The Displacement of Preadsorbed Protein with a Cationic Surfactant at

Department of Physics, UMIST, P.O. Box 88, Manchester M60 1QD, U.K. .... Biological contaminants identified from laryngeal mask airways. J.R. Lu. Brit...
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J. Phys. Chem. B 2001, 105, 9331-9338

9331

The Displacement of Preadsorbed Protein with a Cationic Surfactant at the Hydrophilic SiO2-Water Interface R. J. Green,† T. J. Su, and J. R. Lu* Department of Physics, UMIST, P.O. Box 88, Manchester M60 1QD, U.K.

J. R. P. Webster ISIS, CCLRC, Chilton, Didcot OX11 0QX, U.K. ReceiVed: February 16, 2001

Neutron reflectivity has been used to investigate the interaction between the cationic surfactant dodecyltrimethylammonium bromide (C12TAB) and preadsorbed lysozyme layers at the hydrophilic silica-water interface. Reflectivity measurements were carried out with two different concentrations of lysozyme and a range of C12TAB concentrations at a solution pH of 7. A preadsorbed lysozyme layer was prepared by adsorption from 0.03 or 1 g dm-3 protein solutions. The effect on the adsorbed protein layer structure upon addition of a range of surfactant concentrations, from 0.2 to 14 mM, was determined and the surface excesses of both protein and surfactant within the adsorbed layers calculated. The surface composition of the mixed layers and their structural distributions were identified with the help of the variation of hydrogen/deuterium labeling to the surfactant. It was found that upon increasing surfactant concentration the protein was gradually replaced by the surfactant at the interface. However, a simple replacement mechanism was not observed. Protein removal over the low surfactant concentration range was accompanied by little surfactant adsorption, showing that the desorption was likely to be caused by the formation of highly soluble protein/surfactant complex. At the high surfactant concentration, protein removal was driven by the interplay of the interactions involving the protein, surfactant, and substrate, as evidenced from the coadsorption of surfactant and protein at the interface and the variation of interfacial composition with surfactant concentration. These results, together with previous measurements using sodium dodecyl sulfate (SDS) suggest that for surfactants with the same alkyl chain length the fraction of protein removal is dictated by the nature of the surfactant headgroups. These studies have shown that neutron reflectivity has distinct advantages over other techniques in investigating multicomponent interfacial systems involving biomolecules.

Introduction Study of interactions between surfactants and preadsorbed protein layers has practical implications in the area of surface cleaning. Surfactants are readily used to remove protein foulants from the surfaces of food processing equipment and from the surfaces of porous separation membranes1. The extent of removal of proteins by surfactants from the surfaces of biomaterials is widely used as a criterion in assessing the binding strength of proteins to biomaterials.2,3 These studies have often involved different mixtures of surfactants and proteins, but the predictability of the extent of removal of the adsorbed protein by a given surfactant is poor and in many cases remains highly empirical. This situation is largely caused by the lack of understanding of the interactions between the two species. The interaction between surfactant and protein in bulk solution has been extensively explored over the past four decades. Association of surfactant onto protein molecule usually leads to the deformation and deterioration of protein’s threedimensional structure and the subsequent loss of its biological activity. Tanford et al.4,5 and others6-8 have demonstrated that surfactant binding onto protein molecules usually starts with electrostatic attraction. With increase of surfactant concentration, * To whom correspondence should be addressed. E-mail:[email protected]. † Current address: Department of Chemistry, University of Leicester, University Road, Leicester LE1 7RH, U.K.

hydrophobic interaction tends to prevail. Hydrophobic affinity promotes the association of a substantial amount of surfactant into the hydrophobic domain(s) within a protein molecule and is thus the major factor causing structural breakdown. In many binding processes, both electrostatic and hydrophobic interactions may occur simultaneously and it is therefore difficult to disentangle the two events. Tanford et al. have also shown that for a given surfactant, the strength of binding is affected by the alkyl chain length of surfactant and the relative amount of surfactant bound is limited by its monomer concentration. However, the type of surfactant headgroup usually asserts more significant influence. For example, while anion surfactant such as sodium dodecyl sulfate (SDS) binds to almost all polypeptides, nonionic pentaethylene glycol monododecyl ether (C12E5) shows relatively little affinity to almost any protein in bulk solution. The interaction of surfactant and protein at the solid-solution interface is further complicated by their relative strength of interaction with the solid substrate. A large number of literature studies have been devoted to addressing the issue of elution of preadsorbed protein layers by ellipsometry measurements.2,3,9-12 Because ellipsometry detects the change of refractive index profile across the interface, its main limitation in this type of measurement is its inability to distinguish protein from the bound surfactant. As will be seen later, the binding of cationic surfactant onto preadsorbed lysozyme layers causes partial

10.1021/jp0106247 CCC: $20.00 © 2001 American Chemical Society Published on Web 08/24/2001

