The Interaction between SDS and Lysozyme at the Hydrophilic Solid

The interaction between sodium dodecyl sulfate (SDS) and preadsorbed lysozyme ... using two different solution concentrations of lysozyme and a range ...
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J. Phys. Chem. B 2001, 105, 1594-1602

The Interaction between SDS and Lysozyme at the Hydrophilic Solid-Water Interface R. J. Green, T. J. Su, and J. R. Lu* Department of Chemistry, UniVersity of Surrey, Guildford, Surrey, GU2 7XH, U.K.

J. Penfold ISIS, CCLRC, Chilton, Didcot OX11 0QX, U.K. ReceiVed: October 26, 2000; In Final Form: December 22, 2000

The interaction between sodium dodecyl sulfate (SDS) and preadsorbed lysozyme at the hydrophilic silicon oxide-water interface has been studied using specular neutron reflection. Measurements were carried out using two different solution concentrations of lysozyme and a range of SDS solution concentrations between 0.2 and 2 mM. The surface composition and the adsorbed layer structure were determined by varying H/D labeling of SDS. Initially, a uniform layer or bilayer of protein was formed at the interface by adsorption from either 0.03 or 1 g/L lysozyme solution concentrations. The SDS was then added to the system and the neutron reflectivity measured. It was found that this method of studying the SDS/lysozyme system produced highly repeatable neutron reflectivity profiles. On addition of intermediate SDS concentrations, cooperative binding of the SDS to the protein layer was observed, without any evidence of removal of the preadsorbed protein layer. On increasing the SDS concentration, to above 0.5 mM SDS, partial removal of the protein layer occurred. A concentration of 2 mM SDS was required to completely remove all traces of adsorbed material from the interface. These results suggest that the mechanism for protein elution from the interface is via the coadsorption of SDS to the protein layer and the formation of a SDS/protein complex whose surface activity varies with the extent of SDS binding.

Introduction Interactions between proteins and surfactants in bulk solution have been extensively investigated in recent years.1-5 The results from these studies have shown that the type of surfactant headgroup usually determines the strength of the interaction, with ionic surfactants interacting more strongly than nonionic surfactants. The binding of anionic surfactants has been well exemplified by studies using sodium dodecyl sulfate (SDS).6-10 Its association with many proteins over a wide concentration range results in the formation of precipitates. It is now well established that SDS binding onto protein starts with electrostatic attraction between the negatively charged surfactant ions and the positively charged amino acid residues on the outer surface of protein molecules. Further binding is facilitated by the hydrophobic affinity between the dodecyl chains and the hydrophobic moieties within protein, resulting in the opening up of the hydrophobic domains, and hence the denaturation of the protein molecules. There is less information about the surfactant/protein interaction for analogous systems at the solid/solution interface. This situation is mainly caused by the lack of experimental techniques that can overcome the technical limitations imposed by the in situ state of the interface. The solid/water interface is buried and is difficult to access by X-ray reflection. In addition to this, the coadsorbed layers are associated with quite a large fraction of water and are also untrathin (typically under 100 Å). Furthermore, the layers are disordered and completely lack of in-plane structure. These features together with the fact that two or more components coexist within the interface rule out almost * To whom correspondence should be addressed. E-mail: [email protected].

all of the other established techniques. Thus, although a number of recent studies have been made on the coadsorption of surfactant and protein at the solid/water interface and on the elution of preadsorbed proteins, there is still a lack of direct information about the structure and composition of surfactant and protein under different solution conditions.9-15 Although ellipsometry has been used to address the elution of preadsorbed proteins, its main sensitivity is the overall level of residual adsorption and no distinction can be made between protein and surfactant. Infrared methods can offer information about the physical states of protein molecules, but they cannot identify the amount of adsorption. Techniques such as I125 labeling can in principle distinguish the adsorbed protein from surfactant, but no information about the structural distributions of the protein and surfactant is provided. Neutron reflection is a new technique that is ideally suited for the investigation of protein/surfactant mixed layers at different interfaces.16-18 The same technique has also been shown to be advantageous in studying interfacial layers of polymer/surfactant mixtures.19-22 Because neutrons can pass through many crystalline solids such as quartz, silicon, and sapphire, without much loss of intensity, many experiments at the solid/water interface can now be done using these neutron transparent solids as model substrates. A distinct advantage of neutron reflection is the variation of neutron reflectivity with isotopic substitution. Deuterium labeling to surfactant and water makes it possible to distinguish surfactant from protein within the interface. Our previous neutron reflection study on the binding of SDS to bovine serum albumin (BSA)23,24 has shown that with an appropriate application of isotopic contrasts, very detailed information concerning the structure and composition

10.1021/jp003960g CCC: $20.00 © 2001 American Chemical Society Published on Web 02/06/2001

