Water Interface As Supported by

Langmuir 1998, 14, 1753-1758

1753

Properties of β-Casein at the Air/Water Interface As Supported by Surface Rheological Measurements M. Mellema,* D. C. Clark,† F. A. Husband,‡ and A. R. Mackie§ Food Biophysics Department, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, U.K.

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Received June 20, 1997. In Final Form: December 8, 1997 The properties of air/water adsorbed and spread monolayers of native and dephosphorylated β-casein were monitored using surface pressure (Langmuir trough) and surface rheology (ring trough) techniques. Two stages of rearrangement are observed for native β-casein at surface areas of about 1.0-1.3 and 0.7 m2 mg-1. The first accounts for distinct surface elasticity changes in the film, which are probably due to the expulsion of the most hydrophilic segments of the protein chain. The second accounts for the collapse of the monolayer. The experiments on dephosphorylated β-casein monolayers show that dephosphorylation changes the surface elasticity behavior of the monolayer, in particular between 1 and 1.3 m2 mg-1. We calculated a two-dimensional Flory exponent, ν, for both proteins. This exponent is constant over a (semi-) dilute range of surface tensions, maximally up to a surface area of around 1.3 m2 mg-1. The adsorption of native β-casein is shown to be diffusion-limited up to a surface area of around 1 m2 mg-1. Experiments at high ionic strength show the importance of charge on the typical surface elasticity behavior of β-casein. Experiments with enzymatically treated β-casein show the importance of the presence of a hydrophilic section in the molecule on the surface elasticity behavior. It is assumed and shown that, at surface concentrations below monolayer collapse and at given solvent conditions, native β-casein and dephosphorylated β-casein show irreversible (air/water) adsorption behavior. Furthermore, the proteins in the monolayer are very flexible (i.e. quick relaxations).

Introduction In a wide range of milk products the presence of the protein β-casein at interfaces in the system plays a crucial role in the physical properties of the product. Therefore β-casein has been studied extensively at solid/water1-5 and air/water interfaces. At the air/water interface, the interfacial density can be controlled very well. In addition, the surface pressure6-10 and surface rheology11-15 are experimentally accessible. * To whom correspondence should be addressed. Current address: Food Physics Group, Wageningen Agricultural University, P.O. Box 8129, 6700 EV Wageningen, The Netherlands. E-mail: [email protected]. † Current address: DMV International, P.O. Box 13, 5460 BA Veghel, The Netherlands. E-mail: [email protected]. ‡ E-mail: [email protected]. § E-mail: [email protected]. (1) Brooksbank, D. V.; Davidson, C. M.; Horne, D. S.; Leaver, J. J. Chem. Soc., Faraday Trans. 1993, 89 (18), 319. (2) Dalgleish, D. G.; Leaver, J. J. Colloid Interface Sci. 1991, 141, 288. (3) Dickinson, E.; Horne, D. S.; Phipps, J. S.; Richardson, R. M. Langmuir 1993, 9, 242. (4) Fragneto, G.; Thomas, R. K.; Rennie, A. R.; Penfold, J. Science 1995, 267, 657. (5) Mackie, A. R.; Mingins, J.; North, A. N. J. Chem. Soc., Faraday Trans. 1991, 87 (18), 3043. (6) Benjamins, J.; de Feyter, J. A.; Evans, M. T. A.; Graham, D. E.; Phillips, M. C. Faraday Discuss. Chem. Soc. 1975, 59, 218. (7) Damadoran, S.; Song, K. B. ASC Symp. Ser. 1991, 454, 104. (8) Douillard, R.; Lefebre, J. J. Colloid Interface Sci. 1990, 139 (2), 488. (9) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1979, 70, 415. (10) Hunter, J. R.; Kilpatrick, P. K.; Carbonell, R. G. J. Colloid Interface Sci. 1991, 142 (2), 429. (11) Benjamins, J.; van Voorst Vader, F. Colloid Surf. 1992, 65, 161. (12) Gau, C.-S.; Yu, H.; Zografi, G. J. Colloid Interface Sci. 1994, 162, 214. (13) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1980, 76, 227. (14) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1980, 76, 240. (15) Williams, A.; Prins, A. Colloids Surf. A 1996, 114, 267.

