submitted for publication. (49) Puff, N.; Cagna, A.; Aguie´-Be´ghin, V.; Douillard, R. J. Colloid Interface Sci. 1998, 208, 405.
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Figure 3. Effect of GuHCl concentration on the neutron reflectivity spectra R(q). q is the wave vector, and R is the reflectivity. The continuous line is the spectrum of the buffer. The GuHCl concentrations were (O) 0 M and (0) 2 Μ. The β-casein concentration in the bulk was 100 mg/L, and the temperature was 20 °C.
Figure 2. Variations of the dilational modulus, , as a function of the surface pressure, π, in each of the three regions defined in Figure 1. The slopes of the lines in regions I, II, and III are the y values of eqs 14 or 17 and are the slopes of the isotherms shown in Figure 6 of ref 27 for the corresponding surface pressure values. The dilute regime which is observed at surface pressures below 1 mN/m is not very significant in our experimental conditions. Regime changes are observed at the surface pressures denoted as follows: (a) π/pol, beyond π/pol the / / polymer chains overlap; (b) πblock , beyond πblock the coils of the B blocks overlap and enter a quasi-brush semidilute regime; (c) π/pan, beyond π/pan the pancakes of the A blocks overlap and the interface is dominated by a two-dimensional semidilute // regime; (d) π// pan, beyond πpan the surface concentration of the A blocks is so high that they enter a three-dimensional quasimelt regime.
adsorption layer and especially the first layer are more swollen by the solvent in this study than in the previous one. This should be linked to the buffer which was 10 mM morpholinopropane sulfonic acid in the previous study and which is 100 mM phosphate buffer with 100 mM sodium chloride in this work. Thus, the ionic strength was close to 0.01 in the previous case whereas it is larger than 0.3 in the present study, a value which efficiently screens electrostatic interactions. Thus, an interpretation of the swelling of the adsorption layer is that the ionic attractions are screened by the ionic strength. The effect of increasing the volume concentration of GuHCl is to lower the concentration of adsorbed β-casein, as is visible directly from the spectra which get closer to the buffer spectrum (Figure 3) and from the parameters extracted from the profile adjustment (Table 1). Finally, the concentration decreases and seems to reach a plateau where it is reduced by about 30% when the GuHCl concentration increases from 0 to a value between 1 and 1.5 M, whatever the temperature. The effect of lowering the temperature from 20 to 10 °C is to reduce the total adsorbed amount to 2.2 mg m-2
instead of 2.9 mg m-2 (Table 1). The volume fraction of the first layer is also slightly less than at 20 °C. This fact is consistent with the general view that the first layer comprises a large part of the hydrophobic sequences of the molecule and that these hydrophobic blocks tend to make more hydrophobic interactions when the temperature is increased. A second set of spectra was obtained for adsorption layers formed from a 5900 mg/L solution of β-casein. This much higher volume concentration is close to the value used in neutron scattering experiments46-48 and should be helpful in using the data on the micelle structure to interpret the adsorption experiments.48 A 1 M concentration of GuHCl was chosen as being in the transition region previously determined (Figure 4). Moreover, studies in the bulk showed that this concentration corresponds, in solution, to the transition region between