Water Interface

Langmuir 2003, 19, 2349-2356

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Dilatational Rheology of BSA Conformers at the Air/Water Interface Luis G. Cascaõ Pereira,*,† Olivier Theódoly,‡ Harvey W. Blanch, and Clayton J. Radke* Department of Chemical Engineering, University of California, Berkeley, California 94720-1462 Received August 14, 2002. In Final Form: December 2, 2002 To elucidate the role of protein conformation at the air/water interface, we measured the interfacial dilatational rheology of bovine serum albumin (BSA) and β-casein adsorbed over long time periods using a modified dynamic pendant-drop tensiometer. Companion long-time dynamic surface pressure and ellipsometry measurements are also reported. BSA has well-characterized structural isomers (conformers) whose structural transitions depend on solution pH. It is, therefore, possible to establish a connection between protein structure and interfacial rheology without the ambiguity of comparing different proteins. We also studied β-casein to verify the conclusions obtained from BSA. Adsorbed BSA and β-casein protein layers are primarily elastic with the dilatational elastic modulus arising from two contributions: (1) conformational rearrangement following adsorption that leads to formation of an interconnected, samplespanning interfacial protein network (i.e., an interfacial gel), and (2) the intrinsic structural stability of the individual protein units within the network. The latter component is most important to the interfacial dilatational modulus and explains why adsorbed layers of rigid, globular proteins are more elastic than those of flexible, random-coil proteins. We identify a new surface elasticity relaxation mechanism at the interface due to interprotein conformational rearrangement that is enhanced by electrostatic screening.

Introduction At early times, typically within minutes, equilibrium thin-film forces (i.e., disjoining pressures) are responsible for the longevity of protein-stabilized free, thin films. At longer times, on the order of hours to days, adsorbed protein layers aggregate and macroscopic skins develop.1,2 The aged films are of variable thickness and do not respond to changes in applied force.1 In this circumstance, the stability of aqueous protein films must be understood in terms of the mechanical properties of the two adsorbed protein layers. The semirigid interfacial network that evolves over time is unique to each protein, solution condition, and history. Although much attention has been given in the literature to the rheological characterization of adsorbed protein layers,3-7 the relationship between interfacial rheology and protein structure remains unclear. We address this question by examining bovine serum albumin (BSA), since this protein exists in aqueous solution under different well-characterized structural conformations. We are, therefore, able to investigate the role of protein conforma* To whom correspondence should be addressed. Telephone: (510) 642-5204; fax: (510) 642-4778; e-mail: [email protected] (C.J.R.). Telephone: (650) 846-7685; fax: (650) 621-8186; e-mail: [email protected] (L.G.C.P.). † Current address: Genencor International, 925 Page Mill Road, Palo Alto, CA 94304-1013. ‡ Current address: Rhodia, Inc., 259 Prospect Plains Road, Cranbury, NJ 08512-7500. (1) Cascaõ-Pereira, L. G.; Johansson, C.; Blanch, H. W.; Radke, C. J. Colloids Surf., A 2001, 186, 103. (2) Velev, O. D.; Campbell, B. E.; Borwankar, R. P. Langmuir 1998, 14, 4, 4122. (3) Bos, M. A.; van Vliet, T. Adv. Colloid Interface Sci. 2001, 91, 437. (4) Dickinson, E. Colloids Surf., B 2001, 20, 197. (5) Langevin, D. Adv. Colloid Interface Sci. 2000, 88, 209. (6) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1980, 76, 227. (7) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1980, 76, 240.

Figure 1. pH conditions and helical contents of the five recognized forms of BSA including the crystal structure of the N form and the proposed configurations of the F and E forms.

tion in the rheological properties of the adsorbed layer without the ambiguity inherent to comparison of different proteins. We also measure the interfacial rheology of β-casein to confirm the conclusions obtained from BSA. BSA Conformational Transitions. Serum albumin is one of the most studied proteins, particularly the bovine variant, since it is readily obtained and it displays high structural homology with its human counterpart (HSA).8 As a consequence, BSA is extremely well characterized. BSA has a molecular weight of about 66 kDa.8 The threedimensional structure is available.9,10 Albumin has three homologous domains, and is predominantly helical. As illustrated in Figure 1N, each domain is made of two subdomains sharing a common helical motif. Fatty-acid(8) Peters, T. All About Albumin: Biochemistry, Genetics, and Medical Applications; Academic Press: San Diego, 1996. (9) Carter, D. C.; Chang, B.; Ho, J. X.; Keeling, K.; Krishnasami, Z. Eur. J. Biochem. 1994, 226, 1049. (10) Carter, D. C.; He, X. M. Science 1990, 249, 302