A characteristic feature of β-casein is that the distribution of hydrophilic and charged residues along the polypeptide chain is not uniform; it favors a model similar to a diblock copolymer. The N-terminal block, which accounts for a third or fourth of the molecule, contains most of the charge. When adsorbed on the surface of a dispersed particle, the most hydrophilic parts of adsorbed β-casein molecules can extend into aqueous solution, forming a protective steric barrier.16,17 The second important feature of β-casein is that the majority of the molecule is “random” folded,16,18,19 unlike globular proteins. This kind of structure makes it reasonable to be described by scaling theories developed for polymers.8 Many properties of food dispersions depend on tension gradients due to variations in the surface concentration of adsorbates at the constituent interfaces. We observed that the modulus of elasticity |E| (which is a measure of the dynamic response of the adsorbed molecules to compression and expansion of an interface) of a β-casein monolayer does not simply increase to a plateau with increasing surface coverage or surface pressure. Instead, it shows a maximum, followed by a decrease to an intermediate value. Previously, an apparently related behavior of |E| with β-lactoglobulin was observed. Subsequently, the reason for this was shown to be a lipid contamination of the sample.20 Extensive attempts were made to purify the β-casein sample (chloroform/methanol (16) Andrews, A. L.; Atkinson, A. L.; Evans, D.; Finer, M. T. A.; Green, J. P.; Phillips, M. C.; Robertson, R. N. Biopolymers 1979, 18, 1105. (17) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1979, 70, 403. (18) Chaplin, L. C.; Clark, D. C.; Smith, L. J. Biochem. Biophys. Acta 1988, 959, 162. (19) Tripp, B. C.; Magda, J. H.; Andrade, J. D. J. Colloid Interface Sci. 1995, 173, 16. (20) Clark, D. C.; Husband, F. A.; Wilde, P. J.; Cornec, M.; Miller, R.; Kra¨gel, J.; Wu¨stneck, R. J. Chem. Soc., Faraday Trans. 1995, 91, 1991.

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Mellema et al.

extraction, charcoal extraction, and dialysis, successfully used with β-lactoglobulin). An independent check on the data was carried out by studying a different sample of β-casein, kindly provided by the Hannah Research Institute. We found the β-casein surface dilation properties of the purified and Hannah samples were indistinguishable from the Sigma sample data. This typical dilational behavior of β-casein was first reported by Benjamins et al.6 but was largely ignored. Later, it was carefully proposed12,15,21 that structural changes in the β-casein film, like an expulsion of hydrophilic loops or tails, occur at or around the maximum in |E|. An alternative view is that surface micelles are formed at or around the maximum. However, it recently was shown using atomic force microscopy and LangmuirBlodgett techniques that β-casein probably forms a homogeneous network at the air/water interface.22 In addition, we will show that low-temperature measurements confirm that surface micelle formation is most probably not the reason for the maximum in the modulus. In this paper the typical dilational behavior as described above will be explained in terms of rearrangements resulting from expulsion of the most charged segments into solution. The influence of the amount of charge on this explusion was checked by experiments at high ionic strength and with dephosphorylated β-casein (deph. β-casein). During dephosphorylation most of the five charged phosphate groups are removed from the peptide chain. It should be noted that there is a cluster of four phosphorylated serine residues between positions 15 and 19 of the β-casein molecule. Finally, we performed experiments with a sample of enzymatically treated β-casein. Using this technique, we removed a major portion of the hydrophilic N-terminus of the molecule. We will assume (and justify) that the native and deph. β-casein monolayers can be considered insoluble at given solvent conditions; that is, the proteins are soluble in the bulk but the adsorption process is considered to be irreversible and thus desorption does not occur. Theory Surface Dilation. If molecules adsorb at the air/water interface, the surface pressure π ()γ0 - γ (mN m-1), where γ is the surface tension and γ0 is the surface tension of a clean water surface) increases. The surface pressure is a product of cohesive and electrostatic interactions and kinetic movement and therefore of the orientation of the adsorbate.23 In a dilational experiment, the dynamic response of the adsorbate molecules to compression and expansion of the interface (represented by a change in surface area A (m2 mg-1)) reflects the magnitude of lateral forces present and can be used to provide structural information. This response is dependent on the dilational modulus |E| (mN m-1) of the interface. This modulus is a complex quantity with a storage (elastic) part, d (mN m-1), and a loss (viscous) part, ηdω (mN m-1), resulting from a phase difference that occurs between dπ and dA. The parameter |E| can be expressed as23,24

dπ |E| ) d ln A

(1)

(21) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1979, 70, 427. (22) Gunning, A. P.; Wilde, P. J.; Clark, D. C.; Morris, V. J.; Parker, M. L.; Gunning, P. A. J. Colloid Interface Sci. 1996, 183 (2), 600. (23) Lucassen, J.; van den Tempel, M. Chem. Eng. Sci. 1972, 27, 1283. (24) Lucassen-Reynders, E. H.; Lucassen, J. Adv. Colloid Interface Sci. 1970, 2, 347.