9332 J. Phys. Chem. B, Vol. 105, No. 38, 2001 removal of the polypeptide. Hence, by just determining the total refractive index profile and thus the total amount of the mixture, it is difficult to quantify the extent of protein remaining and the amount of bound surfactant. This shortcoming inevitably constrains the molecular level of information to be accessed. Despite this inherent disadvantage, some of the main trends of interactions between surfactants and proteins have already been outlined from the precedent ellipsometry work. Preadsorbed proteins may be removed by surfactants through either solubilization or replacement mechanisms.2,3,9-12 The former involves the formation of complexes between surfactant and protein, and the latter involves the adsorption of surfactant onto substrate surface. Horbett et al. carried out extensive studies to investigate the strength of binding of proteins adsorbed at interfaces and the subsequent “elutability”.2,3 The effects of surface chemistry, protein concentration and residence time at the interface on the ‘“elutability” of the protein were also explored. In these studies, the interaction between protein and surfactant at the molecular level remained unknown and the surfactant was used simply as a tool to probe the strength of interaction between the protein and biomaterial surface. Similarly, Arnebrant et al. have carried out a series of protein elution experiments using in situ ellipsometry. They considered the effect of the type of surfactant headgroup on the removal of preadsorbed protein, in particular using SDS and alkyltrimethylammonium surfactants (CnTAB).9-13 These authors have shown that the strength of interaction between lysozyme and the anionic SDS is considerably stronger than that of lysozyme with the cationic CnTAB, a trend consistent with that observed in bulk solution.4,5 Likewise, Arnebrant et al.10 showed that, at concentrations well above the critical micellar concentration (CMC), SDS led to the complete removal of all proteins whereas with C18TAB only partial removal was observed. Their conclusion led to the following question: does the C18TAB simply replace the protein or bind to the protein leading to partial removal? Unfortunately, no firm conclusion could be drawn from their results because ellipsometry measurements could not differentiate between the two possibilities. Several recent studies have shown that radiolabeling offers the prospect of identifying the composition of a given component in a multicomponent solution.2,14 The attachment of H3or I125-labeled fragments to proteins has allowed the competitive adsorption from the binary mixtures to be studied. However, because no information can be gained about the distributions of the species involved and their relative location from this method, it is difficult to assess the extent of deformation and denaturation of individual species at the interface. Furthermore, although it is straightforward to probe radioactive signals at the air-water interface, the situation at the solid-solution interface is more complex and the measurement becomes less quantitative. Finally, the attachment of radioactive fragments to proteins may alter their surface activities. Recent development in experimental techniques has highlighted the advantages of neutron reflectivity as a noninvasive means capable of determining the interfacial structure of multicomponent systems, as well as providing quantitative analysis of the adsorbed amount of each surface constituent.15-17 Research using neutron reflectivity has led to the successful determination of the adsorbed layer structures of mixed surfactant systems containing both synthetic polymers and proteins.18-22 These studies have demonstrated the potential of neutron reflection to reveal structural details of adsorbed layers at both the air-water and solid-liquid interfaces. Indeed, we have recently demonstrated the use of neutron reflectivity to

Green et al. quantitatively determine the adsorbed amount and layer structure of protein layers bound by SDS at the hydrophilic solid-liquid interface.22,24 This work is to extend this study to the investigation of interaction between preadsorbed lysozyme and dodecyl trimethylammonium bromide (C12TAB) at the hydrophilic silica-water interface using neutron reflectivity. The combination of neutron reflectivity and the use of deuterium labeling to the surfactant can easily resolve the complexity caused by the presence of the two species at the interface. Thus, any structural changes that occur to the protein layer upon the addition of C12TAB will be detected. The mechanism for the removal of lysozyme by C12TAB is considered and also directly compared with previous reflectivity data where SDS was used. Experimental Section Neutron reflection measurements were performed on the white beam reflectometers SURF and CRISP at the Rutherford Appleton Laboratory, ISIS, Didcot, U.K.,25 with neutron wavelengths from 1 to 6 Å. The sample cell configuration used was similar to that used by Fragneto et al.26 A Teflon trough, containing the aqueous solution, was clamped against a silicon block of dimensions 12.5 × 5 × 2.5 cm3. For the CRISP and SURF instruments, the collimated beam entered, at a fixed angle, through the end of the silicon block and was reflected at a glancing angle at the solid-water interface before exiting through the opposite end of the block. Neutron measurements were carried out at three different glancing angles, 0.35°, 0.5°, and 0.8°, and the three reflectivity curves merged manually. The beam intensity was calibrated by taking the intensity below the critical angle at the silicon-D2O interface to be unity. A flat background was determined by extrapolation to high values of momentum transfer, κ (κ ) (4π sin θ)/λ, where λ is the wavelength and θ is the glancing angle of incidence). For all the measurements the reflectivity profiles were essentially flat at κ > 0.2 Å2. The typical background for D2O runs was 2 × 10-6 (measured in terms of reflectivity). Hen egg white lysozyme was purchased from Sigma (Cat. No. L6876) and used as supplied. Lysozyme has a molecular weight of 14 600 Da, with an isoelectric point around pH 11. Thus, at pH 7, lysozyme is positively charged.27 Lysozyme is one of more stable globular proteins whose dimensions are approximately 30 × 30 × 45 Å3. Hydrogenated C12TAB (h-C12TAB) was purchased from Polysciences (99%+) and was recrystalized several times till its surface tension around its CMC showed no minimum. For the deuterated surfactant, a fully deuterated version (dC12dTAB, abbreviated to d-C12TAB) was used and made by the method outlined by Simister et al.28,29 All protein and surfactant solutions were prepared using a phosphate buffer with the pH controlled at pH 7 and keeping the total ionic strength fixed at 0.02 M. The glassware and the Teflon troughs were cleaned using an alkaline detergent (Decon 90) followed by repeated rinsing with ultrapure Elgastat (UHQ) water. The large (111) face of the silicon block was polished using an Engis polishing machine as described previously.22,23 Polished blocks were cleaned using piranha solution and exposing the surface to UV/ozone to remove any traces of organic impurities.30,31 This procedure produces surfaces that have repeatable thickness and roughness values of the oxide layer and that are completely wetted by water. Finally, the blocks are clamped against the neutron Teflon cells in contact with UHQ water until the beginning of the neutron experiment.