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free albumin has a triangular or heart like shape.11 Moreover, BSA undergoes several well-recognized conformational changes depicted in Figure 1, usually under nonphysiological conditions. Foster investigated isomerizations obtained by varying pH;12 these are the focus of the present study. Four isomers of the normal or N form, found close to the isoelectric point and up to neutral pH (see Figure 1N), have been recognized and are depicted in Figure 1:12 F, or fast migrating, at pH 4 (Figure 1F); E, or extended, below pH 3 (Figure 1E); B, or basic, near pH 8 (Figure 1B); and A, or aged, near pH 10 (Figure 1A). All structural transitions are reversible. These structural changes are predicted based on physicochemical evidence; their relationship to the tertiary configuration (Figure 1N) has only been tentatively established.11 Although studied with BSA, such conformational transitions apparently also occur with HSA.8 In the presence of fatty acids, other conformational changes have been identified.8 Acid Transitions. The acid-induced structural changes of albumin have been investigated by a wide range of methods, and are characterized by changes in secondary as well as tertiary structure between the N (Figure 1N) and F (Figure 1F) forms. Foster demonstrated that the abrupt discontinuity in the titration curve13 at pH 3-5 coincided with the appearance of a faster migrating species, the F form, as seen with gel electrophoresis. The F form is characterized by a longer, less compact (11% volume increase) and increasingly asymmetric molecule, followed by a decrease in helical content from 55% in the N form (Figure 1N) to 45% in the F form (Figure 1F).12 The decrease in helical content is attributed to helix f β-sheet and helix f coil transitions.14,15 Carter and Ho interpreted the transition as separation of the two halves in heart-shaped albumin due to a net positive charge increase of each half below the pI at 4.74.9.11 Figure 1F pictures their proposed structure for the F form.11 The result is an abrupt opening of the molecule, involving breakup of salt bridges and of attractive hydrophobic interactions. Foster demonstrated that this transition proceeds in two steps through a well-defined intermediate, F′, characteristic of a cooperative mechanism.12 At pH values below 4, albumin undergoes another expansion to the E form.12 Figure 1E illustrates the structure proposed by Carter and Ho for the E form.11 Apparently, the F f E transition is to the full extent allowed by the disulfide bond structures. Yet another decrease in helical content from the F form is observed for the E structure. Base Transitions. Under slightly alkaline conditions, between pH 7 and 9, albumin undergoes another conformational change, known as the basic or N f B transition from the N form (Figure 1N) to the B form (Figure 1B).8 This transition is more subtle and more gradual in onset than the N f F transition.12 It is often described as a “structural fluctuation”, or a loosening of the molecule with loss of “rigidity”.8 There is evidence that discrete steps are associated with this transition,16 characteristic (11) Carter, D. C.; Ho, J. X. Adv. Protein Chem. 1994, 45, 153. (12) Foster, J. F. In Albumin Structure, Function and Uses; Rosenoer, V. M., Oratz, M., Rothschild, M. A., Eds.; Pergamon: Oxford, 1977; pp 53-84. (13) Tanford, C.: Swanson, S. A.: Shore, W. S. J. Am. Chem. Soc. 1955, 77, 6414. (14) Era, S.; Sogami, M. J. Peptide Res. 1998, 52, 431. (15) Era, S.; Itoh, K. B.; Sogami, M.; Kuwata, K.; Iwama, T.; Yamada, H.; Watari, H. Int. J. Peptide Protein Res. 1990, 35, 1. (16) Hart, B. J.; Wilting, J.; De, G. J. J. Biochem. Pharmacol. 1986, 35, 1005.

Casca˜ o Pereira et al.

of a noncooperative mechanism. Considerably less structural information is known about this isomer, and there are no proposed structures available. However, transition to the B form involves a volume increase and a decrease in helical content, albeit less than that of the N f F transition.12 Because this transition happens at neutral pH, it is thought to have physiological significance. If aqueous solutions of albumin are maintained at pH 9 and low ionic strength at 3 °C for 3-4 days, yet another isomerization occurs which is known as the A form, or aged.8 There is also no proposed structure for the A form. Figure 1 shows the interrelation of the five recognized forms,8 the crystal structure of the N form, and the proposed configurations of F and E isomeric forms,11 as well as the changes in helical content.12 In this study, we focus on the rigid N form, and either its acid F form or basic B form that are readily accessible with small changes in solution pH. Experimental Section Reagents and Materials. Bovine serum albumin (BSA), 99% pure with essentially no fatty acid (Sigma Chemical Co., St. Louis, MO, A-0281, Lot No. 89H7604), and β-casein from bovine milk, minimum 90% purity (Sigma Chemical Co., St. Louis, MO, C-6905, Lot Nos. 108H7812 and 25H9550), were stored at 4 and -20 °C, respectively, prior to use without further purification. All aqueous solutions were prepared with distilled water further purified with a four-stage Milli-Q reagent grade water system (Millipore, resistivity greater than 18.2 mΩ‚cm). Proteins were dissolved under mild agitation. Solution pH was adjusted to desired values by minute additions of concentrated NaOH (Fisher Scientific, Pittsburgh, PA, Certified ACS S318-500) or HCl (Fisher Scientific, Pittsburgh, PA, Certified ACS plus A144C-212). pH was measured both before and after the experiments and exhibited little variation. At higher pH, experiments were conducted both under air and under an inert nitrogen atmosphere without observing any difference. An aqueous stock solution of 1.0 M NaCl (Fisher Scientific, Pittsburgh, PA, Certified ACS S271-500) was prepared from salt roasted at 400 °C for 24 h to drive off organic contaminants, and aliquots were used throughout all experiments. Solution concentrations were determined by UV spectroscopy at 280 nm, using extinction coefficients of 40 260 M-1 cm-1 for BSA8 and 11 040 M-1 cm-1 for β-casein.17 Solutions were used immediately after preparation, unless otherwise indicated. Aliquots from the same solution prepared according to these guidelines were used for all measurements. All glassware was first cleaned with distilled/deionized water (DI H2O), submerged in a 120 °C solution of a 4:1 v/v mixture of concentrated H2SO4 (J. T. Baker, Phillipsburg, NJ), and H2O2 (J. T. Baker, Phillipsburg, NJ), i.e., Piranha solution, for 15 min, and then thoroughly rinsed with DI water. All experiments were performed at ambient temperature, 22 °C. Interfacial Tension. Pendant-bubble tensiometry was used to determine the dynamic interfacial tension at the air/water interface with the air bubble formed upward at the tip of a U-shaped stainless steel needle (7.9 mm i.d.) immersed in the aqueous protein solution. To prevent water from entering the capillary and thus modifying bubble volume, the needle tip has a Teflon ring machined to fit tightly around it. The same apparatus was used for both interfacial tension and interfacial dilatational rheology measurements, as illustrated in Figure 2. The imaging system included a video camera manufactured by Rame´-Hart, Inc., a Cole-Parmer fiber optic illuminator, and two polarizers. The polarizers eliminated stray light reflections and permitted fine-tuning of the light intensity. The positions of the camera, sample holder, and drop-dispensing capillary were adjusted in the x, y, and z planes by means of multimovement Oriel Instruments translation stages. Likewise, fine positioning of the optical glass cell (Hellma Model 700.00) was obtained using an Oriel Instruments vertical and horizontal translation stage. The optical cell was filled with 30 mL of protein solution and covered with an acetate sheet to prevent evaporation of the water (17) Swaisgood, H. E. Adv. Dairy Chem. 1992, 1, 63.