Interaction between SDS and Lysozyme of the protein and surfactant within the adsorbed layer can be obtained. We found that addition of SDS causes expansion of the preadsorbed BSA layers and hence the denaturation of the adsorbed BSA molecules. The removal does not start until a critical SDS concentration is reached. Once the removal starts the mixtures are dissolved into solution within a very narrow SDS concentration range. These results indicate that the removal is manifested via the interaction between SDS/BSA complexes and the solid substrate. The present work is part of our continuing interests in the nature of protein/surfactant systems at interfaces. Neutron reflection has been used to explore how the stability and the chemical nature of the protein affect its interaction with SDS at the solid/water interface. In contrast to the less rigid BSA used in our previous work, lysozyme has been used in this work because of its robust globular structure. While the main objective is to see if the two proteins produce a similar pattern of complex formation and removal there are two specific points of interest. First, our previous neutron reflection work has shown25,26 that, upon adsorption onto the hydrophilic silicon oxide/water interface, lysozyme molecules retain their globular structure but the adsorbed BSA molecules are somewhat deformed. This may well imply that lysozyme is more effective at resisting denaturation and desorption by SDS than BSA. Second, as the two proteins have different primary structures, it is useful to know if the equilibrated binding ratio of surfactant to protein is comparable. These results will allow a direct comparison to be made with the measurements in bulk solution, and to identify the specific role of the interface. Experimental Section Neutron reflection measurements were performed on the white beam reflectometers SURF and CRISP at the Rutherford Appleton Laboratory, ISIS, Didcot, U.K., using neutron wavelengths from 1 to 6 Å. The sample cell configuration is almost identical to that used by Fragneto et al.27 The collimated beam enters, at a fixed angle, through the end of the silicon block and reflected at a glancing angle at the solid/water interface before exiting through the opposite end of the block. Neutron measurements were carried out for three different glancing angles, 0.35°, 0.8°, and 1.8, to produce a single reflectivity profile from 0.012 to 0.5 Å-1. The beam intensity was calibrated by taking the intensity below the critical angle for silicon/D2O 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 Å-1. 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.28 Lysozyme is one of most stable globular proteins whose globular dimension is approximately 30 × 30 × 45 Å3. Hydrogenated SDS (h-SDS) was purchased from Polysciences (99%+) and was recrystallized several times until its surface tension around the critical micellar concentration (cmc) showed no minimum. The surface tension for this particular batch of sample was found to be in good agreement with previous measurements.29 The deuterated SDS (d-SDS) was made by reacting deuterated dodecanol with chlorosufonic acid in dry ether below 5 °C as described previously.23 The extent of deuteration of the dodecyl chain was found to be 96 ( 2% by

J. Phys. Chem. B, Vol. 105, No. 8, 2001 1595 NMR. The deuterated SDS sample was also recrystallized several times before use and its surface tension plot was within an error of (1 mNm-1, identical to that of the fully hydrogenated. All samples were prepared in a buffered solution, with its pH controlled using a phosphate buffer at pH 7 while 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 in Elgastat ultrapure water (UHQ). The large (111) face of the silicon block was polished using an Engis polishing machine as described previously.23 Polished blocks were cleaned using piranha solution and exposing the surface to UV/ozone to remove any traces of organic impurities.30 This procedure produces surfaces that have repeatable thickness and roughness values of the oxide layer and which are completely wetted by water. Finally, the blocks were clamped into the neutron Teflon cells in contact with UHQ water until the beginning of the neutron experiment. This kept the freshly treated oxide surface fully hydrated. Neutron Reflection. Specular neutron reflection offers reliable information about the structure and composition of layers present at different interfaces.18,31,32 Neutron reflectivity varies with the scattering length density distribution along the surface normal and this is in contrast to the variation of X-ray reflectivity with the electron density distribution. Unlike electron density profiles, the scattering length density distributions can be varied by isotopic substitution without altering the chemical composition of the interfacial system. For the protein/surfactant systems concerned in this work, isotopic labeling can be made by deuterium substitution to surfactant and water although it is less convenient for labeling the protein. Because hydrogen and deuterium have opposite signs in scattering lengths, these substitutions create sufficient isotopic contrasts for separating surfactant from protein in the mixed interfacial layers. The total layer scattering length density (F) is related to the scattering lengths of the components within the layer and their compositions through

F ) npbp + nsbs + nwbw

(1)

where np, ns, and nw are the number densities of protein, surfactant, and water and bp, bs, and bw are their corresponding scattering lengths. Neutron reflectivity profiles are usually analyzed by means of the optical matrix formalism, which has been described in detail elsewhere.33,34 A typical modeling procedure begins with an assumption of a structural model for the adsorbed layer, followed by calculation of the reflectivity based on the optical matrix formula. The calculated reflectivity is then compared with the measured data and 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 for each layer. The fitting of a set of reflectivity profiles measured under different isotopic compositions can be used to differentiate between adsorbed species at an interface and will also reduce greatly the ambiguity of the model interpretation. The choice of the number of sublayers used in the model 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. For the mixed protein/surfactant system studied here the volume fraction of each component in a uniform layer is expressed as

F ) φpFp + φsFs + φwFw

(2)

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where Fp, Fs, and Fw are the scattering length densities of protein, surfactant, and water and φp, φs, and φw their respective volume fractions. Thus

φ p + φs + φw ) 1

(3)

