Langmuir 1998, 14, 6457-6469
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Role of Dispersion Interactions in the Adsorption of Proteins at Oil-Water and Air-Water Interfaces Tapashi Sengupta and Srinivasan Damodaran* Department of Food Science, University of WisconsinsMadison, Madison, Wisconsin 53706 Received March 6, 1998. In Final Form: July 13, 1998 The adsorption kinetics of β-casein, BSA, and lysozyme at the air-water interface were compared with those at the triolein-water interface. The rates of adsorption of β-casein, BSA, and lysozyme to the oil-water interface were consistently higher by an order of magnitude, compared to the respective bulk diffusion coefficients. The rates of adsorption of β-casein and BSA to the air-water interface were, however, closer to the respective bulk diffusion coefficients, while that of lysozyme to the air-water interface was an order of magnitude smaller than its bulk diffusivity. Also, the equilibrium concentrations of the three proteins at the oil-water interface were several times higher than those at the air-water interface. The remarkable differences in the rates of adsorption of the three proteins, particularly lysozyme, at these interfaces were unequivocally explained by differences in the potential energies of interaction of the proteins with these two interfaces. Potential energy calculations showed that dispersion interactions between proteins and the oil-water interface is attractive, whereas those between proteins and the air-water interface were generally repulsive. Thus, the slower rate of adsorption of proteins to the air-water interface may be attributed to the dominating repulsive dispersion interactions. In addition, the existence of an energy barrier in the potential energy profile of lysozyme as it approached the air-water interface conclusively explained its unusually slow adsorption rate and the presence of a long lag period (60 min) for its adsorption to commence at the air-water interface.
Introduction Adsorption of proteins at fluid-fluid interfaces and their behavior in the adsorbed state play an important role in the formulation and stabilization of different foam-based and emulsion-based products in food industry. Therefore, an understanding of the kinetics of protein adsorption and the properties of adsorbed protein films is essential for comprehending the important physical and chemical factors that affect the formation and stability of food colloids. It is generally accepted that the rate of adsorption of proteins or small molecular surfactants to an interface is diffusion controlled. Several studies have been conducted to elucidate the diffusion-limited kinetics of adsorption of proteins to the air-water interface.1-13 However, most of those studies have shown that the rate of diffusion of proteins to the air-water interface deviated significantly from the bulk diffusion coefficients of proteins. The * To whom correspondence should be addressed. Telephone: 608263-2012. Fax: 608-262-6872. E-mail:
[email protected]. (1) Graham, D. E.; Phillips, M. C. J Colloid Interface Sci. 1979, 70, 403. (2) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1979, 70, 415. (3) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1979, 70, 427. (4) Benjamins, J.; De Feijter, J. A.; Evans, M. T. A.; Graham., D. E.; Phillips, M. C. Discuss. Faraday Soc. 1975, 59, 218. (5) De Feijter, J. A.; Benjamins J. In Food Emulsions and Foams; Dickinson, E., Ed.; Royal Society of Chemistry: London, 1987; pp 7285. (6) MacRitchie, F.; Alexander, A. E. J. Colloid Sci. 1963, 18, 453. (7) Xu, S.; Damodaran, S. Langmuir 1992, 8, 2021. (8) Cao, Y.; Damodaran, S. J. Agric. Food Chem. 1995, 43, 2567. (9) Xu, S.; Damodaran, S. J. Colloid Interface Sci. 1993, 157, 485. (10) Xu, S.; and Damodaran, S. J. Colloid Interface Sci. 1993, 159, 124. (11) Anand, K.; Damodaran, S. J. Colloid Interface Sci. 1995, 176, 63. (12) Anand, K.; Damodaran, S. J. Agric. Food Chem. 1996, 44, 1022. (13) Hunter, J. R.; Carbonell, R. G.; Kilpatrick, P. K. J. Colloid Interface Sci. 1991, 143, 37.