Rheology of BSA Conformers at Air/Water Interface

Langmuir, Vol. 19, No. 6, 2003 2351 imaginary components of the Gibbs elasticity: E ) E′ + iE′′.22 Throughout this work, the word “modulus” refers to the interfacial dilatational modulus, E, and not to the interfacial shear modulus that can be similarly defined. A periodic strain was applied by differentially oscillating the bubble area, and the stress response was measured from bubble shape analysis. For sinusoidal variations in bubble surface area at a given oscillation frequency, E′ and E′′ are independently determined from the following relations:23

Ao cos φ E′ ) ∆σ ∆A

(2)

Ao E′′ ) ∆σ sin φ ∆A

(3)

and

Figure 2. Schematic of the combined pendant-bubble dynamic tensiometer and surface dilatational rheometer. phase. Prior to protein dissolution, water was saturated with inert nitrogen gas to minimize CO2 uptake into the solution. The entire apparatus was mounted on a pressurized vibration isolation table from Newport (Model VW-3046-OPT-2). To avoid impurities, all apparatus parts were cleaned with saturated NaOH in ethanol and washed extensively with DI water. After formation of a fresh air bubble at the needle tip, the interfacial tension was followed in time using axisymmetric bubble shape analysis.18 This technique determines the interfacial tension of an air/water or oil/water boundary by the shape of a gravity distorted air bubble or liquid drop. Image acquisition and regression of the interfacial tension was performed with commercially available Dropimage software (Rame´-Hart, Inc., Mountain Lakes, NJ) by fitting the Laplace equation to the drop shape. Dropimage software also controls an automatic pipetting system (Rame´-Hart, Inc., Mountain Lakes, NJ) that maintains constant bubble volume over long time periods (1 day) during which dynamic tensions are measured. Typical precision in tension measurements is (0.5 mN m-1. Throughout this work we report tension data in terms of the surface pressure, Π ) γo - γ, where γo is the surface tension of the clean water/air interface. Interfacial Dilatational Rheology. Simple visual observation of protein skins obtained by continued exposure at the air/ water interface for hours1,2,19,20 provides no quantitative information on interfacial mechanical properties.1 By using dynamic interfacial rheology measurements, interfacial aggregation/ gelation progress can be determined to establish the strength of the interfacial aggregate and to characterize its viscoelastic nature. Because of relevance to the coalescence of protein dispersions,4 we examined interfacial dilatational rheology, rather than the shear rheology. The linear viscoelastic surface dilatational storage modulus E′ was determined together with the surface dilatational loss modulus E′′, by subjecting the air/water interface to infinitesimal periodic expansion and contraction. As originally proposed by Gibbs,21 the surface dilatational modulus is defined as

E)

dσ dA/A

(1)

where A is the air-water interfacial area and σ is the air/water interfacial stress. Since the bubble area oscillates periodically, the dilatational modulus exhibits two contributions: an elastic part accounting for the recoverable energy stored in the interface (storage modulus, E′) and a viscous part accounting for energy lost through relaxation processes (loss modulus, E′′). The interfacial storage and loss moduli correspond to the real and (18) Mobius, D.: Miller, R. Drops and Bubbles in Interfacial Research; Elsevier: Amsterdam, 1998. (19) Proust, J. E.; Tchaliovska, S. D.; Ter-Minassian-Saraga, E. J. Colloid Interface Sci. 1984, 98, 319. (20) Clark, D. C.; Mackie, A. R.; Wilde, P. J.; Wilson, D. R. Faraday Discuss. 1994, 98, 253. (21) Gibbs, J. W. Collected Works; Longmans: New York, 1928.