With eqs 2 and 3, the volume fractions of each constituent cannot be solved, since there are three unknown components. However, repeating the SDS binding measurements using hydrogenated (h-SDS) and deuterated SDS (d-SDS), will create two independent equations from eq 2, allowing the volume fraction of both lysozyme and SDS in the adsorbed layer to be determined. If the scattering length for d-SDS is adjusted to equal to that of D2O, then the surfactant is invisible and eq 2 becomes

F ) φpFp + (1 - φp)Fw

(4)

and the surface excess of the protein in the layer (Γ) is

Γ)

τ(1 - φp) n′wVwNa

(5)

where n′w is the number of water molecules associated with each protein molecules, Vw is the volume of water and is taken to be 30 Å3, and n′w contains contribution from d-SDS and can be assessed from the volume restriction requirement. More details on this treatment can be found from our previous work.23 Although eqs 1-5 have been developed under the condition of uniform layer distribution, they are directly applicable to each sublayer 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. Results and Discussion The measurement of the silicon oxide layer present on the freshly polished block was characterized by neutron reflectivity immediately prior to the protein adsorption experiments. 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 all previous protein adsorption studies.23,24 The oxide layer was fitted using a layer thickness of 17 ( 3 Å and F ) 3.41 × 10-6 Å2, and this fit was used for all subsequent adsorption experiments carried out using this block. The interaction between SDS and a preadsorbed lysozyme layer was studied over the SDS solution concentration range of 0.2 to 2 mM SDS and at two fixed lysozyme concentrations of 0.03 and 1 g/L. For each SDS binding measurement, the preadsorbed lysozyme layer was characterized by neutron reflectivity immediately before the addition of the surfactant solution to the system. Figure 1 shows an example of the neutron reflectivity profiles obtained for the adsorption from 0.03 and 1 g/L lysozyme solutions at the silicon oxide interface. These measurements were performed in D2O so as to highlight the adsorbed protein layer. The amount of adsorbed lysozyme was determined by fitting the data using the optical matrix formalism as already outlined. The volume fraction and surface excess values for the adsorbed layers were calculated using eqs 2-5 and a scattering length density for the pure protein of 3.66 × 10-6 Å-2.25 Adsorption from 0.03 g/L lysozyme solution led to the formation of a single uniform layer of adsorbed lysozyme. From the data fitting, the thickness of the layer was found to be 30 ( 3 Å with a volume fraction of 0.32. Thus, with respect

Figure 1. Neutron reflectivity profiles obtained for the adsorption of lysozyme from 0.03 (O) and 1 g/L (∆) solutions at the silica/D2O interface (pH 7, I ) 0.02 M). The continuous lines indicate the best fits resulting in a uniform layer fit for 0.03 g/L lysozyme and a two layer fit for 1 g/L lysozyme.

to the dimensions of a lysozyme molecule, a thickness of 30 Å implies that the protein molecule is lying sideways-on with the long axis parallel to the surface. For 1 g/L lysozyme, a twolayer model for the adsorbed protein layer was required to obtain a successful fit of the data with a thickness of 30 ( 3 Å for both layers and the volume fractions of 0.46 and 0.32, respectively. This fit suggests a bilayer structure, containing a dense inner layer and more diffuse outer layer, with the protein molecules again adsorbed sideways-on in both layers. These results are in agreement with the data reported in our previous work.25 In calculating the adsorbed amount of protein, complete exchange of labile hydrogens with the surrounding D2O was assumed. This is not necessarily the case given that lysozyme is very stable and exchange of hydrogens within the core of the protein will be hindered. However, Fourier transform infrared spectroscopy and NMR studies have shown that, in solution, the rate of exchange is rapid and that the majority of the labile hydrogens exchange within an hour of making the lysozyme/D2O solution.35,36 Also, as discussed previously, detailed experiments investigating lysozyme adsorption by neutron reflectivity showed no evidence of further H/D exchange occurring during adsorption.25 Therefore, when considering the time scales of the neutron experiments, the assumption of complete H/D exchange, although not strictly true, is reasonable and within error. Finally, information about the extent of H/D exchange within lysozyme can be assessed independently by comparing the surface excesses of lysozyme with and without SDS before the removal starts and the details will be described later. It should be remembered that any uncertainty in the extent of H/D exchange only affects surface excess calculations and not the determination of layer thickness. Once the protein layer has been characterized, an SDS solution is introduced to the liquid cell and the new surface measured. After each SDS binding experiment, the solid surface was cleaned by rinsing the cell with a solution of high SDS concentration at its cmc (8 mM in pure water). This was followed by copious rinsing of the cell with UHQ water before a final rinse in D2O. The silicon block in D2O was then measured by neutrons and compared with the previous SiO2/ D2O measurement to ensure 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 SDS binding measure-