micelles and monomers.48 As for the previous set of experiments, the data were satisfactorily fitted with a two-layer model (Table 2). However, the total surface concentration was smaller at 10 and 20 °C than that calculated with a weaker volume concentration of β-casein in the same solvent conditions (Table 1). A similar observation has been made previously using X-ray reflectivity when the bulk concentration of β-casein increases from 100 to 1000 mg/L. The only interpretation found for this phenomenon was that a part of the hydrophobic blocks are expelled out of the water phase and that the reflectivity data must be computed assuming that the refractive index of that layer is that of a medium composed of air and of protein (no solvent in the layer). Such a treatment at a bulk concentration of 1000 mg/L gave a coherent surface concentration, larger than that measured at a concentration of 100 mg/L.26 In the present neutron reflectivity experiments, the wave vector range available (0.0170.095 Å-1) practically precludes the use of a three-layer model to improve the χ2 of the fit to the experimental data. Thus, to check the hypothesis that a part of the protein is in the air instead of in water a fixed layer in air with the characteristics previously determined (9.5 Å thick, Φ ) 0.64)26 has been added to the interface model and the spectrum calculated by fitting the thickness and the volume fraction of the two layers in the buffer. This procedure gives a very good fit to the experimental data, and the χ2 of the adjustment to the experimental data is practically the same as that obtained by fitting a twolayer model without a layer of protein in the air (Table 2). This procedure gives the same result as long as the layer
Adsorption Layers of β-Casein
Langmuir, Vol. 17, No. 6, 2001 1901
Table 1. Effect of GuHCl on the Structure of the Adsorption Layer Formed by β-Casein at the Air-Buffer Interfacea GuHCl (mol L-1)
Nbs 10-6 (Å-2) ((0.05)
Nb1 10-6 (Å-2) ((0.05)
Nb2 10-6 (Å-2) ((0.05)
0 0.5 0.7 1.0 1.5 1.7 2.0
6.13 6.32 6.20 6.02 6.33 6.34 6.37
5.37 5.43 5.15 5.37 5.33 5.20 5.31
6.00 6.13 5.95 5.81 6.08 6.18 6.19
0 0.5 0.7 1.0 1.5 1.7 2.0
6.23 6.30 6.33 6.34 6.41 6.42 6.43
5.18 5.23 5.28 5.14 5.23 4.86 4.67
6.04 6.18 6.06 6.12 6.22 6.30 6.40
Th1 (Å) ((0.5)
Th2 (Å) ((0.5)
φ1 ((0.01)
φ2 ((0.01)
Γ1 (mg m-2)
Γ2 (mg m-2)
Γt (mg m-2)
10 °C 49.5 115.0 32.5 75.5 30.5 83.0 26.5 69.5 18.5 68.5 17.5 71.5 18.5 65.5
0.24 0.26 0.32 0.21 0.29 0.34 0.31
0.04 0.06 0.08 0.07 0.07 0.05 0.05
1.58 (0.08 1.15 (0.06 1.32 (0.05 0.75 (0.05 0.74 (0.05 0.79 (0.05 0.77 (0.05
0.6 (0.2 0.6 (0.1 1.0 (0.1 0.7 (0.1 0.7 (0.1 0.5 (0.1 0.5 (0.1
2.2 (0.2 1.7 (0.2 2.3 (0.2 1.4 (0.2 1.4 (0.2 1.2 (0.1 1.3 (0.2
20 °C 52.5 86.0 44.0 85.5 32.5 72.0 27.5 70.5 24.5 86.5 24.0 103.0 21.0 150.0
0.32 0.32 0.31 0.35 0.34 0.45 0.51
0.06 0.04 0.08 0.07 0.06 0.04 0.01
2.25 (0.09 1.89 (0.08 1.36 (0.06 1.31 (0.06 1.12 (0.06 1.45 (0.06 1.43 (0.06
0.7 (0.1 0.4 (0.1 0.8 (0.1 0.6 (0.1 0.6 (0.1 0.5 (0.1 0.2 (0.2
2.9 (0.2 2.3 (0.2 2.1 (0.2 1.9 (0.2 1.8 (0.2 1.9 (0.2 1.6 (0.2
a The neutron reflectivity spectra were analyzed using the two-layer model described in the text. Nb is the scattering length density, Th is the thickness of a layer, φ is the volume fraction of protein in the layer, and Γ is the concentration per surface unit area. The subscripts s, 1, and 2 refer to the substrate, the layer close to air, and the layer between the previous layer and the bulk, respectively. The total surface concentration is Γt ) Γ1 + Γ2. The bulk β-casein concentration is 100 mg/L.