Protein-Surfactant Interactions at the SiO2-H2O Interface

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TABLE 1: Structural Parameters Obtained from the Fitting of Reflectivity Profiles for the Adsorption of Pure h-C12TAB/ D2O, pH7 at the SiO2 Interface [C12TAB], mM τ1 ((2), Å 106F ((0.2), Å-2 0.05 0.2 1 7 14

0 6 14 21 22

0 5.8 5.4 4.2 3.6

A, Å2

Γ, mg m-2

0 967 ( 80 240 ( 20 71 ( 4 53 ( 3

0 0.05 ( 0.02 0.21 ( 0.02 0.72 ( 0.04 0.97 ( 0.05

Results and Discussion Two different silicon blocks were used in this work. Their surfaces were initially characterized by neutron reflectivity measurements to determine the thickness and uniformity of the native silicon oxide surface layers. These measurements were performed, using the liquid cell arrangement described in the Experimental Section, with the silicon oxide layer in contact with D2O in a manner identical to that used in previous protein adsorption studies.22-24 The resultant reflectivity profiles were analyzed using the model fitting on the basis of the optical matrix formula, the details of which have been outlined elsewhere.32,33 In this fitting procedure, a structural model is first assumed and the reflectivity is calculated using the optical matrix formula. The calculated reflectivity is then compared with the measured data. The structural parameters are then varied in a least-squares iteration until a best fit is found. The structural parameters used in the fitting are the number of layers, thickness (τ), and the corresponding scattering length density (F) for each layer. The choice of the number of sublayers is dependent upon the extent of inhomogeneity across the interface; however, in general, the minimum number of layers that will successfully fit the data is chosen. The two reflectivity profiles were fitted using the thicknesses of 18 ( 3 and 14 ( 3 Å for the oxide layers. In both cases F was found to be 3.41 × 10-6 Å2 and was the same as the value for bulk silicon oxide, suggesting little defects within the oxide layers. No roughness was necessitated in the fitting, again showing the good uniformity of the oxide layers. These fits were then used for all subsequent adsorption experiments carried out using the relevant blocks. Cationic surfactants, such as C12TAB, will adsorb directly to the negatively charged silicon oxide surface. Therefore, to effectively analyze the interaction of C12TAB with a preadsorbed protein layer, the adsorption of C12TAB to the bare oxide needed to be determined. In the literature, studies of the adsorption of CnTAB have been carried out by both ellipsometry and neutron reflectivity.34-37 However, neutron reflectivity studies of C12TAB in pH 7 buffer conditions have not previously been performed. Therefore, these reflectivity profiles were measured at the bare silicon oxide-water interface under a range of surfactant concentration and the resultant structural parameters are shown in Table 1. To maximize the contrast between the surfactant layer and the aqueous solution, the measurements were performed using hydrogenated C12TAB in D2O. The data were fitted using the optical matrix formula to determine the adsorbed amount of the surfactant at the interface. Reflectivity curves and their corresponding best uniform layer fits for the adsorption at 7 and 14 mM are given in Figure 1. The thickness and surface excess of the adsorbed layer increased with the surfactant concentration up to the CMC of C12TAB (the CMC decreased to ca. 12 mM as a result of addition of phosphate buffer). At the CMC, a uniform layer fit was used with a thickness of 22 Å and F ) 3.6 × 10-6 Å-2. Thicknesses above 25 Å or below 20 Å were found to produce poor fitting. This observation thus indicates that the thickness could be determined

Figure 1. Neutron reflectivity profiles for the adsorption of 0 (O), 7 (b) and 14 mM (*) hydrogenated C12TAB at the hydrophilic SiO2D2O interface. The continuous lines were calculated reflectivity profiles based on the optical matrix formula with the structural parameters given in Table 1.