Interaction between SDS and Lysozyme

Figure 2. The neutron reflectivity profiles obtained for the addition of 0.5 mM hydrogenated SDS solution to a preadsorbed lysozyme layer (from 1 g/L solution), via experimental (O) Method 1 and (∆) Method 2. The continuous lines represent the best fits using a two layer model. The best fit for Method 1 corresponds to inner layer τ1 ) 29 Å and outer layer τ2 ) 31 Å with F1 ) 5.0 × 10-6 A-2 and F2 ) 4.7 × 10-6 A-2, and for Method 2, τ1 ) 18 Å and τ2 ) 27 Å with F1 ) 5.1 × 10-6 A-2 and F2 ) 4.9 × 10-6 A-2.

ment was performed. This method for studying the effect of SDS binding on the preadsorbed lysozyme layer was used as the primary method for this investigation and will be referred to as Method 1. In the literature, different methods are often described for the study of adsorption processes. It is, therefore, important to determine whether small changes in experimental procedure affect the structure of the adsorbed layer. In an alternative method (Method 2), the first preadsorbed protein surface was prepared as in Method 1. However, the surfactant solutions were then added sequentially, in increasing SDS concentration order, to the solution cell without refreshing the surface with a new protein layer between each SDS binding measurement. For both methods, no evidence of any time effect was found and, therefore, the surface layers are regarded as in equilibrium. As an example, the reflectivity profiles obtained on addition of 0.5 mM h-SDS to the preadsorbed lysozyme layer via both methods are compared (Figure 2). In Method 1, the SDS solution was added to a freshly prepared protein layer and, in Method 2, a 0.2 mM SDS solution was placed in contact with the preadsorbed lysozyme surface, followed by the replacement of this solution by the 0.5 mM h-SDS solution. The second method is widely used in the literature to investigate protein elution by surfactants, for example, by Wahlgren and Arnebrant, to study the removal of T4 lysozyme by SDS.9 Figure 2 clearly shows a difference in the reflectivity profiles resulting from the two different methods. When using Method 1 the reflectivity profile has a sharper interference fringe at lower κ than for Method 2, suggesting that the adsorbed layer is thicker in Method 1. Thus, it appears that when using Method 2 the protein layer has underwent less expansion. This is possibly because in Method 2 the previous addition of a lower SDS concentration has disrupted the protein layer as a result of the formation of a SDS/lysozyme complex. Therefore, a further addition of 0.5 mM SDS solution to this surface does not tell us about the interaction between SDS and a pure lysozyme layer, but rather the further interaction between SDS and the SDS/ lysozyme complex. In this case, the extent of interaction and protein removal is dependent on the surface history. In the present study, we are interested in quantifying the adsorbed amounts of SDS and lysozyme, resulting from the interaction

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Figure 3. A neutron reflectivity profile showing the binding of 0.5 mM deuterated SDS to the lysozyme layer preadsorbed from a 0.03 g/L lysozyme solution in D2O. The continuous line is the best uniform layer fit with structural parameters listed in Table 1. For comparison, the dashed line indicates the reflectivity fitted to the preadsorbed lysozyme layer in D2O.

of SDS with the pure protein layer and, therefore, the use of Method 1 is more relevant. However, the results shown in Figure 2 highlight the difficulty in designing these adsorption experiments and question the validity of comparing results between different researchers where their experimental procedures differ. SDS Binding to Preadsorbed Lysozyme. Initially, the interaction of SDS with preadsorbed protein from a 0.03 g/L lysozyme concentration solution was considered. In this system, the equilibrium adsorbed lysozyme layer could be modeled as a single uniform layer of thickness of 30 Å as shown in Figure 1. Binding of both h-SDS and d-SDS to this preadsorbed layer was studied using SDS concentrations from 0.2 to 2 mM using Method 1 and neutron measurements were measured at different water contrasts. The addition of d-SDS in D2O had an effect on the shape of the neutron reflectivity profile as compared with the pure protein reflectivity curve. This is seen in Figure 3, where the reflectivity profile for 0.5 mM d-SDS bound to lysozyme in D2O is compared to that of the pure protein layer. Since the scattering length density of d-SDS is about 6.8 × 10-6 Å-2, similar to that of D2O, its presence at the interface is largely masked by the D2O. Therefore, the observed change in reflectivity (Figure 3) outlines the structural change of lysozyme within the adsorbed layer. The best fit, as shown by the solid line in Figure 3, was calculated with a layer thickness of 46 ( 3 Å and F ) 5.2 × 10-6 Å-2. This single uniform layer model fits the data well and shows that the addition of SDS to the system has increased the thickness of the layer. However, the distribution of lysozyme along the surface normal is still uniform. The volume fraction of the protein within the layer was calculated to be 0.2 as compared to 0.32 for the original preadsorbed lysozyme layer. The surface excess value was calculated as 1.36 mg/m2, which was the same as that before the addition of SDS. Therefore, for a 0.5 mM SDS solution, the addition of SDS did not lead to the removal of any lysozyme from the interface, although it did have an effect on the structure of the adsorbed layer. By also considering a different contrast, h-SDS/D2O, the amount of SDS in the adsorbed layer can be determined. Figure 4 shows the reflectivity profile of the h-SDS contrast for 0.5 mM SDS in D2O. When comparing this reflectivity profile with that of the d-SDS contrast the results showed that the different labeling of SDS had little effect on the reflectivity curves. The reflectivities of h-SDS and d-SDS are almost identical suggest-