Figure 4. Effect of GuHCl concentration and of temperature on the total surface concentration, Γ, of β-casein. The bulk protein concentration was 100 mg/L. The temperatures were (O) 10 °C and (9) 20 °C (data are from Table 1).
in the air is no more than 11.5 Å thick at 5 °C, 9.5 Å thick at 10 °C, or 13.1 Å thick at 30 °C with a fixed volume fraction of 0.64 or has a volume fraction less than 0.64 at 5.5 °C, 0.56 at 10 °C, or 0.64 at 20 °C or 30 °C with a fixed thickness of 9.5 Å. Finally, the determination of the concentration profile and of the surface concentration by neutron or X-ray reflectivity may give correct values only if an accurate layer model is available. In the case of synthetic block copolymers adsorbed at the air-water interface, it has been possible to use deuterated blocks and to deduce from these measurements that a polymer layer about 20 Å thick is in the air.50-52 Thus, the interpretation that a part of β-casein may be in the air is supported by these studies with synthetic organic molecules. At the end of this part, it can be concluded that GuHCl modifies, as expected, the solvent properties of the buffer. As shown elsewhere,48 there is in the bulk a transition from the micelle to the monomer state of β-casein at a (50) An, S. W.; Thomas, R. K.; Baines, F. L.; Billingham, N. C.; Armes, S. P.; Penfold, J. J. Phys. Chem. B 1998, 102, 387-393. (51) An, S. W.; Thomas, R. K.; Baines, F. L.; Billingham, N. C.; Armes, S. P.; Penfold, J. J. Phys. Chem. B 1998, 102, 5120-5126. (52) An, S. W.; Thomas, R. K.; Baines, F. L.; Billingham, N. C.; Armes, S. P.; Penfold, J. J. Macromolecules 1998, 31, 7877-7885.
GuHCl concentration between 1 and 1.5 M. This should indicate that the whole polypeptide chain is then in “good solvent conditions”. This probably reflects the fact that the free energy of a protein molecule in the micelle, including the terms of the core, of the interface, and of the corona,27 is larger than the energy of solvation by the solvent. In other words, the hydrophobic side chains do not constitute any more structures which impose their behavior to the molecule in solution (formation of hydrophobic clusters in the chain or between chains). A transition is also observed in the adsorption layer at the same GuHCl concentration; the surface concentration and the thickness of the first layer decrease until about 1 M and remain constant thereafter. Concerning the adsorption of β-casein at GuHCl concentrations larger than 1.5 M, the occurrence of a first layer which is rather narrow and which has a volume fraction much larger than that of the second layer is clearly in favor of the adsorption of blocks of the polypeptide chain which have affinity for the interface. This situation should happen because of the occurrence in these blocks of numerous amino acids exhibiting an attractive energy excess toward the interface.53 Surface Tensiometry. In dynamic measurements, and π (eq 3) were simultaneously determined during adsorption kinetics. The area of the bubble was submitted to a sine wave deformation ensuring the calculation of . γ was taken as the mean value of the surface tension during one period. γ0 was the surface tension of the buffer without protein. The relation between and π was determined with a standard bulk β-casein concentration of 100 mg/L. Experiments were also performed with a protein bulk concentration of 10 mg/L. The kinetics is then slower, and the surface pressure reached after 20 min is only 15 mN/m instead of 22 mN/m (without GuHCl). However, the plot of versus π is exactly the same as with a larger protein concentration but over a restricted range of data. Moreover, in these conditions no change of the slope y was noticed in the frequency range between 0.05 and 0.2 Hz. As previously shown,27,49 the measurement of the dilational modulus as a function of surface pressure may give relevant information on the structure of the protein molecules in the successive regimes occurring when the (53) Bouchaud, E.; Daoud, M. J. Physique 1987, 48, 1991-2000.