to an accuracy of (3 Å. The area per molecule (A) could be derived from the optimally fitted F, and the value was subsequently found to be 53 Å2. It should be noted that the orientation of the surfactant molecules within the adsorbed layers could be investigated using partially deuterated C12TAB, e.g., the alkyl chain deuterated (dC12hTAB) and the head deuterated (hC12dTAB). However, since the main objective of this work was to explore the binding of the cationic surfactant with preadsorbed lysozyme layers, only the surface excesses and total thickness values for C12TAB were required. Fragneto et al.34 have studied the detailed structure of C16TAB at the hydrophilic silicon oxide-water interface using neutron reflection combined with partially labeled surfactants. They suggested that the adsorption of the cationic surfactant leads to the formation of flattened micellar structures, which in terms of modeling to neutron reflectivity were approximated to defective bilayers. Inside these bilayers, the surfactant molecules project their headgroups toward the outside to face the hydrophilic solid substrate and water and the hydrophobic tails are excluded from direct contact with the aqueous environment. It is likely that the layers formed by C12TAB take similar structural conformation. The C16TAB layer at its CMC is about 32 Å thick, and the area per molecule is 31 Å2 (62 Å2 for a pair), as compared with a thickness of 22 Å and an area per molecule 52 Å2 for C12TAB. These results show that increase in n in CnTAB results in the increase of surface excess and the bilayer thickness, implying that as n is shortened, the bilayer becomes less densely packed. A decrease in the concentration of C12TAB below the CMC leads to the increase in the area per molecule and hence a reduced surface coverage, as expected. However, the thicknesses of the C12TAB layers also drop drastically with concentration. For example, at 1 mM the thickness of the adsorbed C12TAB layer is about 14 ( 3 Å. This trend of thickness reduction is in contrast with the rather constant layer thicknesses observed by Fragneto et al. for C16TAB34 over a wide surfactant concentration. It should be remembered that as the adsorbed amount of C12TAB decreases fast below the CMC, the reflectivity measurements quickly become insensitive to the decoupling of the thickness from its corresponding scattering length density. Alternatively, it can be said that as surface excess decreases, layer thickness and scattering length density become more

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Green et al.

interrelated. The errors quoted in Table 1 only refer to the range of sensitivity when the scattering length density is fixed at the optimal value. For the mixed protein/surfactant layer system considered here, the surface excesses of the protein and surfactant within the adsorbed layer were determined by the procedure outlined below. The total scattering length density of the adsorbed layer (as determined from fitting of the neutron reflectivity profiles) can be expressed as

F ) φpFp + φsFs + φwFw

(1)

where Fp, Fs, and Fw, the scattering length densities of protein, surfactant, and water, are known, and φp, φs, and φw, their respective volume fractions, are not known.38,39 In addition, the volume fractions of all constituents must add up to one (φp + φs + φw ) 1). With these two equations the volume fractions of each constituent cannot be solved, since there are three unknown components among them. However, repeating the C12TAB binding measurements using two different isotopic contrasts involving both h-C12TAB and d-C12TAB will lead to the independent resolution of eq 1 and the determination of the volume fraction of both lysozyme and C12TAB in the adsorbed layer. Once the volume fractions are known, the area per molecule and surface excess of each surface constituent can easily be determined. The area per molecule of lysozyme can be calculated using

A)

∑bp

φpFpτ

(2)

where ∑bp is the total scattering length for lysozyme and τ the thickness of the layer. The surface excess is then calculated by

Γ)

MW 6.02A

(3)

where MW is the molecular weight of the protein. Equations 2 and 3 can also be used to determine A and Γ for C12TAB. Although eqs 1-3 have been developed under the condition of uniform layer distribution, they are directly applicable to each of the sublayers when more than one layer is required to model the density distribution profiles. The total adsorbed amounts are obtained by summing over the sublayers used in the fitting procedure. Surfactant/protein interactions were studied using a range of surfactant solution concentrations, from 0.05 to 14 mM, and at two fixed lysozyme concentrations (0.03 and 1 g dm-3). For each surfactant binding measurement, the preadsorbed lysozyme layer was characterized by neutron reflectivity immediately before the addition of the surfactant solution to the system. These measurements were performed in D2O so as to highlight the adsorbed protein layer. The volume fraction and surface excess values for the adsorbed layers were calculated using eqs 1-3 and the scattering length density for the pure protein was taken as 3.66 × 10-6 Å-2.40 Adsorption from 0.03 g dm-3 lysozyme solution led to the formation of a single uniform layer of adsorbed lysozyme at the SiO2-D2O interface, with thickness of 32 ( 3 Å and F ) 5.1 × 10-6 Å-2. Area per molecule for lysozyme was calculated to be 1400 Å2, suggesting the formation of sideways-on monolayer. For adsorption from a 1 g dm-3 lysozyme solution, a bilayer structure was produced containing a dense first layer and more diffuse outer layer, with the protein molecules adsorbed sideways-on in both layers (with