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Figure 4. A neutron reflectivity profile showing the binding of 0.5 mM hydrogenated SDS to the lysozyme layer preadsorbed from a 0.03 g/L lysozyme solution in D2O. The continuous line is the best uniform layer fit for the binding of d-SDS onto the lysozyme layer in D2O as shown in Figure 3. The dashed line indicates the best fit for the h-SDS profile assuming 0.1 mg/m2 of SDS is bound at the interface.

ing that there is little SDS present at the interface, as shown in Figure 4 where the solid line indicates the best fit calculated for the d-SDS data. The dashed line assumes a SDS surface excess of 0.1 mg/m2 bound at the interface. This fits the measured data well, thus confirming that the amount of SDS present at the interface is less than 0.2 mg/m2. Indeed, the observed expansion of the lysozyme layer indicates that some SDS molecules must have penetrated into the protein layer, causing some disruption of the compact lysozyme structure. The data shown so far considers exclusively the binding of 0.5 mM SDS to a single uniform layer of preadsorbed lysozyme and suggests that a small amount of SDS binding occurs, inducing changes to the thickness and structure of the protein layer. Some ambiguity remains since the adsorbed amount of SDS at the interface could not be precisely determined. However, further experiments using a higher lysozyme concentration of 1 g/L produced larger differences between the h- and d-SDS reflectivity profiles and enable us to illustrate the binding of SDS at the interface more clearly. The adsorption of lysozyme from a 1 g/L solution led to the formation of a two-layer structure at the interface, both layers of thickness 30 Å as already discussed. The addition of SDS to this surface had a large effect on the neutron reflectivity, as shown by the large variation of the reflectivity profiles when using different H/D labels of SDS. The comparison is given, for 0.5 mM SDS in D2O, in Figures 5 and 6. While the use of d-SDS did not significantly change the reflectivity profile from that of the preadsorbed pure protein surface (Figure 5), h-SDS generated a pronounced interference fringe (Figure 6). This shift in the interference fringe toward lower κ is due to an increased amount of hydrogenated species, indicating h-SDS binding. However, the bound SDS cannot be distinguished from the protein layer when both species are hydrogenated. The reflectivity profile using d-SDS highlights the change in the protein structure upon addition of SDS to the system. Figure 5 shows very little change in the reflectivity upon addition of d-SDS, which suggests that the overall dimensions of the protein layer remains unchanged. The best fit (solid line in Figure 5) indicates a two layer fit of thicknesses 29 and 31 Å and F ) 5.15 × 10-6 and 5.5 × 10-6 Å-2 respectively. The corresponding best fit for the h-SDS reflectivity profile (solid line in Figure 6) gives F ) 5.0 × 10-6 and 4.7 × 10-6 Å-2 respectively. These fits show that, for the two layer model used, very little SDS resides

Green et al.

Figure 5. A neutron reflectivity profile showing the binding of 0.5 mM deuterated SDS to the lysozyme layer preadsorbed from a 1 g/L lysozyme solution in D2O. The continuous line is the best two-layer fit with structural parameters listed in Table 1. For comparison, the dashed line indicates the reflectivity fitted to the preadsorbed lysozyme layer in D2O.

Figure 6. A neutron reflectivity profile showing the binding of 0.5 mM hydrogenated SDS to the lysozyme layer preadsorbed from a 1 g/L lysozyme solution in D2O. The continuous line is the best twolayer fit with structural parameters listed in Table 1. For comparison, the dashed line indicates the reflectivity fitted to the preadsorbed lysozyme layer in D2O.

in the layer nearest to the silicon oxide substrate, since the F values for the first layer are very similar for both h- and d-SDS contrasts. Therefore, the bound SDS is predominantly within the outer layer, with surface excesses within the inner and outer layers are calculated to be 0.08 and 0.46 mg/m2 respectively. The total surface excess of lysozyme was found to be 3.4 mg/ m2, approximately equal to that of the original preadsorbed protein layer. This indicated that, as for the above single uniform protein layer (Figures 3,4), 0.5 mM SDS does not facilitate the removal of any lysozyme from the oxide surface. If should be noted, however, that although the overall layer thickness did not increase, there is a clear shift in protein fragments from the inner to the outer layer, as indicated by the change in the two F values and the layer surface excesses (see Table 1). There are two possible reasons why SDS binds preferentially to the outer lysozyme layer. First, the density of protein in the outer layer is less and this may have facilitated the incorporation of SDS within the layer. Second, it may be that the nature of the oxide substrate effects the binding of SDS. Both silicon oxide and SDS are negatively charged at pH 7 and this prevents SDS adsorbing directly to the oxide layer. The repulsion between

Interaction between SDS and Lysozyme

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TABLE 1: Composition of the Mixed Protein/Surfactant Interfaces Obtained from the Fitting to Reflectivity Profiles [LSZ] (a) 0.03 g/l

(b) 1.0 g/l

[SDS] (mM)

τ1 ((3/Å)