where ∆σ is the amplitude of periodic interfacial-stress variation, Ao is the unperturbed interfacial area of the bubble, ∆A is the amplitude of periodic interfacial area variation, and φ is the phase angle between the periodic stress and strain curves. To determine the surface storage and loss moduli from eqs 2 and 3, the surface area and interfacial stress are best fit to the following functions:

A ) Ao + ∆A cos ωt

(4)

σ ) σo + ∆σ cos(ωt+φ)

(5)

and

where σo is the unperturbed tension and ω is the set frequency in radians per second. The unknown parameters ∆σ, ∆A, and φ are regressed using a least-squares method. Once the fitting procedure is complete, the surface storage and loss moduli follow from eqs 2 and 3. Mobius and Miller18 provide a comprehensive review of oscillatory bubble tensiometry. The volume-dispensing unit provided with the bubble tensiometer permits sinusoidal variations of the bubble surface area from low frequencies (thousandths of hertz) up to 0.2 Hz reliably. We modified the bubble tensiometer in Figure 2 to enable investigation of a wider range of frequencies. New oscillation hardware consisted of a pneumatic air cylinder (McMaster-Carr, Los Angeles, CA, Part No. 6604K21) mechanically coupled to a voice-coil linear actuator (Bei Kimco, San Marcos, CA, Model LA13-12-OOA). Actuator motion was forced using a function synthesizer (Hewlett-Packard, Palo Alto, CA, Model 3325A). Prior to driving the linear actuator, the function signal was amplified using a brushless PWM servo-amplifier (Advanced Motion Controller, Camarillo, CA, Model B12A6) connected to a dc power supply (Hewlett-Packard, Palo Alto, CA, Model 6281A). The function synthesizer was computer controlled via LabVIEW software (National Instruments, Austin, TX, Version 4.0). Similar to the dynamic tension measurements above, surface rheological behavior was followed over 24 h. To avoid continuously oscillating the bubbles for such long times, we imposed bubblearea perturbations intermittently. For the first 3 h, an area perturbation was imposed every 20 min; subsequently, area perturbations were imposed hourly. Each perturbation consisted of a frequency sweep of sine waves over 2 orders of magnitude in frequency. We chose six frequencies nearly equally spaced on a logarithmic scale: 0.01, 0.0178, 0.0316, 0.0562, 0.1, and 0.2 Hz. Each frequency sweep lasted 6 min. In this manner, we measure slowly evolving surface tensions and dilatational moduli simultaneously. Dynamic surface tensions obtained with intermittent bubble oscillation and those without any oscillation are indistinguishable. Equations 2 and 3 follow from linear response theory and demand small strains so that the interface lies in the linear viscoelastic regime. We set ∆A/Ao equal to 4.5%, since above a (22) Lucassen, J.; Van den Tempel, M. Chem. Eng. Sci. 1972, 27, 1283. (23) Lucassen, J.; Barnes, G. T. J. Chem. Soc., Faraday Trans. 1 1972, 68, 2129.

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Figure 3. Surface pressure (left) and ellipticity (right) histories for 0.1 g L-1 BSA, pH 5.0, 1 mM NaCl. relative strain of about 7.0% nonlinear effects were seen. Below 4.5% relative strain, we found that the surface dilatational moduli were independent of strain. Ellipsometry. Ellipsometry experiments at the air/water interface employed a compensator-and-rotating-analyzer ellipsometer (Sentech 400, Berlin, Germany) using the procedures of Theódoly et al.24 The trough containing the solution was mechanically isolated from the ellipsometer and connected to a pneumatic vibration isolation table (Newport Inc., Irvine, CA Model VW-3046-OPT-2) to minimize fluctuations of the air/water interface. The angle of incidence was set at 55°, close to the Brewster angle, where sensitivity is the highest. Assuming a single layer model for the adsorbed protein, its thickness d and its refractive index n were obtained from the ellipsometric angles ∆ and Ψ. For the case of adsorbed layers of several nanometers in thickness, ∆ varies linearly with layer thickness, whereas Ψ varies quadratically.25 In the thickness range reported in this work, ellipsometric detection is sensitive only to ∆. We are, therefore, left with an undetermined system of one equation and two unknowns. However, for reasonable values of the index of refraction of the adsorbed protein layer, variations in ∆ are directly proportional to the adsorbed amount.25 In the absence of a known adsorption density profile and a precise value for the index of refraction of the protein layer, ellipsometry data are used here to establish qualitative comparisons between adsorbed amounts.

Results and Discussion BSA Adsorption and Interfacial Rheology. Figure 3 reveals the typical pattern of transient adsorption for the protein solutions considered in this work. This particular figure reports the adsorption kinetics as reflected by dynamic surface pressure, plotted on the left ordinate, and by dynamic ellipticity (i.e., the angle ∆ in degrees), plotted on the right ordinate, for 0.1 g L-1 aqueous BSA in 1 mM NaCl and pH 5.0 adsorbed at the air/water interface. pH 5 corresponds to the N or “normal” form where BSA in the bulk aqueous solution is in its most stable and rigid state. Both measurement techniques yield similar adsorption kinetics. Therefore, below we compare protein adsorption of different BSA conformers in terms of their surface pressure data only. The adsorption kinetics reported in Figure 3 is characteristic of most proteins and polymers.26,27 At early times there is a sharp rise in adsorbed amount to a plateau (24) Theódoly, O.; Ober, R.; Williams, C. E. Eur. Phys. J. E 2001, 5, 51. (25) Azzam, R. M. A.; Bashara, N. M. Ellipsometry and Polarized Light; Elsevier: New York, 1987. (26) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 2001, 70, 403. (27) de Feijter, J. A.; Benjamins, J.; Tamboer, M. Colloids Surf. 1987, 27, 243.