Figure 2. Neutron reflectivity profiles for the addition of 0.2 (O), 7 (∆), and 14 mM (b) deuterated C12TAB (pH 7/D2O) to a preadsorbed lysozyme layer (adsorbed from 1 g/L lysozyme solution). For comparison, the reflectivity curves for the pure lysozyme layer (+) and from the bare SiO2-D2O interface (*) is also shown for comparison. The continuous lines are the model fits with structural parameters given in Table 2.

structural parameters of 30 ( 3 Å, F ) 4.9 × 10-6 Å-2 and 32 ( 3 Å, F ) 5.6 × 10-6 Å-2, respectively). These results have been discussed in greater detail previously, where the adsorbed layer structures of lysozyme at the SiO2-D2O interface has been studied in depth.41,42 In calculating the adsorbed amount of protein, complete exchange of labile hydrogens with the surrounding D2O was assumed. Although such an assumption is not strictly true, we have found from our previous results that the small fraction of unexchanged hydrogens present are undetectable and well within the errors of the experiment.22-24,43 The method used to investigate surfactant binding to the preadsorbed protein layer was as follows. After characterization of the protein layer, a surfactant solution was introduced to the liquid cell and then analyzed by neutron measurements. The solid surface was then cleaned by rinsing the cell with an 8 mM solution of SDS to remove all adsorbed material at the oxide surface. This was followed by copious rinsing of the cell with UHQ water before a final rinse in D2O. The block was then measured and the resulting reflectivity curve compared with the previous SiO2-D2O measurement to check that all the adsorbed material had been removed and that the SiO2 surface had returned to its original state. The protein solution was then reintroduced to the cell and the protein binding measured and compared with previous measurements before the next surfactant binding measurement was performed. The adsorption of lysozyme from a 1 g dm-3 solution leads to the formation of a two-layer structure at the interface, both layers of thickness approximately 30 Å, as already discussed. The reflectivity profiles for the introduction of 0.2-14 mM d-C12TAB/D2O solutions to this preadsorbed lysozyme layer is given in Figure 2. The addition of d-C12TAB had an effect on the shape of the neutron reflectivity profile, as shown by the change in reflectivity in comparison to that of the pure protein reflectivity curve. Since the scattering length density of d-C12TAB is 6.83 × 10-6 Å and is similar to that of D2O, its presence at the interface is largely masked by the D2O. Therefore, the observed changes in reflectivity seen in Figure 2 outlined changes to the structure and adsorbed amount of lysozyme left within the layers. As the concentration of surfactant was increased, the gradient of the reflectivity curves gradually

Protein-Surfactant Interactions at the SiO2-H2O Interface

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TABLE 2: Composition of the Mixed Protein/Surfactant Interfaces Obtained from the Fitting to the Reflectivity Profiles [LSZ], g dm-3 Γ1LSZΓ2LSZΓC1 12TAB ΓC2 12TAB(a) 0.03

(b) 1

mg m-2

[C12TAB], τ1 ((2), τ2 ((2), mM Å Å 0 0.2 1 7 0 0.05 0.2 1 7 14

32

2.1

28 29 28 30 31 31 34 36 38

1.73 1.48 0.65 2.34 2.34 2.09 2.14 1.64 1.03

32 24 23 24

0 0 0 0.35 1.29 0 0.58 0 0.43 0 0.2 0.1 0.26 0.33

0 0 0 0.025

decreased, indicating the removal of material from the interface. However, for all surfactant concentrations measured, the addition of d-C12TAB did not lead to complete removal of protein from the interface. This can be seen in Figure 2 by the fact that none of the surfactant/protein reflectivity profiles coincide with that of the bare SiO2 surface. At low C12TAB concentrations, from 0.05 to 1 mM, the reflectivity profiles were fitted to a twolayer model with dimensions similar to those of the pure protein layer. For example, at 0.2 mM C12TAB, the model fits for each layer were 31 ( 3 Å, F ) 5.1 × 10-6 Å-2 and 23 ( 3 Å, F ) 6.0 × 10-6 Å-2, respectively. In comparison to the fits from the pure protein layer, these parameters show that the inner protein layer was not significantly affected by the addition of surfactant, but the thickness was reduced and material lost from the outer layer. For the higher C12TAB concentrations, from 7 to 14 mM, a single uniform layer could be used to successfully fit the data. For example, the introduction of a 14 mM d-C12TAB solution led to a uniform layer fit of 38 ( 3 Å and F ) 5.95 × 10-6 Å-2 suggesting that, even when at the concentration above the surfactant’s CMC, some protein remained adsorbed at the interface. Reflectivity measurements using h-C12TAB (F ) -0.231 × 10-6 Å-2), as well as d-C12TAB, allow sufficient isotopic contrasts between the surfactant and D2O and determination of the adsorbed amounts of both the surfactant and protein at the interface. The results from fitting all the neutron reflectivity profiles for the addition of h- and d-C12TAB to the preadsorbed protein surfaces are summarized in Table 2. The detailed description of the neutron data analysis for the addition of 0.2, 1, and 14 mM surfactant solutions to the preadsorbed protein surface is given in Figures 3-5. The effect of introducing low solution concentrations of surfactant to the adsorbed protein layer (preadsorbed from a 1 g/L protein solution) is shown in Figure 3, for the addition of 0.2 mM h- and d-C12TAB solutions in D2O. The use of the solution contrast d-C12TAB/D2O has an effect on the shape of the neutron reflectivity profile, as shown by the change in reflectivity in comparison to that of the pure protein reflectivity curve (dotted line in Figure 3). As discussed in Figure 2, a twolayer model was required to successfully fit this reflectivity curve. The volume fractions of the protein within each layer were calculated to be 0.50 and 0.13, as compared to 0.54 and 0.28 for the original preadsorbed lysozyme layer. The calculated total surface excess was 2.5 mg m-2 as opposed to 3.6 mg m-2 prior to surfactant addition, suggesting that significant quantities of protein had been removed from the interface. By also considering the second solution contrast, h-C12TAB/D2O, the amount of C12TAB present at the interface is highlighted. Comparison of this reflectivity profile with that of the corresponding d-C12TAB reflectivity curve showed that the different