0 0.2 0.5 1.0 2.0 0 0.2 0.5 1.0 2.0

30 46 46 60 0 30 27 29 25 0

τ2 ((3/Å)

Γ1LSZ ((0.2/mgm-2)

30 28 31 30 0

1.37 1.36 1.36 1.13 0 2.02 2.12 1.89 1.49 0

Figure 7. The variation of surface composition with bulk SDS concentration shown as the surface excess of lysozyme (a), SDS (b), layer distribution of SDS (c), and the total layer thickness (d). For Figures 7a, b, and d, the symbols indicate (9) 0.03 g/L and ([) 1 g/L lysozyme concentrations, and for Figure 7c (2) inner and (b) outer layers for the two layer lysozyme system (1 g/L lysozyme).

the oxide layer and SDS may also inhibit the incorporation of SDS within the protein layer close to the oxide substrate. The SDS distribution throughout the protein bilayer agrees with our earlier observations for the single uniform layer of adsorbed protein at 0.03 g/L lysozyme, with a very low surfactant volume fraction within the inner protein layer. Similar experiments were also carried out using 0.2, 1, and 2 mM SDS solution concentrations at both the uniform and bilayer preadsorbed lysozyme surfaces. The results from the fitting of all the data are given in Table 1. Figure 7 plots the total surface excesses calculated for both lysozyme and SDS with respect to SDS solution concentration. The presence of SDS at the interface is seen clearly for the bilayer protein system, where the preadsorbed protein layer was formed from 1 g/L solution (Figure 7a). Figure 7b shows that a maximum in

Γ2LSZ ((0.2/mgm-2)

Γ1SDS ((0.05/mgm-2)

Γ2SDS ((0.05/mgm-2)

1.37 1.35 1.53 1.47 0

0 0-0.1 0-0.1 0-0.1 0 0 0.05 0.08 0.02 0

0 0.31 0.46 0.08 0

the surface excess of SDS at an intermediate SDS solution concentration of 0.5 mM. Above this concentration, the surface excess of both SDS and lysozyme decreased until, at a 2 mM SDS concentration, all the material was removed from the interface. This trend is seen clearly for the bilayer lysozyme system where the presence of SDS at the interface is quite well defined. In Figure 7c, the distribution of SDS throughout the preadsorbed protein layer is highlighted for the bilayer system. This shows that, at all SDS concentrations studied, less than 0.1 mg/m2 of SDS resides in the inner lysozyme layer, with the majority of SDS bound within the outer protein layer. Although the presence of SDS is less well defined for the uniform protein layer system preadsorbed from a 0.03 g/L solution, an expansion of the lysozyme layer is clearly seen if we consider the overall thickness of the layer (Figure 7d). This suggests that there is indeed some interaction and binding of SDS to the adsorbed protein. As discussed previously,24-26 to calculate the scattering length density of protein we have assumed 100% H/D exchange of all labile hydrogens (F°p ) 3.66 × 10-6 Å-2 for lysozyme). However, in its native conformation a small percentage of hydrogens do not easily exchange due to their encapsulation within the hydrophobic core of the globular protein’s structure. Because the typical accuracy of neutron reflectivity measurements is around 5%, any deviation greater than this is expected to be detected. If the H/D exchange is incomplete by more than 5% (Fp < F°p), eq 4 would expect an overestimation of the surface excesses of lysozyme by almost a similar extent. Upon addition of SDS, the globular nature of lysozyme is deteriorated and Fp f F°p. One would therefore expect a decrease of lysozyme surface excess with the bulk concentration of SDS. From Figure 7 we can see that on addition of up to 0.5 mM SDS, ΓLSZ remains constant within the experimental error, hence suggesting that the fraction of unexchanged hydrogens is small and that its influence to surface excess is within the experimental error of (0.2 mg/m2. We have also carried out similar studies investigating the interaction between SDS and preadsorbed bovine serum albumin (BSA), at the silicon oxide surface using neutron reflectivity.23,24 There are two main similarities between SDS removal of BSA and lysozyme. First, in both cases, SDS induces the deterioration of the protein structure as indicated by the expansion of the adsorbed protein layer prior to protein removal. Second, for both the lysozyme and BSA systems, the distribution of SDS throughout the adsorbed protein layer is skewed toward the outer part of the layer. However, there is a significant difference between the two protein systems when considering the extent of SDS binding to the protein layer. For the SDS/BSA system, the number of grams of SDS per gram of protein adsorbed at the interface (weight ratio) was calculated as 0.43 (for 0.1 mM SDS). Whereas, the observed weight ratio for SDS/lysozyme was calculated as only 0.07 (assuming ΓSDS ) 0.1) for the

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Figure 8. A schematic representation of the elution of lysozyme by SDS, for both uniform and bilayer preadsorbed protein structures.