Figure 4. Surface dilatational storage (filled circles) and loss (open circles) moduli histories at 0.01 Hz for 0.2 g L-1 BSA, pH 5.0, 1 mM NaCl. Lines connect the data points.

region, followed by a small increase in adsorbed amount over very long times. For the time scales and solution conditions investigated, i.e., up to 1 day, the amount of adsorbed protein continues to increase. The initial surface pressure increase is associated with population of the bare interface by protein.28,29 Surface pressure increases at extended times are attributed to conformational changes of the adsorbed protein and/or the development of interfacial aggregates.28,29 Apparently, proteins aged at an interface rearrange to expose hydrophobic chain segments to air28,29 and hydrophilic segments to the aqueous solution subject to the fixed primary sequence. This partial unraveling increases the surface pressure. Continued protein adsorption and insertion at an already populated interface, and even multilayer formation, further increase surface pressure, as suggested by the ellipsometry data in Figure 3. Washout experiments in which the protein solution is replaced by the solvent indicate no change in surface pressure or ellipticity after about 1 h of exposure to the air/water interface both for the BSA conformers and for β-casein (discussed later). Thus, not long after a protein molecule arrives at the interface, it is irreversibly attached. Such irreversible adsorption for proteins at fluid/fluid interfaces is consistent with our previous studies.29-34 Figure 4 illustrates the dilatational rheology behavior of the N form of BSA corresponding to the adsorption kinetics presented in Figure 3. We plot both the storage (closed circles) and loss (open circles) moduli obtained at 0.01 Hz for up to 20 h. Lines simply connect data points. Starting already at the first datum obtained at 20 min, both moduli are large and equal in magnitude to their final values within the experimental scatter of (4 mN m-1. Similarly, at 20 min, there is significant protein adsorption, as seen in Figure 3. The storage component in Figure 4, E′ ) 55 mN m-1, is larger than the loss component, E′′ ) 18 mN m-1, implying that the adsorbed (28) Tripp, B. C.; Magda, J. J.; Andrade, J. D. J. Colloid Interface Sci. 1995, 173, 16. (29) Beverung, C. J.; Radke, C. J.; Blanch, H. W. Biophys. Chem. 1999, 81, 5980. (30) Tupy, M. J.; Blanch, H. W.; Radke, C. J. Ind. Eng. Chem. Res. 1998, 37, 3159. (31) Anderson, R. E.; Pande, V. S.; Radke, C. J. J. Chem. Phys. 2000, 112, 9167. (32) Hickel, A.; Radke, C. J.; Blanch, H. W. Biotechnol. Bioeng. 2001, 74, 1. (33) Cascaõ-Pereira, L. G.; Hickel, A.; Radke, C. J.; Blanch, H. W. Biotechnol. Bioeng. 2002, 78, 595. (34) Svitova, T. F.; Weatherbee, M.; Radke, C. J. J. Colloid Interface Sci.; to appear, 2003.


Figure 5. Frequency dependence of the surface dilatational storage (filled circles) and loss (unfilled circles) moduli at 1 h for 0.1 g L-1 BSA, pH 5.0, 1 mM NaCl.

BSA layer is primarily elastic in nature. As a consequence, the value of the storage component dominates the magnitude of the dilatational modulus E ()xE′ 2+E′′ 2). Elastic behavior of the air/water interface is general for all protein solutions studied here. Elastic rheological behavior is also evident over the entire range of frequencies investigated. Figure 5 highlights a frequency sweep from 0.01 to 0.2 Hz at 1 h adsorption time for the same N-form BSA solution discussed in Figures 3 and 4. Storage (closed circles) and loss (open circles) moduli are plotted as a function of frequency on a semilogarithmic scale. The elastic component of the dilatational modulus is consistently larger than its viscous counterpart over the frequency range considered. Consequently, we present below dilatational moduli obtained only at one frequency, namely 0.01 Hz. Our conclusions are independent of this frequency choice within the range studied. As noted above, we likewise discuss our data only in terms of the dynamic storage modulus, E′. Adsorption of BSA Conformers. Three BSA conformers were investigated: the native N form as well as the F and B forms, obtained with small variations in solution pH (see Figure 1). In Figure 6, we report the adsorption kinetics of the three conformers at the air/ water interface. The N, F, and B conformers all display similar adsorption histories, consisting of a sharp rise at early times followed by a steadily increasing surface pressure over long times. Surface pressures in Figure 6 of both the normal (pH 5) and basic (pH 7) forms are nearly identical within the experimental error of (0.5 mN m-1. Both curves, however, exhibit higher surface pressures than the fast (pH 4) form at all times. Therefore, although all conformers adsorb strongly at the air/water interface, adsorption of the N and B forms is greater than that of the F form over the 10 h period studied. Structural characteristics, discussed in the Introduction and summarized in Figure 1, of the BSA conformers in solution are the origin of the difference in interfacial coverage. From Figure 1N, the N form occurs near the BSA isoelectric point and is the most compact structure. Although a protein crystal structure is not available, transition to the B form under basic conditions leads to a small volume increase compared to the N form.12 This result is in contrast to the acid transition leading to the more elongated and flexible F form. Therefore, the more compact N and B forms pack at the interface with a smaller molecular projected area, giving rise to higher surface