Figure 3. Neutron reflectivity profiles obtained for the addition of 0.2 mM deuterated (O) and hydrogenated (+) C12TAB in D2O to a preadsorbed lysozyme layer (adsorbed from 1 g dm-3 lysozyme solution). The continuous line is the best two-layer fit for the d-C12TAB system with structural parameters given in Table 2. This calculated profile also fits the hydrogenated C12TAB curve well, and the result shows that the different isotopic labeling of the surfactant has no effect on the reflectivity, thus suggesting that very little surfactant is present at the interface. For comparison, the dotted line indicates the reflectivity fitted for the preadsorbed protein layer and the dashed line corresponds to the fit for the bare SiO2-D2O interface.

Figure 4. Neutron reflectivity profiles obtained for the addition of 1 mM deuterated (O) and hydrogenated (+) C12TAB in D2O to a preadsorbed lysozyme layer (adsorbed from 1 g dm-3 lysozyme solution). The continuous lines are the best two-layer fits with structural parameters given in Table 2. The difference between these two reflectivity curves indicates the extent of C12TAB association. For comparison, the dashed line corresponds to the fit to the bare SiO2D2O interface.

labeling of C12TAB had no effect on the reflectivity, which suggested that no C12TAB was present at the interface. Thus it appears that the introduction of low surfactant solution concentrations to the preadsorbed protein surface leads to partial removal of protein, mainly from the outer layer, but does not lead to any coadsorption of C12TAB. The reflectivity profiles for the addition of a 1 mM C12TAB solution in D2O to the preadsorbed protein layer is given in Figure 4. At this intermediate surfactant concentration, the reflectivity profile for the d-C12TAB contrast shows that the lysozyme layer structure has been disrupted by the presence of

9336 J. Phys. Chem. B, Vol. 105, No. 38, 2001

Figure 5. Neutron reflectivity profiles obtained for the addition of 14 mM deuterated (O) and hydrogenated (+) C12TAB in D2O to a preadsorbed lysozyme layer (adsorbed from 1 g/L lysozyme solution). The continuous lines are the best two layer fits with structural parameters given in Table 2. The extent of C12TAB association is indicated by the difference between the two reflectivity curves. For comparison, the dashed line corresponds to the fit to the bare SiO2D2O interface.

surfactant in solution. As in the case of low surfactant concentrations, this is largely manifested as a reduced thickness and adsorbed amount of protein within the outer layer. The thickness of the outer layer is reduced to 24 ( 3 Å and the surface excess calculated as 0.16 mg m-2 compared to 1.3 mg m-2 for the pure protein layer. However, by also considering the h-C12TAB/D2O contrast, the presence of surfactant at the interface is also evident. Fitting to the reflectivity profiles revealed the volume fraction of surfactant at approximately 0.03 within both protein layers and surface excesses of C12TAB of 0.1 and 0.07 mg m-2 in the inner (34 ( 3 Å) and outer (24 ( 3 Å) protein layers, respectively. Thus, an almost even distribution of surfactant throughout the whole protein layer structure was observed under this condition. As the concentration of surfactant increased, the amount of protein at the interface further decreased. Consistent with this trend is the corresponding increase in the amount of adsorbed surfactant. However, as seen in Table 2, the complete removal of protein from the interface was not achieved, even when the surfactant solution concentration was increased to 14 mM, just above the CMC of C12TAB in buffer solution. Figure 5 shows the reflectivity profiles for the addition of 14 mM h- and d-C12TAB/D2O to the preadsorbed lysozyme layer. The reflectivity curves of the mixed protein/surfactant system differed from that obtained from the bare silica-D2O interface and those after hand d-C12TAB adsorption onto the bare silica-D2O interface, suggesting that some protein remained adsorbed to the interface. In fact, the large difference in the reflectivity profiles of the two isotopic contrasts of C12TAB in the presence of lysozyme clearly highlighted a significant adsorption of C12TAB at the mixed interface. A uniform layer model was used to fit these reflectivity profiles with a thickness of 38 ( 3 Å and the surface excesses for the protein and surfactant calculated as 1.03 and 0.33 mg m-2, respectively. It was found that for these higher surfactant concentrations, a single uniform layer was appropriate for the distributions of both the protein and surfactant. This observation, together with the fact that the determined thickness was greater than that of a single protein layer or a pure surfactant layer, suggested that surfactant molecules had become inter-

Green et al.