uniform protein layer and 0.16 for the bilayer system, at 0.5 mM SDS solution. Therefore, it appears that on average the SDS binds more readily to a preadsorbed BSA layer than to lysozyme. It is useful to compare the detailed SDS distribution within the SDS/lysozyme two layer system with that obtained from the SDS/BSA layer under 0.1 mM SDS.23 The former has a weight ratio of SDS to lysozyme of 0.07 in the inner layer and 0.3 in the outer layer while the latter has a ratio of 0.42 in the inner layer and 0.68 in the outer layer. The SDS/BSA system was studied at pH 5, matched to the isoelectric point of the protein, whereas the lysozyme molecule was positively charged in the present study at pH 7. Proteins are known to undergo a maximum adsorption to an interface at a pH corresponding to its isoelectric point, due to the minimization of electrostatic repulsion. Similarly, SDS appears to bind at the interface to a higher degree to BSA at pH 5 than to lysozyme at pH 7 and the reduced charge of the interfacial system may play some role here. This trend is opposite to the apparent intuition that the positive charges on lysozyme should attract more negatively charged SDS ions. But it is unclear how the positive charges on lysozyme interact with the negative charges on the substrate surface and how the overall charge-related interaction is to strike a balance with the dominant hydrophobic affinity. Tanford et al.6-8 have extensively examined the binding of SDS onto both native proteins and denatured polypeptides in bulk solution. Among some 10 different proteins studied, they

found no obvious difference either between different types of proteins or between native proteins and denatured ones. Between pH 5 and 7 SDS binding to protein does not show any significant variation with pH. At the SDS concentration of around 0.5 mM, the molar binding ratio of SDS to protein is about 20 and this is equivalent to the weight ratio of 0.4. Thus, the average weight ratio of 0.43 for SDS binding to BSA at the solid/solution interface is, within experimental error, the same as the value found from their association in bulk solution, suggesting that the presence of the solid substrate has no apparent effect in this case. However, the weight ratio is significantly lower for SDS binding to lysozyme at the solid/liquid interface, which would indicate that the type of protein does effect its extent of binding at the interface, an apparent inconsistency with the finding of Tanford et al. from the binding in bulk solution. As already discussed, the charge density of the solid surface and the number of net charges on protein vary with solution pH. These effects will affect the tendency for SDS to bind at the interface. The combined action has already been visualized from the skewed SDS distributions perpendicular to the solid surface. The binding of SDS to BSA was measured at pH 5 and was close to the isoelectric point of BSA. In comparison, SDS binding to lysozyme was studied at pH 7 and the isoelectric point for lysozyme is about pH 11. The subtle charge distributions within the adsorbed protein layers may be responsible for the observed difference. Further work will thus be needed to examine the

Interaction between SDS and Lysozyme effect of pH on the extent of SDS binding for a given protein. This information together with the isoelectric point of the protein will lead to an improved understanding of SDS binding to different proteins at the solid/liquid interface. We may also be able to draw comparisons between the present SDS/lysozyme system and our previous study carried out at the air/liquid interface.37 For the air/liquid system, at intermediate SDS concentrations the SDS-lysozyme complex dominated the adsorption process which resulted in the enhanced adsorption of both SDS and lysozyme. In the present study, evidence of this complex formation is again apparent at intermediate SDS concentrations. At 0.5 mM SDS, there is significant adsorption of SDS with no loss in the amount of adsorbed lysozyme, suggesting that the complex formed between the two species has a preference to remain adsorbed at the interface. Future studies on the coadsorption of SDS and BSA at the air/water interface may offer a more convenient examination of the effect of solution pH on the relative strength of binding between SDS and the two proteins without the complication of the solid substrate. The present study suggests that the initial binding of SDS to the preadsorbed protein layer does not initially lead to a reduction in the adsorbed amount of protein. Desorption of the lysozyme is induced at higher SDS concentrations via the formation of a surfactant/protein complex at the interface. Froberg et al. have also shown that the addition of lower SDS solution concentrations does not induce large scale desorption of lysozyme adsorbed at a mica surface.38 This observation is consistent with our previous work of removal of BSA by SDS on the surface of silicon oxide. The results show that the eventual dissolution of the complexes is triggered by a combined effect of structural reorganization of peptides and increased electrostatic repulsion caused by SDS binding. The biophysical pathway leading to the removal of the protein/surfactant complexes is summarized in a schematic representation shown in Figure 8. For SDS interaction with a single uniform layer of preadsorbed protein, minimal SDS binding is observed. As the SDS solution concentration is increased, the protein layer initially expands before being completely removed from the interface. However, SDS addition to a bilayer of adsorbed protein results in significant binding of SDS to the outer layer of protein. In both cases, a SDS concentration of greater than 0.5 mM is needed before any reduction in the amount of adsorbed protein occurs and a SDS concentration of 2 mM required to completely remove all traces of adsorbed material. The threshold concentrations coincide well with those obtained for BSA/SDS binding. The mechanism for the removal of protein by SDS at the solid/liquid interface appears to involve the binding of SDS to the adsorbed protein layer, via predominantly hydrophobic interactions, leading to structural changes within the protein layer. Since these structural changes to the protein layer occur at SDS concentrations lower than that required for any protein removal to occur, it appears that they are a necessary prerequisite to protein elution. Summary The pattern of removal of preadsorbed lysozyme from the hydrophilic silicon oxide/water interface by addition of anionic surfactant SDS is broadly similar to that of BSA/SDS system studied in our previous work. Initial addition of SDS causes the expansion of the preadsorbed lysozyme layer, indicating the binding of SDS into the interior of lysozyme layer and the denaturation of its globular structure. The removal does not start