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Table 2. Effect of Temperature on the Structure of the Adsorption Layer Formed by β-Casein at the Air-Buffer Interface with 1 M GuHCl in the Bulka temp (°C)
Nb1 10-6 (Å-2) ((0.05)
Nb2 10-6 (Å-2) ((0.05)
Th1 (Å) ((0.5)
5.5 10.0 20.0 30.0
5.78 5.79 5.74 5.39
6.28 6.26 6.22 6.15
40.6 44.4 50.8 51.6
5.5 10.0 20.0 30.0
5.89 5.91 5.83 5.48
6.28 6.26 6.22 6.15
two-layer model plus one layer in air 38.7 119.8 0.13 0.02 44.6 109.6 0.13 0.03 49.7 76.8 0.15 0.04 49.6 88.2 0.25 0.06
Th2 (Å) ((0.5)
φ1 ((0.01)
two-layer model 120.9 0.17 113.3 0.16 81.6 0.18 90.7 0.28
φ2 ((0.01)
Γ1 (mg m-2)
Γ2 (mg m-2)
Γt (mg m-2)
χ2
0.02 0.03 0.04 0.06
0.93 ( 0.07 0.96 ( 0.07 1.23 ( 0.08 1.95 ( 0.09
0.3 ( 0.2 0.5 ( 0.2 0.4 ( 0.1 0.7 ( 0.1
1.3 ( 0.2 1.4 ( 0.2 1.7 ( 0.2 2.7 ( 0.2
0.2084 0.0169 0.1491 0.0060
0.68 (0.06 0.78 (0.07 1.00 (0.08 1.64 (0.08
0.3 (0.2 0.4 (0.2 0.4 ( 0.1 0.7 (0.1
1.8 (0.3 2.0 (0.3 2.2 (0.3 3.2 (0.3
0.2081 0.0170 0.1486 0.0059
a The neutron reflectivity spectra were analyzed using the two- and three-layer models described in the text. Nb is the scattering length density, Th is the thickness of a layer, φ is the volume fraction of the protein in the layer, and Γ is the concentration per surface unit area. The subscripts s, 1, and 2 refer to the substrate, the layer close to air, and the layer between the previous layer and the bulk, respectively. The total surface concentration is Γt ) Γ1 + Γ2. The bulk β-casein concentration is 5900 mg/L. In the case of the three-layer model, a fixed layer of thickness 9.5 Å (Φ ) 0.64, Γ) 0.82 mg m-2) as found by X-ray reflectivity for a β-casein layer adsorbed from a 1000 mg/L solution26 was added in the air to the two-layer model with adjustable parameters.
Figure 5. Effect of GuHCl concentration on the relationship between the dilational modulus, , and the surface pressure, π. The dilational modulus and the surface pressure were recorded simultaneously during the adsorption of β-casein from a 100 mg/L solution. The temperature was 10 °C. The GuHCl concentrations were (+) 0 M, (0) 1 M, (O) 2 M, and (]) 4 M.
surface concentration and pressure increase. To understand the features of the /π curves, it should be remembered that the relevant parameter is the slope of the line joining the origin and any point of the curve (Figure 5). Temperature Effect. The effect of temperature on the /π curves measured without GuHCl in the solution is not very pronounced in the range explored (Figure 6). However, it is clear that the line going through the origin has a constant slope at low surface pressure and that this slope decreases significantly when temperature increases. Referring to the theoretical model, this slope variation is indicative of a two-dimensional pancake with a decreasing fractal dimension27 when temperature increases. Therefore, the interactions between the monomers are ruptured by a temperature increase. This is suggestive more of a contribution of hydrogen bonding in the monomer interactions than of a contribution of hydrophobic interactions, which should have been increased by a temperature rise. The other features of these curves are similar to those of the curves of Figure 5 and are discussed in the next section. GuHCl Concentration Effect. The effect of the GuHCl concentration on the /π relations is shown in Figure 5. The main features of these curves may be described as a function of increasing surface pressure (the x-axis) and
Figure 6. Effect of temperature on the relationship between the dilational modulus, , and the surface pressure, π. The dilational modulus and the surface pressure were recorded simultaneously during the adsorption of β-casein from a 100 mg/L solution without GuHCl. The temperatures were (b) 10 °C, (×) 20 °C, and (]) 30 °C.