apparent diffusion coefficient value of the positively charged protein, lysozyme, was particularly intriguing because it was about an order of magnitude smaller than the bulk diffusion coefficient (this work and refs 10 and 11). Moreover, it exhibited a long lag time (about 60 min) to commence adsorption at the air-water interface, tentatively suggesting existence of an energy barrier to its adsorption. The negatively charged proteins, β-casein and BSA, on the other hand exhibited no lag time to commence adsorption and their apparent diffusion coefficients were slightly higher than or equal to their respective bulk values (this work and refs 8, 9, 11, and 12). The data in the literature tentatively suggest that the rate of adsorption of proteins at an interface is not simply limited by its diffusivity but rather by the nature of interaction of various molecular forces of the protein molecule with the force field of the interface.7 It appears that as the protein molecule approaches the interfacial force field in the course of its Brownian diffusion, the chemical potential gradient at a finite distance from the interface is no longer a simple function of concentration gradient between the subsurface and the interface but is also governed by the sum of the interaction potentials between the protein and the interface. These interaction potentials might arise from the interaction of the electrostatic, van der Waals, and hydrophobic forces of the molecule with the force field of the interface per se, as well as with the apolar phase. In the present study, we investigated the kinetics of adsorption of lysozyme, β-casein, and BSA at the airwater and at a planar triolein-water interface under identical experimental conditions. The differences between the kinetics of adsorption of each of these proteins at the air-water interface and triolein-water interface were analyzed in terms of the energetics of interaction of various molecular forces of the proteins with these interfaces. Specifically, we have considered the following
10.1021/la980275g CCC: $15.00 © 1998 American Chemical Society Published on Web 10/02/1998
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contributions to the total potential energy of the protein molecule as it approached the interface. 1. The first is the electrostatic energy arising from repulsive Coulombic interactions between the charged protein molecule in the aqueous phase and its image charge generated in the low dielectric apolar phase and also the interaction between the electrical double layers of the protein and the interface. It is well established that the air-water interface has a positively charged inner double layer as a result of water orientation at the interface,14-16 while the inner layer of the oil-water interface is generally negatively charged due to the preferential adsorption of anions to the interface.17 The electrical double layer interaction therefore may be attractive or repulsive depending on the sign and magnitude of the surface potential of the proteins and the interface. 2. The second is the attractive hydrophobic interaction energy between protein molecules and the interface. This is primarily entropic in nature, originating from the rigid structuring of water around hydrophobic patches on protein surface with an associated loss in entropy. 3. The third is the ubiquitous van der Waals interaction energy. This consists of the attractive Debye-Keesom interactions arising from dipole-induced dipole interactions and dipole orientation effects and the London dispersion contribution arising from induced dipole-induced dipole interactions. The contributions from the dispersion interactions may be considerable in magnitude, besides being attractive or repulsive depending on the polarizabilities of the phases under consideration. On the basis of these potential energy calculations, we show that lysozyme experiences an energy barrier of about 13kT, whereas both BSA and β-casein experience only an attractive potential for adsorption at the airwater interface. On the other hand, none of these proteins experience any energy barrier for adsorption at the planar triolein-water interface. These theoretical calculations agreed very well with the experimental data. Materials and Methods Triolein (99% pure), lyophilized and salt free bovine β-casein (minimum 90%), crystallized and lyophilized chicken egg white lysozyme (95%), and lyophilized bovine serum albumin (9699%) were obtained from Sigma Chemical Co. (St. Louis, MO). Methyl oleate (99% pure), sodium cyanoborohydride (95% pure), ultrapure Na2HPO4 and NaH2PO4, and NaCl were purchased from Aldrich Chemical Co. (Milwaukee, WI). 