J. Phys. Chem. B, Vol. 105, No. 8, 2001 1601 until a critical SDS concentration is reached. These observations suggest that the removal process is administered via surfactant/ protein complexes whose surface activities decrease with increasing the amount of SDS inside the complexes. A further common feature is the skewed distributions of SDS molecules across the interfaces, as a result of depletion possibly caused by the repulsive effect of negative charges on the solid substrate to SDS ions. The main differences between the two proteins studied are the extent of skewness of SDS distributions at the interface and the total amount of bound SDS molecules, as reflected in the relative weight ratios between surfactant and protein. Further studies will need to be carried out to establish why much less SDS is bound with preadsorbed lysozyme at the hydrophilic solid/water interface. It will also be of interest to characterize the effect of SDS binding to the protein layers preadsorbed onto hydrophobed solid surface and to compare the extent of removal with the findings from the hydrophilic solid/solution interface. References and Notes (1) Ananthapadmanabhan, K. P. In Interactions of Surfactants with Polymer and Protein; Goddard, E. D., Ananthapadmanabhan, K. P., Eds.; CRC Press: 1993; p 320. (2) Mattice, W. L.; Riser, J. M.; Clark, D. S. Biochem. 1976, 15, 4264. (3) Mascher, E.; Lundahl, P. J. Chromatogr. 1989, 476, 147. (4) Gimel, J. C.; Brown, W. J. Chem. Phys. 1996, 104, 8112. (5) Valstar, A.; Brown, W.; Almgren, M. Langmuir 1999, 15, 2366. (6) Tanford, C. J. Mol. Biol. 1972, 67, 59. (7) Reynolds, J. A.; Gallagher, J. P.; Steinhardt, J. Biochem. 1970, 9, 1232. (8) Reynolds, J. A.; Tanford, C. J. Biol. Chem. 1970, 245, 5161. Also see: Reynolds, J. A.; Tanford, C., Proc. Natl. Acad. Sci. U.S.A. 1970, 66, 1002. (9) Wahlgren, M.; Arnebrant, T. Langmuir 1997, 13, 8. (10) Rapoza, R. J.; Horbett, T. A. J. Colloid Interface Sci. 1990, 136, 480. (11) Wahlgren, M.; Arnebrant, T. J. Colloid Interface Sci. 1991, 142, 503. (12) McGuire, J.; Wahlgren, M. C.; Arnebrant, T. J. Colloid Interface Sci. 1995, 170, 193. (13) Elwing, H.; Askendal, A.; Lundstrom, I. J. Colloid Interface Sci. 1989, 128, 296. (14) Horbett, T. A.; Brash, J. L. Proteins at Interfaces II: Fundamentals and Applications; Horbett, T. A., Brash, J. L., Eds.; ACS Symposium Series 602; American Chemical Society: Washington, DC, 1995. (15) Yada, R. Y.; Jackman, R. L. Protein Structure Function Relationships in Foods; Blackie Academic & Professional: Glasgow, 1994. (16) Horne, D. S.; Atkinson, P. J.; Dickinson, E.; Pinfield, V. J.; Richardson, R. M. Int. Dairy J. 1998, 8, 73. (17) Lee, L. T.; Jha, B. K.; Malmsten, M.; Holmberg, K. J. Phys. Chem. B 1999, 103, 7489. (18) Lu, J. R.; Thomas, R. K. J. Chem. Soc., Faraday Trans 1998, 94, 995. (19) Cooke, D. J.; Dong, C. C.; Lu, J. R.; Thomas, R. K.; Simister, E. A.; Penfold, J. J. Phys. Chem. B 1998, 102, 4912. (20) 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. (21) Purcell, I. P.; Lu, J. R.; Thomas, R. K.; Howe, A. M.; Penfold, J. Langmuir 1998, 14, 1637. (22) Purcell, I. P.; Thomas, R. K.; Penfold, J.; Howe, A. M. Colloid Surf. 1995, 94, 125. (23) Lu, J. R.; Su, T. J.; Thomas, R. K.; Penfold, J. Langmuir 1998, 14, 6261. (24) Lu, J. R.; Su, T. J.; Thomas, R. K. J. Phys. Chem. B 1998, 102, 10307. (25) Su T. J.; Lu, J. R.; Thomas, R. K.; Cui, Z. F.; Penfold, J. Langmuir 1998, 14, 438. (26) Su, T. J.; Lu, J. R.; Thomas, R. K.; Cui; Z. F.; Penfold, J. J. Phys. Chem. B 1998, 102, 8100. (27) Fragneto, G.; Lu, J. R.; McDermott, D. C.; Thomas, R. K.; Rennie, A. R.; Gallagher, P. D.; Satija, S. K. Langmuir 1996, 12, 477. (28) Alderton, G.; Ward, W.; Febold, H. J. Biol. Chem. 1945, 157, 45.

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