Langmuir, Vol. 19, No. 6, 2003 2353

Figure 6. Surface pressure history of BSA isomers at 0.01 g L-1, 1 mM NaCl.

pressures than the F form. Conversely, the maximum interfacial coverages for the more compact N and B forms are higher than that for the F form. In addition, an electrostatic barrier for adsorption likely exists at pH values away from the isoelectric point, as for the F and B forms. Dilatational Rheology of BSA Conformers. Figure 7 presents the dynamic interfacial dilatational rheology for each conformer in terms of the storage modulus, E′. The top curve replots the rigid N form of BSA presented previously in Figure 4. Almost immediately following adsorption at the air/water interface, there is a large storage modulus of the N form of about 55 mN m-1 that remains constant throughout the experiment, up to 10 h, within experimental error of (4 mN m-1. The decaying curve in Figure 7 corresponds to the storage modulus of the less rigid B form. Similar to the more rigid N form, following initial adsorption at ca. 20 min, there is a quick rise from 0 to E′ ) 48 mN m-1. For the B conformer, however, the storage modulus steadily falls to a value of about 18 mN m-1 over 10 h. The third curve in Figure 7 reflects the F form, obtained under acidic conditions. Up to about 2 h, only a small but nonzero storage modulus of around 3 mN m-1 is detected. After 2 h, corresponding to a surface pressure of ca. 12 mN m-1 in Figure 6, the storage modulus increases to a value of 30 mN m-1. Although the storage modulus of the F form eventually increases beyond that of the B form, at 9 h, it is still half the value of the more rigid N form. Comparison of the strikingly different interfacial rheology of BSA conformers in Figure 7 compared to their adsorption kinetics in Figure 6 reveals that interfacial rheology measurements are diagnostic of protein conformational stability at an interface. Although each BSA conformer adsorbed layer evolves to a lower free energy state, indicated by an increase in surface pressure with time, this evolution is accomplished by the adsorbed protein exhibiting drastically different rheological behavior. Adsorption amounts alone are clearly not sensitive to the structural state of adsorbed proteins. Following adsorption, the storage modulus of each conformer in Figure 7 increases from an initial value of zero. Despite population of the air/water interface by all three conformers, the storage modulus increase is immediate and more pronounced for the more stable structures, the N and B forms, than for the open structure of the F form. Therefore, interfacial dilatational moduli are largest for those protein molecules possessing higher internal structural rigidity.

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Figure 7. Dilatational storage modulus history of BSA isomers at 0.1 g L-1, 1 mM NaCl. Lines connect the data points.

To explain the slow transient rise in storage modulus in Figure 7 for the F species, we argue that an increasing adsorption density (see Figure 6) decreases the nearest neighbor protein separation distances at the interface and favors the development of interprotein bridges. Thus, an interfacial network or gel forms and slowly strengthens as a result of the connections between adjacent adsorbed molecules. This process is evidenced by the continuous increase in the storage modulus and is consistent with the experimental observations of protein skins or interfacial aggregate formation after prolonged interfacial exposure.1,2 Similar network formation likely applies to the N and B forms, but occurs on a time scale beyond that of the initial rapid rise in E′ evidenced in Figure 7. Increasing development of surface interprotein linkages is likely for adsorbed protein undergoing conformational change after prolonged interfacial exposure. Relaxation Mechanism under Basic Conditions. We now turn to the maximum in storage modulus for the B form in Figure 7. Following an almost immediate increase to about 50 mN m-1, the storage modulus of the less stable, loose B form slowly falls. Although not shown, the loss modulus decayed as well, indicating an overall decrease of the dilatational modulus E. Reduction of the storage modulus is due to a relaxation mechanism at or near the interface such as a diffusion/kinetic exchange22 with the bulk solution and/or conformational changes in the adsorbed layer.6,7,35 To understand the nature of the relaxation mechanism responsible for slow decay of the storage dilatational modulus of the B species in Figure 7, we investigated the role of electrostatic forces by varying the solution ionic strength over 1 order of magnitude. With 1.0 mM NaCl added electrolyte, the Debye screening length, κ-1, is 9.6 nm, whereas with 0.1 mM NaCl added electrolyte it is 30.4 nm. Figure 8 displays the resulting adsorption histories in terms of the surface pressure of the B species at these two ionic strengths, showing identical behavior within experimental scatter. In Figure 9, we report the corresponding storage dilatational moduli measured over 10 h at each ionic strength. The lower curve corresponds to an ionic strength of 1.0 mM NaCl; the top curve corresponds to 0.1 mM NaCl added electrolyte. Although the adsorption histories portrayed in Figure 8 are essentially the same, the dynamic storage moduli of these two salt-containing solutions are markedly different. At both ionic strengths, there is an initial rise in the storage (35) Serrien, G.; Geeraerts, G.; Ghosh, L.; Joos, P. Colloids Surf. 1992, 68, 219.


Figure 8. Surface pressure history of the “basic” form of 0.1 g L-1 BSA in 0.1 mM NaCl (top curve) and in 1.0 mM NaCl (bottom curve).