14C-formaldehyde, with a specific radioactivity of 10 mCi/mmol, was purchased from New England Nuclear Co. (Boston, MA). Extreme care was taken in purifying water for adsorption studies. A Milli-Q Ultrapure water purification system (Millipore Corp., Bedford, MA) with a Qpak1 cartridge package (composed of activated charcoal and reverse osmosis, ion-exchange, and ultrafiltration cartridges), capable of removing inorganic and organic impurities, was used to purify the water. The resistivity of the water used was 18.2 mΩ.cm. The surface tension of this water at 25 °C was 71.9 ( 0.1 mN/m. Radiolabeling of proteins was carried out by the reductive methylation of the amino groups using sodium cyanoborohydride and 14C-formaldehyde, as described elsewhere.7 The concentration of proteins was determined using E1% values of 4.6 at 280 nm for β-casein, 26.3 at 281 nm for lysozyme, and 6.67 at 279 nm for bovine serum albumin, respectively. The specific radioactivities of 14C-radiolabeled β-casein, lysozyme, and BSA were (14) Weyl, W. A. J. Colloid Sci. 1951, 6, 389; Good, R. J. J. Phys. Chem. 1957, 61, 810. Fletcher, N. H. Philos. Mag. 1962, 7, 255. (15) Jarvis, N. L.; Schieman, M. A. J. Phys. Chem. 1968, 72, 74. (16) Frumkin, A. Electrochim. Acta 1960, 2, 351. Kochurova, N. N.; Rusanov, A. I. J. Colloid Interface Sci. 1980, 81, 297. Brodskaya, E. N.; Eriksson, J. C.; Laaksonen, A.; Rusanov, A. I. J. Colloid Interface Sci. 1996, 180, 86. (17) Sengupta, T.; Papadopoulos, K. J. Colloid Interface Sci., 1994, 163, 234.
Sengupta and Damodaran 0.795, 1.245, and 1.116 µCi/mg, respectively. These specific radioactivity values correspond to incorporation of 1.9, 1.8, and 7.3 mol of 14C per mole of protein, respectively. Apparatus Used for Adsorption Studies at the OilWater Interface. A noninvasive new methodology employing the radiotracer technique for monitoring the adsorption of 14Clabeled proteins at a planar triolein-water interface18 was used in this study. The essential features of this technique are as follows. The entire system for protein adsorption studies consisted of a plexiglass housing placed on a vibration free surface. The temperature inside the housing was maintained at 25 ( 2 °C, by circulating water from a constant temperature water bath. Humidity was maintained almost constant by placing reservoirs of water inside the housing as well as by spreading wet towels. The system was setup such that the surface tension as well as the protein concentration at the interface could be monitored simultaneously during the entire course of the experiment. The surface tension was measured by the Wilhelmy plate technique using a ST 9000 surface Tensiometer (Nima Technology Ltd., Coventry, England), interfaced with an IBM computer. A thin sand-blasted platinum plate was used as the sensor. A teflon trough having inner dimensions of 17.45 cm length, 5.5 cm width, and 4 cm depth was used as the Langmuir trough. One side of the trough had a small hole (1 mm diameter) capped tightly with a septum for injecting protein into the bulk phase. The ST 9000 unit and the Teflon trough were placed on a plexiglass platform, which was designed to damp any vibrations. In each experiment, 350 mL of a solution consisting of 20 mM phosphate buffer (pH 7.0) adjusted to an ionic strength of 0.1 M with NaCl was used. A very thin (3 mm diameter, 12.7 mm length) Teflon-coated magnetic stir bar was placed at the center of the trough prior to spreading the triolein film on the water surface. A stock solution of triolein was freshly prepared every day by dissolving 2 µL of triolein in 10 mL of hexane-chloroform solvent, taken in a ratio of 85:15 by volume. A 1000 Å oil film was formed on the water surface, which remained stable over the entire duration of protein adsorption lasting for 15-20 h. Incorporation of methyl oleate at a low concentration of 0.5% by weight of triolein into the oil phase increased the stability and uniformity of the oil film. At this concentration, methyl oleate exerted no noticeable surface pressure when spread alone on the water surface. In a typical experiment, a stock solution containing 3 µL of methyl oleate in 1 mL of chloroform was prepared. A 12.5 µL aliquot of this was further diluted to 1 mL with chloroform. To 156 µL of this stock solution was added 4.8 mL of triolein stock solution, and the resulting mixture