|E| ) x2d + (ηdω)2

(2)

where ηd (mN s m-1) is the surface dilational viscosity and ω (s-1 or cycles s-1) is the frequency of the applied dA. Assuming an insoluble monolayer, the parameter |E| can be derived from a Langmuir trough π-A curve, for which a surface was compressed and expanded. More commonly an oscillatory experiment with variable frequency is applied. When the characteristic time of one compression or expansion is very large or small compared to the time of rearrangements, a viscous component is not detectable. If the characteristic time of deformations is very large compared to the time of rearrangements, |E| is called a static modulus and an increase in E during compression is expected until a plateau is reached at saturation.24 If adsorbate rearrangements take place at the same rate as the deformations, |E| is called a dynamic modulus.12 If a protein monolayer can be considered insoluble, rearrangements take place only within the monolayer. Polymer Scaling. Flexible, random coil polypeptides can be described using equations developed for synthetic polymers.25,26 The protein β-casein is however an amphiphilic polyelectrolyte. According to some3 using neutron reflectometry, for an adsorbed monolayer of this protein, this property accounts for the formation of a dense inner layer of the most hydrophobic segments and a more extended outer layer of hydrophilic segments. On the other hand, self-consistent field theory calculations,27 neutron reflectometry data using higher neutron intensities and alternative models,28 and SAXS experiments5 show that the formation of these layers is not that clear at all. In water, probably the most hydrophobic segments of the chain will be more densely assembled near hydrophobic interfaces or each other. However, a separation into distinct regions with strongly differing densities is unlikely. The molecule β-casein has been shown to possess hardly any secondary structure.18,19 The RdD of a coil in a space of dimension d is the average distance of the collection of atoms from their common center of mass. (So R2D is the radius of the molecule in two dimensions, R3D is the radius in three dimensions, etc.) If the monolayer is in a 2D (semi-) dilute regime, we can apply

R2D ∝ Nν

(3)

where N is the number of segments or amino acid residues and ν is a Flory (“swelling”) exponent, determined by a volume exclusion factor but also by the solvent quality and system dimensionality d following

ν)

3 d+2

(4)

ν)

2 d+1

(5)

1 d

(6)

ν)

for (respectively) the situations of good, Θ, and poor solvent. Thus, for a 2D chain in good solvent (or in a state of “self-avoiding walk”25), ν is theoretically predicted to be 0.75. The surface pressure can be expressed as

π ∝ A-y

(7)

where (25) Bijsterbosch, H. D.; de Haan, V. O.; de Graaf, A. W.; Mellema, M.; Leermakers, F. A. M.; Cohen Stuart, M. A.; van Well, A. A. Langmuir 1995, 11, 4467. (26) Vilanove, R.; Poupinet, D.; Rondulez, F. Macromolecules 1988, 21, 2880. (27) Leermakers, F. A. M.; Atkinson, P. J.; Dickinson, E.; Horne, D. S. J. Colloid Interface Sci. 1996, 178, 681. (28) Wagemaker, M.; van Well, A. A.; de Graaf, L. A. Unpublished results.

Properties of β-Casein at the Air/Water Interface -y )

2ν 1 - 2ν

Langmuir, Vol. 14, No. 7, 1998 1755 (8)

The scaling parameters ν and y for d ) 2 can be derived from a double-logarithmic plot of a π-A isotherm in a (semi-) dilute regime. Adsorption Kinetics. An apparent diffusion coefficient D (m2 s-1) for the adsorbate can be derived from an A-1-t0.5 curve.7,17,29 The structure-dependent reversibility of adsorption is not taken into account. We mean that conformational changes after adsorption could change the rate of desorption (if this rate cannot be neglected). The following expression can be applied in the determination of D:

1 D ) t0.5 × 2C0 A Π

0.5

()

(9)

where Π is 3.14... and C0 is the bulk phase concentration (g m-3, considered constant as the adsorption proceeds). Under conditions of diffusion-limited adsorption, D describes the rate of arrival of the adsorbate at the interface and the ratio A-1/t0.5 is constant over quite a range of time. At relatively high surface concentrations, more protein is present, which hinders proteins from the solution from adsorbing. In this way, D provides information on the general development of the adsorption behavior with time.

Experimental Section Preparation of Protein. Solutions were prepared in 0.01 M phosphate buffer (pH ) 7.0, γ0 ) 72.7 mN m-1), made up by dissolving appropriate amounts of NaH2PO4, H2O, and Na2HPO4 (BDH, AnalaR) in double-glass-distilled water. β-casein (Sigma, C6905 Lot 12H9550, from bovine milk) or 80% deph. β-casein (Sigma, C-8157 Lot 81H9620, from bovine milk, enzymatically prepared) was used without further purification. A stock solution was prepared at a β-casein concentration of 0.1 g L-1, which is below the cmc.16 The protein concentration was checked by UV spectroscopy, using an absorption coefficient of E278 ) 0.46 mg mL-1 cm-1. The enzyme treatment was carried out by forming an emulsion of 40% n-tetradecane in buffer stabilized by β-casein and adding trypsin (Sigma, T8642) at a concentration of 1:500 trypsin/βcasein. This was stirred at room temperature for 6 min before trypsin inhibitor (Sigma T4385) was added. Brij35 was added and the system was centrifuged (1500g, 10 min) before separation of the methanol/water (upper) phase and precipitation, freezedrying, and storage under nitrogen. The Brij35 and very low molecular weight (