Figure 6. Variation of surface excess of lysozyme (a) and C12TAB (b) with bulk C12TAB concentration. The symbols indicate the lysozyme solution concentrations, (×) 0 g dm-3, (s) 0.03 g dm-3, and (l) 1 g dm-3, used to prepare the preadsorbed protein layer.

spersed within a slightly expanded protein layer, which was indicative of binding between the surfactant and protein. The surface excess of protein at the interface remained relatively high and so was the level of surfactant, with both contents well within measurable range. The reflectivity profiles described above consider the interaction of C12TAB with a bilayer of adsorbed protein, preadsorbed from a 1 g dm-3 solution. As already outlined previously, a lysozyme concentration of 0.03 g dm-3 was also used and this resulted in the formation of a single uniform layer of protein. The results of the interaction of C12TAB with this surface are also summarized in Table 2, which shows a trend similar to that of the bilayer system already described. For the uniform protein layer system, low concentrations of surfactant ranging from 0.2 to 1 mM C12TAB, lead to the gradual removal of protein with no evidence of surfactant adsorption. Relatively high surfactant concentrations, e.g., at 7 mM C12TAB, were required to achieve substantial protein removal. This trend again coincides with the noticeable adsorption of C12TAB. To summarize these results, graphs of the surface excesses calculated for both lysozyme and C12TAB with respect to C12TAB solution concentration are given in Figure 6. As already described earlier, both sets of measurements indicate a progressive replacement of lysozyme by C12TAB, but no complete removal was achieved in either case. At low surfactant concentrations, the surface excess of lysozyme gradually decreases, with little evidence of surfactant binding to the protein at the interface. As the initial removal of protein molecules is mainly from the outer protein layer, it appears that this process is facilitated by the direct contact of the protein molecules in the outer layer with C12TAB. It is hence likely that the removal is via the binding between the protein and surfactant. Since there was no evidence of coadsorption or increased adsorption upon addition of surfactant, the newly formed protein-surfactant complex must have little affinity for the surface and is of a highly soluble nature in aqueous environment. When the surfactant concentration is increased to an intermediate surfactant concentration, both the surfactant and protein begin to coexist at the interface. Although the protein content keeps decreasing, in all cases the surfactant was distributed uniformly throughout the whole protein layer with respect to the surface normal. The even distribution of surfactant throughout the layer suggested the formation of a highly interwoven protein-surfactant mixture at the interface. This idea of an

Protein-Surfactant Interactions at the SiO2-H2O Interface

Figure 7. Comparison of the protein elution capabilities of SDS (×) and C12TAB (b) shown as the variation of surface excess of lysozyme (a) and surfactant (b) with bulk surfactant concentration.

interwoven structure persists upon further increase in surfactant concentration. When the CMC is approached, the overall structure of the surfactant layer becomes close to that obtained for the pure surfactant system although its surface excess is much lower. This observation is broadly consistent with the fact that the surface excess of protein within the layer remains reasonably high. At pH 7, both C12TAB and lysozyme are positively charged and the substrate surface is negatively charged. The SiO2 surface thus promotes the adsorption of both species. However, the preadsorbed lysozyme may partially hinder the adsorption of the surfactant and the adsorption of C12TAB may be administered via hydrophobic association with the preadsorbed protein, resulting in a mixed layer of protein fragments and surfactant. It can thus be summarized that although the overall observation is the gradual displacement of lysozyme by C12TAB over the entire surfactant concentration range, a displacement mechanism alone does not describe all the molecular events. Protein removal at low surfactant concentrations appears to proceed via the formation of highly soluble complex between surfactant and lysozyme molecules adsorbed in the outer layer. As the surfactant concentration increases, the protein molecules removed from the interface are from the inner layer. This process appears to occur through the association of C12TAB with lysozyme molecules adsorbed in the inner layer, followed by the dissolution of the complex into aqueous solution. This mechanism is well supported by the coadsorption of the two species at the interface. We have also carried out studies investigating the interaction of SDS with a preadsorbed lysozyme layer.24 A comparison of that study with the present one is given in Figure 7, where the surface excesses of protein and surfactant with respect to increasing surfactant concentration for both SDS and C12TAB are presented. A significant difference in the mechanisms for protein removal by the two surfactants is immediately evident from this graph. For SDS, protein is removed via two distinct steps. Initially, binding of SDS to the preadsorbed layer leads to enhanced adsorption of SDS at the interface with no loss in the amount of adsorbed protein. In contrast, C12TAB is able to partially remove the adsorbed protein at relatively low surfactant concentrations, although with no evidence of coadsorption of the C12TAB. At intermediate surfactant concentrations, and within a relatively small concentration range well below the CMC, SDS completely removes all the protein from the interface via a coadsorption mechanism whereby complexation occurs