mixed thoroughly and spread over a period of 2 h on 96 cm2 water surface in the Langmuir trough. The thickness of the triolein film thus formed after evaporation of the solvent was about 1000 Å. The film was allowed to equilibrate for another hour or until no further change in surface tension occurred. To initiate protein adsorption, a known volume (0.5-2.0 mL) of the protein stock solution was injected through the hole in the side of the trough, without disturbing the oil film. The bulk protein concentration was 1.5 × 10-4% (w/v). The surface pressure and surface cpm measurements at the oil-water interface were started simultaneously as soon as the injection of the protein was completed. At the same time, gentle stirring of the protein solution by a magnetic stir bar was initiated. The rotation of the stir bar was preadjusted to about 60 rotations per minute in order to avoid ripples on the triolein film. The stir bar was moved very slowly along the length of the trough for proper mixing of the injected protein by moving the magnetic stirrer beneath the plexiglass platform. Proper care was taken to prevent breakage of the oil film. To the naked eye, the oil-covered surface of the water remained perfectly calm under this gentle stirring motion. The stirring was continued only for the first 15 min, after which it was stopped. The protein concentration at the oil-water interface was monitored by measuring surface radioactivity using a rectangular gas proportional counter with a Mylar window (8 × 4 cm) (Ludlum Measurements, Inc., Sweetwater, TX). The counts per minute were integrated using a rate meter (Model 2200, Ludlum (18) Sengupta, T.; Damodaran, S. J. Colloid Interface Sci., in press.
Role of Dispersion Interactions
Figure 1. Variation of surface concentration, Γ (0), and surface pressure (O) of β-casein with time during its adsorption to the (a) triolein-water interface and the (b) air-water interface. The bulk concentration of β-casein was 1.5 µg/mL in 20 mM phosphate buffer, pH 7.0, I ) 0.1 at 25 °C. Measurements) and printed out on a strip chart recorder interfaced with the rate meter. The Mylar window was suspended over the middle of the trough at a distance of about 4-5 mm from the water surface, without touching the trough. A carrier gas composed of 98.0% argon and 2.0% propane was passed continuously through the gas proportional counter at the rate of 20 mL/ min. The counts per minute measurements were made at 1 min intervals for the first hour of the experiment, followed by measurements at 10 min intervals during the rest of the experiment. Calibration curves required to convert counts per minute readings at the oil-water and air-water interfaces into protein surface concentrations (mg/m2) were constructed, as described elsewhere.18 The surface cpm of the equilibrated oil film (before initiating any protein adsorption), together with the cpm of the bulk solution with 14C-labeled protein injected into it, was taken as the background radioactivity. This background cpm was subtracted from the actual surface cpm values collected during protein adsorption at the oil-water interface. The background corrected cpm was then divided by the slope of the cpm versus surface radioactivty calibration curve to obtain the surface radioactivity (µCi/m2) at every time interval. By division of the instantaneous surface radioactivity (µCi/m2) by the specific activity of the protein (µCi/mg), the transient surface concentration of the protein (mg/m2) was obtained. The kinetics of adsorption of proteins at the air-water interface was performed as described elsewhere.9
Results Figures 1a, 2a, and 3a, respectively, show the rate of accumulation of β-casein, lysozyme, and BSA at the triolein-water interface. In preliminary experiments, a lag period of 9-18 min for the commencement of adsorption to the oil-water interface was observed for all three proteins. This lag phase was found to be due to the time required for proper mixing in the bulk phase of the injected protein solution. This is because when a small volume of radiolabeled Na214CO3 was injected in a similar manner into the bulk phase, it required about 15-18 min for the
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Figure 2. Variation of surface concentration, Γ (0), and surface pressure (O) of lysozyme with time during its adsorption to the (a) triolein-water interface, and (b) air-water interface. The bulk concentration of lysozyme was 1.5 µg/mL in 20 mM phosphate buffer, pH 7.0, I ) 0.1 at 25 °C.