interpreted according to the theory summarized in the theoretical section. First, a linear relation from π ) 0 to 5-6 mN/m is indicative of a power law relation between and π, as predicted by the theory,27 and should correspond to the first semidilute regime (the gaseous regime normally ends around 1 mN/m). The slope of that line is larger than 3, as predicted by theory in the case when the properties of the interface are dominated by a twodimensional behavior. This slope steadily decreases to a value close to 3 for a GuHCl concentration between 2 and 4 M (Figures 5 and 7). The values of the slope (y) are linked to the Flory exponent and are indicative of conformations of “pancakes” between θ conditions (y ) 8) and excluded volume conformations (y ) 3). Thus, the results show that at GuHCl concentrations larger than 2 M, the attractions between the statistical units of the twodimensional (hydrophobic) sequences are weak whereas at smaller GuHCl concentrations they are significant (Figures 5 and 7). Moreover, the effect of temperature on the value of y indicates that the interactions are larger at 10 °C than at 20 °C and are even weaker at 30 °C. Thus, they do not seem to be hydrophobic in nature and the possibility of hydrogen bonding on the polypeptide backbone may be suspected. In fact, structures such as helices or β-sheets may be hypothesized as previously done for adsorbed peptides or proteins.54,55
Adsorption Layers of β-Casein
Figure 7. Effects of the GuHCl concentration and temperature on the exponent y of the first semidilute regime of the β-casein adsorption layer. The exponent was measured as shown in Figure 5. The bulk β-casein concentration was 100 mg/L. The temperatures were (O) 10 °C and (b) 30 °C.
At surface pressures between 6 and 10-12 mN/m a crossover is observed which is not as sharp as predicted by the model (this crossover will be referred to later as the first crossover). This is probably an expected difference between the model and the experiment: the model is a sharp asymptotic description of the regimes and of their crossovers whereas the experiment made on a “real smallsized polymer” must be a smooth approximation of the model. However, the crossover seems to begin at the same surface pressure (the same correlation length) whatever the GuHCl concentration and the swelling of the twodimensional blocks in the dilute regime. This corresponds to the end of the two-dimensional semidilute regime where the two-dimensional interface space is saturated by twodimensional coils. This saturation should depend only on the energy gained by each monomer (approximated by the surface pressure) and should not depend on the initial conformation of the blocks (the slope of the /π curve in the first semidilute regime), which is determined by the GuHCl concentration. This is exactly what is observed experimentally. At a surface pressure close to 11 mN/m, the lowest value of the ratio /π in this crossover is approximately 0.6, a value significantly smaller than 1 (the smallest value expected if the polymer is characterized by an intermediate value of R (region II on Figure 2)). Thus, in the frame of the model this low value would mean that the protein has a “high” value of R (region III of Figure 2). However, in such a case the theoretical value of the ratio /π should reach 0. But as discussed previously, the difference between 0 and 0.6 may be due to a “smoothing” effect of the real polymer as compared to the theoretical model where the down and up variations of do occur at constant π. Nevertheless, it can be observed that after going through that minimum the /π ratio tends toward 1, as in the model. Finally, the value of the /π ratio goes down whereas the model predicts that it should remain at unity (such a value corresponds to a quasi-melt on the air side of the interface and quasi-brushes on the liquid side). This discrepancy points to some limitation of the model, but this is not (54) Puggelli, M.; Noncentini, M.; Gabrielli, G.; Poletti, L. Nuovo Cimento 1994, 16D, 1529-1536. (55) Clegg, R. S.; Reed, S. M.; Hutchison, J. E. J. Am. Chem. Soc. 1998, 120, 2486-2487.