J. Phys. Chem. B, Vol. 105, No. 38, 2001 9337 between the two species. However, although C12TAB continues to gradually displace protein from the interface, as the concentration is increased to its CMC, complete protein removal is not reached. These two contrasting mechanisms for surfactantinduced protein removal at interfaces highlight significant differences in the nature of the interaction between lysozyme and the two surfactants carrying opposite charges. It is known from the literature that the strength of interaction between proteins and surfactants is highly dependent on the alkyl chain length of the surfactant, the type of its headgroup, and in particular, the charge on the surfactant. In this study, we have fixed the structure of alkyl chains and have illustrated two distinct mechanisms by which surfactants remove preadsorbed lysozyme from the SiO2-D2O interface resulting from the two different headgroups. The work has shown that complex formation between surfactants and proteins play an important role in the elution mechanism. For C12TAB, protein-surfactant complexes lead to partial removal of lysozyme from the interface at low surfactant concentration with no evidence of coadsorption. Coadsorption of the two species does occur if the surfactant concentration is increased, and this is followed by progressive replacement of the preadsorbed protein as the surfactant is further increased. However, complete removal of protein from the surface is not achieved, even if the concentration of C12TAB is increased to its CMC. Comparison of the elution methods of SDS and C12TAB highlighted the significant roles played by the charge types on the surfactants during protein removal. References and Notes (1) Arnebrant, T.; Wahlgren, M. C. Proteins at Interfaces II: Fundamentals and Applications; Horbett, T. A., Brash, J. L., Eds.; ACS Symposium Series 602; American Chemical Society: Washington, DC, 1995; pp 239-254. (2) Rapoza, R. J.; Horbett, T. A. J. Colloid Interface Sci. 1990, 136, 480. (3) Bohnert, J. L.; Horbett, T. A. J. Colloid Interface Sci. 1986, 111, 363. (4) Tanford, C. J. Mol. Biol. 1972, 67, 59. (5) Reynolds, J. A.; Tandford, C. J. Biol. Chem. 1970, 245, 5161. (6) Reynolds, J. A.; Herbert, S.; Polet, H.; Steinhardt, J. Biochemistry 1967, 6, 937. (7) Reynolds, J. A.; Gallagher, J. P.; Steinhardt, J. Biochemistry 1970, 9, 1232. (8) Tanner, R. E.; Herpigny, B.; Chen, S. H.; Rha, C. K. J. Chem. Phys. 1982, 76, 3866. (9) Wahlgren, M.; Arnebrant, T. J. Colloid Interface Sci. 1991, 142, 503. (10) McGuire, J.; Wahlgren, M. C.; Arnebrant, T. J. Colloid Interface Sci. 1995, 170, 193. (11) Wahlgren, M.; Arnebrant, T. Langmuir 1997, 13, 8. (12) McGuire, J.; Wahlgren, M. C.; Arnebrant, T. J. Colloid Interface Sci. 1995, 170, 182. (13) Maulik, S.; Dutta, P.; Chattoraj, D. K.; Maulik, S. P. Colloids Surf. B 1998, 11, 1. (14) Anand, K.; Damodaran, S., J. Colloid Interface Sci. 1995, 176, 63. (15) Horne, D. S.; Atkinson, P. J.; Dickinson, E.; Pinfield, V. J.; Richardson, R. M. Int. Dairy J. 1998, 8, 73. (16) Lee, L. T.; Jha, B. K.; Malmsten, M.; Holmberg, K. J. Phys. Chem. B 1999, 103, 7489. (17) Lu, J. R.; Thomas, R. K., J. Chem. Soc., Faraday Trans 1998, 94, 995. (18) Cooke, D. J.; Dong, C. C.; Lu, J. R.; Thomas, R. K., Simister, E. A.; Penfold, J. J. Phys. Chem. B 1998, 102, 4912. (19) Cooke, D. J.; Blondel, J. A. K.; Lu, J. R.; Thomas, R. K.; Wang, Y.; Han, B.; Yan, H.; Penfold, J. Langmuir 1998, 14, 1990 (20) Purcell, I. P.; Lu, J. R.; Thomas, R. K.; Howe, A. M.; Penfold, J. Langmuir 1998, 14, 1637. (21) Purcell, I. P.; Thomas, R. K.; Penfold, J.; Howe, A. M. Colloid Surf. 1995, 94, 125. (22) Lu, J. R.; Su, T. J.; Thomas, R. K.; Penfold, J. Langmuir 1998, 14, 6261.

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