Figure 3. Variation of surface concentration, Γ (0), and surface pressure (O) of BSA with time during its adsorption to the (a) triolein-water interface, and (b) air-water interface. The bulk concentration of BSA was 1.5 µg/mL in 20 mM phosphate buffer, pH 7.0, I ) 0.1 at 25 °C.
surface cpm to reach an equilibrium value. When this time period was subtracted from the adsorption curves, as shown in Figures 1a, 2a, and 3a, then none of these three proteins exhibited a lag period for the commencement of adsorption at the triolein-water interface. Figures 1b, 2b, and 3b, respectively, show the adsorption curves for β-casein, lysozyme, and BSA at the air-water interface. The air-water adsorption studies were carried
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Table 1. Selected Properties of β-Casein, Bovine Serum Albumin (BSA) and Lysozyme property
β-casein
BSA
lysozyme
refs
amino acid residues per molecule molecular weight percentage helix disulfide bridges per molecule tertiary structure hydrophobicity (cal/mol of residue) size Å × Å × Å hydrodynamic radius, Å charge at pH 7.0 ∞, high-frequency relative permittivity i, static dielectric constant polar group density, g/mL nonpolar group density, g/mL
209 23 980 10 0 disordered coil 1330
575 66 306 50 17 globular 1120 140 × 38 × 38 35.79 -17 2.717 3.1317 1.547 0.86
129 14 315 44 4 globular 970 45 × 30 × 30 19.67 +9 2.7847 3.2328 1.545 0.886
36, 38, 39 36, 38, 39 1 1 1 46 32, 35 this work 38, 42 this work this work this work this work
35.79 -13 2.70 3.1073 1.534 0.903
Table 2. Selected Data from Surface Pressure and Surface Concentration Curves of This Work and Others property apparent diffusion coefficient,
β-casein
interface cm2/s
O/W A/W
bulk diffusion coefficient cm2/s Γeq, mg/m2
O/W A/W O/W A/W O/W A/W
projected area, Å2/molecule surface pressure, mN/m
10-5
1.4 x (5.9, 10, 11.7) x 10-7 6.1 x 10-7 6.64 2 600 1991 12.9 19.5
out by the pouring method;7-12 in this case the protein solution was first poured into the trough, the surface was cleaned of proteins by sweeping with a capillary tube attached to an aspirator, and then the protein from the bulk phase was allowed to adsorb at the interface. Since the solution was homogeneous from the very beginning, one would expect no mixing-related lag period in the beginning of adsorption of proteins. However, the data in Figures 1b, 2b, and 3b show that while β-casein and BSA exhibited no lag period for adsorption, lysozyme exhibited a lag period of about 60 min for the commencement of adsorption at the air-water interface. The molecular properties of the three proteins are listed in Table 1. According to Ward and Tordai,19 the initial rate of change of surface concentration, Γ, is a linear function of the inverse of square root of time, as given by eq 1. The
dΓ D ) Cb dt πt
( )
1/2
(1)
concentration of protein accumulated at an interface at any time t is obtained by integrating the above equation from time 0 to time t, as shown in eq 2. All other symbols
Γ)
∫0t(dΓ/dt)dt ) 2Cb(Dt/π)1/2
(2)
have been described in the Nomenclature section. The apparent diffusion coefficients of proteins may be calculated from the initial slopes of the Γ vs xt curves. This equation was used by Graham, Phillips, and co-workers,1-5 MacRitchie and Alexander,6 and Damodaran and coworkers7-12 for describing the diffusion of proteins to the air-water interface during the initial period of adsorption, when Γ was