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Figure 8. Kinetics of surface pressure, π, of adsorbed layers of β-casein as a function of guanidine hydrochloride concentration. The protein bulk concentration was 100 mg/L, and the temperature was 30 °C. The GuHCl concentrations were (0) 0 M, (∆) 0.5 M, (]) 1 M, (×) 1.5 M, and (O) 2 M. The inset shows the quasi-equilibrium value of the surface pressure as a function of the GuHCl concentration. The experimental error is smaller than the size of the symbols.
completely surprising because it is built on such a simplifying hypothesis as the location of the junctions between hydrophilic and hydrophobic sequences being strictly located in the interface plane. Equilibrium Values. Surface pressure has been measured in conditions of quasi-equilibrium after 15 h of adsorption, as a function of GuHCl concentration and for a bulk protein concentration of 100 mg/L. It was observed that the equilibrium surface pressure, which is 24.5 mN/m when there is no GuHCl in solution, decreases when the GuHCl concentration increases from 0 to 1.5 M and that it remains constant at 19.5 mN/m beyond this GuHCl value (Figure 8). A very simple interpretation of this observation is that the correlation length (ξ) increases and because the surface pressure is proportional to the power -2 of the correlation length it can easily be calculated that in the plateau region the ratio of the correlation lengths at high GuHCl concentration and without GuHCl is (24.5/19.5)1/2 ) 1.12. This value should be correlated with a variation of the volume fraction in the quasi-brush layer, which is expected to dominate the properties of the adsorption layer in this equilibrium situation. From Table 1, such a correlation cannot be found in the volume fractions of the second layer where the quasibrush structure is expected to occur. The small values and inaccuracies of the calculated volume fractions in this layer probably preclude any conclusion. However, the expected increase of the correlation length is consistent with a loosening of the structure of the molecules previously noticed by a decrease of the value of the exponent y when the GuHCl concentration increases (Figure 7). Conclusion The effect of GuHCl on β-casein adsorption was studied by measurements of the structure and of the properties of layers formed at the air-buffer interface. Neutron reflectivity showed that the adsorbed amount of protein decreases when GuHCl increases, as expected from the denaturing and solubilizing effects of this agent on proteins. A rise of temperature increases the adsorbed amount. This fact indicates that the hydrophobic effect is strongly involved in the energetics of adsorption. The bulk
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concentration effect on the surface concentration leads to the interpretation that a part of the adsorbed material is in air when concentration is high enough. This feature of the adsorption layer was previously deduced from X-ray reflectivity measurements in the case of β-casein and from neutron reflectivity in the case of organic block copolymers. It seems now that the occurrence of a part of the molecule in air should be included in any model of block copolymer adsorption at the gas-liquid interface. The surface tension measurements performed on the adsorption layers are interpreted with a model of block copolymer. They show that the effect of GuHCl concentration in the bulk is to change the conformation of the two-dimensional blocks from a moderately collapsed twodimensional structure toward an excluded volume coil. This change is indicative of the rupture of noncovalent bonds between the monomers of the two-dimensional blocks, as was expected from the properties of GuHCl. An
Aschi et al.
increase of temperature also seems to break noncovalent interactions and to lead to more swollen adsorbed molecules. Thus, the conformation of adsorbed molecules is strongly dependent on noncovalent interactions which may not be only hydrophobic. Finally, it can be concluded that numerous noncovalent bonds contribute to the structure of the hydrophobic blocks of β-casein when this protein is adsorbed at the air/buffer interface from a nondenaturing solvent. Acknowledgment. Our thanks to B. Monties for his interest in adsorption layer structure and properties and to MENRT (France) and SERST (Tunisia) for supporting the stay of A.A. in France in the frame of the research and teaching network 07/317/TN. LA001159S