Figure 9. Dilatational storage modulus histories at 0.01 Hz for 0.1 g L-1 BSA in the “basic” form in 0.1 mM NaCl (top curve) and in 1 mM NaCl (bottom curve). Lines connect the data points.

moduli to ca. 48 mN m-1, followed by a decrease over the 10 h period. However, with less electrostatic screening, the storage dilatational behavior remains larger for longer times. As the protein species studied here display irreversible adsorption, diffusion/kinetic exchange with the bulk solution is not a candidate relaxation process. Rather, the operative modulus-relaxation mechanism for BSA likely involves rearrangement of the adsorbed molecule into a more unfolded and flexible conformation, which is in agreement with the notion of the interface acting as a denaturing agent.33 As the adsorbed protein layer relaxes to a less compact configuration, its intrinsic intraprotein rigidity weakens, and the storage dilatational modulus falls with time. Unfolding at the interface involves rearrangement of charged protein segments into a charged environment. Thus, rearrangement is favored under conditions of electrostatic screening. As a consequence, unfolding from a compact to a flexible molecule occurs faster with 1.0 mM NaCl added electrolyte compared to 0.1 mM NaCl. Comparison with β-Casein. The behavior of BSA conformers was compared with that of β-casein at conditions near their isoelectric points to avoid possible influence on the interfacial dilatational rheology due to charge effects. β-Casein contains no disulfide bonds and has little secondary structure.35 Native β-casein in aqueous solution is highly expanded with a radius of gyration of


Figure 10. Surface pressure (left) and ellipticity (right) histories for 1.0 g L-1 β-casein, pH 5.8, 10 mM NaCl, and for 1.0 g L-1 BSA, pH 5.0, 1 mM NaCl.

Figure 11. Dilatational storage modulus histories at 0.01 Hz for 1.0 g L-1 BSA, pH 5.0, 1 mM NaCl (open circles), and for 1.0 g L-1 β-casein, pH 5.8, 10 mM NaCl (filled circles). Lines connect the data points.

4.6 nm.35 It has a molecular weight of 23-24 kDa with 209 amino acid residues and an isoelectric point (pI) of 4.9-5.2.17 Figure 10 reports adsorption histories up to 10 h for 1.0 g L-1 BSA in 1.0 mM aqueous NaCl, pH 5.0 (the N state, at a 10-fold concentration increase compared to that in Figure 7), and 1.0 g L-1 β-casein in 10 mM aqueous NaCl, pH 5.8. Surface pressure is indicated on the left ordinate, while ellipsometry data are presented on the right ordinate. The adsorption history of both proteins is identical when gauged by these two techniques: dynamic adsorption density is characterized by a sharp rise followed by a slow steady increase at longer times. At the same concentration, 1.0 g L-1, β-casein adsorbs more strongly at the air/water interface than does BSA, as indicated in Figure 10 by larger surface pressures and ellipticities, in agreement with the notion of β-casein being an excellent foaming agent.37 Figure 11 presents the corresponding interfacial dilatational rheology measured at 0.01 Hz for BSA in the N state (open circles) and β-casein (closed circles). The storage dilatational modulus of BSA is at all times larger than that of β-casein, despite less BSA being adsorbed. This situation is analogous to that shown in Figure 7, (36) Andrews, A. L.; Atkinson, D.; Evans, M. T. A.; Finer, E. G.; Green, J. P.; Phillips, M. C.; Robertson, R. N. Biopolymers 1979, 18, 1105. (37) Dalgleish, D. G. J. Soc. Dairy Technol. 1989, 42, 91.

Langmuir, Vol. 19, No. 6, 2003 2355

Figure 12. Schematic of adsorbed protein layers subjected to an interfacial area compression, as in a dilatational rheology experiment. Adsorbed protein molecules, represented as spheres, are connected by interprotein contacts, shown as dark lines. Upon compression, globular proteins, such as BSA, respond as hard spheres, resisting compression and yielding high storage moduli. Flexible proteins, such as β-casein, respond as soft spheres, favoring compression and yielding low storage moduli.

where the rigid N-state BSA conformer exhibits a larger storage dilatational modulus than the elongated, flexible F species. We conclude that the interfacial dilatational modulus reflects this difference in adsorbed molecular structure between the two proteins. Storage dilatational moduli of both BSA and β-casein slowly increase with time. This is also analogous to Figure 7, where the storage dilatational modulus of the flexible, elongated F species also increases with time due to the development of interprotein contacts between neighboring adsorbed molecules. To explain these observations, we consider the adsorbed protein layer as a two-dimensional network of connected beads (proteins)38 on a plane representing the air/water interface, as indicated schematically in Figure 12. At early time, the adsorbed proteins remain as individual entities. However, upon prolonged exposure to the interface, conformational changes result in the development of an interconnected network (represented in Figure 12 by horizontal bars between individual units). The interprotein linkages may arise from electrostatic and/or hydrophobic interactions. Conformational change also leads to an increase in structural flexibility of the adsorbed proteins. Thus, there are two contributions to the measured dilatational moduli: (1) the intraprotein flexibility and (2) the aggregated interprotein network strength. In general, less flexible adsorbed proteins, such as BSA (top of Figure 12), are more effective in restoring the previous state of the interface (i.e., they are more elastic) than the more flexible proteins, such as β-casein (bottom of Figure 12). Thus, the early rise in storage modulus in Figure 7 for the N- and B-state conformers is postulated to arise from increasing adsorption at the interface (see Figure 6) to a coverage permitting interprotein contact and deformation. Area compression/expansion in this state alternately deforms and relaxes the individual adsorbed protein molecules. Since the N- and B-state conformers are relatively rigid molecules, an early large storage modulus is found. The same process occurs for the F-state conformer, but this molecule is relatively soft and does not (38) Wijmans, C. M.; Dickinson, E. Langmuir 1998, 14, 7278.

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resist compression/expansion. For the relatively soft F-state conformer in Figure 7, the intraprotein contribution to the storage modulus is small. Conversely, the second contribution of the slowly building interconnected network dominates and gives rise to the slow increase seen in E′. Aggregated structures also build with the B-state conformer in Figures 7 and 9, but this contribution to E′ is apparently smaller than the growing flexibility of the individual B conformers caused by partial unfolding. At this point, the overall storage modulus declines and a transient maximum is observed. The rigid N state lies between these two situations. The processes of network building and loss of internal structural rigidity are slower and offsetting. Hence, a somewhat constant dilatational storage modulus is found in Figures 7 and 11 for up to 10 h of interface exposure. Conclusions Linear viscoelastic interfacial dilatational moduli diagnose the onset and strength of interfacial aggregation and the dynamic conformation changes of proteins adsorbed at the air/water interface. BSA is studied in this work since it exists in several well-characterized conformations in bulk aqueous solution. We are thus able to probe the influence of solution molecular structure on dynamic interfacial moduli for the same protein species, but with differing degrees of intrinsic rigidity. β-Casein, a flexible protein, is also studied for comparison with BSA. BSA and β-casein are irreversibly adsorbed at the air/ water interface. The adsorbed layers of both proteins exhibit larger storage moduli than loss moduli and thus are primarily elastic in nature. Unlike low-molecularweight surfactants whose surface dilatational moduli are dictated by diffusion/kinetic exchange with the bulk solution22 or by Gibbs-Marangoni surface pressure gradients,39 the surface dilatational moduli of adsorbed protein arise primarily from aggregated network structures at the interface.38 For BSA and β-casein, the interfacial dilatational storage modulus reflects two contributions, both initiated by exposure to the air/water interface: (1) the strength of interprotein linkages formed upon conformational rearrangement and (2) the intraprotein structural rigidity at the interface that opposes interface compression and/or expansion. This latter contribution is not usually considered.38 Surprisingly, for the two proteins studied here the structural stability of the adsorbed species appears to be the most important. This may explain why adsorbed layers of globular BSA are more elastic than those of flexible β-casein and why the rigid N-state conformer of adsorbed BSA is more elastic than either the F-state or B-state conformer.


Interprotein links build between adsorbed protein due to rearrangement of the protein structure at the interface, thereby increasing the storage modulus in time. Concomitantly, intraprotein hydrophobic and electrostatic interactions may be disrupted due to the presence of the interface, partially unfolding the protein and reducing its structural rigidity. This process lowers the dilatational storage modulus. If the individual protein molecules are “soft” and flexible, then the strength of linked interprotein network formed upon conformational rearrangement is the dominant contribution to interfacial elasticity. Here a slow rise in storage modulus is obtained due to the slow development of interprotein bridges. This is indeed observed for the flexible and intrinsically “soft” F state of BSA and for β-casein. Conversely, if the structural rigidity of the protein is the dominant contribution to the dilatational modulus, then a maximum in dynamic interfacial storage modulus arises. The initial rise in storage modulus coincides with the initial rise observed in adsorption kinetics (see the knees in Figure 6). The subsequent decrease in storage modulus is due to intraprotein unfolding. We clearly observe such a maximum for the rigid and charged B-state conformer. The case of the most rigid N state is intermediate, since the characteristic intraprotein unfolding time is close to the characteristic interprotein linkage time and these two contributions partially offset. Electrostatic screening increases the rate of interfacial unfolding. Consequently, a possible strategy for immobilizing enzymes at the air/water or oil/ water interface while maintaining their structural stability and activity involves strong adsorption to favor the compact state, followed by minimal electrostatic screening to retard the intraprotein relaxation mechanism. Interfacial dilatational storage moduli are more sensitive to adsorbed molecular structure than are the adsorbed amounts from either surface tension or ellipsometry. Interfacial rheometry is, therefore, a valuable tool for investigating protein conformational stability at an interface. In addition to characterizing the interfacial rheological behavior of proteins used in food emulsions and foams, dilatational interfacial rheology is useful in the design of interfacial enzyme catalysis strategies.33 Acknowledgment. The authors thank Nestle´ Inc. and DOE (Grant FG03-94ER14456) for financial support. We acknowledge the contribution of Lisa Biese as a summer intern under support by the ERC Program of the National Science Foundation (Award EEC-9731725) and the National Sea Grant College Program of the U.S. Department of Commerce’s National Oceanic and Atmospheric Administration under NOAA Grant NA66RG0477, Project No. R/E-59PD, through the California Sea Grant College Program.

(39) Lucassen-Reynders, E. H.; Wasan, D. T. Food Struct. 1993, 12, 1.

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Water Interface

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