Binding of Sodium Dodecyl Sulfate to Bovine Serum Albumin Layers

Adsorption of bovine serum albumin (BSA) from a 0.15 g dm-3 solution onto a hydrophilic silicon oxide surface produced a densely packed uniform layer ...
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Langmuir 1998, 14, 6261-6268

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Binding of Sodium Dodecyl Sulfate to Bovine Serum Albumin Layers Adsorbed at the Silica-Water Interface J. R. Lu* and T. J. Su Department of Chemistry, University of Surrey, Guildford GU2 5XH, U.K.

R. K. Thomas Physical and Theoretical Chemistry Laboratory, South Parks Road, Oxford OX1 3QZ, U.K.

J. Penfold ISIS, CCLRC, Chilton, Didcot OX11 0QX, U.K. Received March 3, 1998. In Final Form: August 4, 1998 One of the main difficulties in studying the interaction of protein and a surfactant at an interface is to establish the structural distributions of each component in the mixed layer. We demonstrate in this work that the distributions of the surfactant, protein, and water adsorbed at the hydrophilic solid/aqueous solution interface can be separately and unambiguously determined by using specular neutron reflection combined with deuterium labeling of the surfactant and water. Adsorption of bovine serum albumin (BSA) from a 0.15 g dm-3 solution onto a hydrophilic silicon oxide surface produced a densely packed uniform layer with a thickness of 35 ( 3 Å. The binding of sodium dodecyl sulfate (SDS) onto this preadsorbed BSA layer was studied at a constant SDS concentration of 1 × 10-4 M with different isotopic compositions of SDS and water. The surface excesses of BSA, SDS, and the structural distributions of BSA, SDS, and water were obtained by simutaneously fitting a single structural model to the set of measured neutron reflectivity profiles. The results show that at this SDS concentration the structural profiles of the interfacial components are approximated well as uniform layer distributions. The binding of SDS results in an expansion of the preadsorbed BSA layer from 35 ( 3 Å in the absence of SDS to 50 ( 5 Å, suggesting considerable structural deformation of the protein. The weight ratio of SDS to BSA in the mixed layer was found to be 0.43, in close agreement with the literature value for the binding of SDS onto denatured protein in the bulk, suggesting that the protein in the adsorbed complex is also denatured.

Introduction The deposition of proteins at solid interfaces is of widespread occurrence and is a problem, for example, in the fouling of a food processing plant, in the deposition of blood proteins onto cardiovascular implants, and in biomedical separations such as the isolation of individual proteins from mixtures using ceramic membranes. Adsorption of proteins often results in a heterogeneous layer which may decompose with adverse biological consequences.1 Adsorbed proteins are usually removed using surfactant formulations which may act by coadsorption into the protein layer. Although protein removal is important in technological applications, and protein-surfactant interactions are also of considerable fundamental interest, few direct experiments have been made on the characterization of coadsorbed proteins and surfactants at interfaces and consequently there is no sound understanding of the nature of the interaction of surfactants and proteins in these circumstances. The experimental difficulties arise partly because the protein layer itself is complex and the addition of a surfactant makes it more so, and partly because there are few techniques that can probe the interfacial structure with the necessary selectivity and resolution. Several authors have used ellipsometry to follow the dynamics of protein removal from a solid surface, but ellipsometry * To whom all correspondence should be addressed. (1) Horbett, T. A.; Brash, T. A. Protein at Interfaces II: Fundamentals and Applications; Horbett, T. A., Brash, J. L., Eds.; ACS Symposium Series 602; American Chemical Society: Washington, DC, 1995.

cannot clearly identify structural changes, nor can it easily cope with the additional complexity of a coadsorbed surfactant.1-6 We have recently shown with the higher resolution of neutron reflection that it is easy to overinterpret ellipsometric data and draw misleading conclusions.7 Radiolabeling, for example, with I125 can be used to follow adsorption but gives no structural information8 and the attachment of tracer containing fragments to protein or surfactant molecules may often change their surface activity, as has been seen in some examples from the protein engineering field.9 In contrast, the interaction of proteins with surfactants in bulk solution has been well-characterized. Binding is generally considered to be a competitive process involving the partitioning of the surfactant between protein/surfactant aggregates and surfactant micelles.10 The binding of surfactants onto globular proteins usually induces (2) Wahlgren, M.; Arnebrant, T. J. Colloid Interface Sci. 1991, 142, 503. (3) Ruardy, T. G.; Schakenraad, J. M.; Van der Mei, H. C.; Busscher, H. J. Surf. Sci. Rep. 1997, 29, 1. (4) McGuire, J.; Wahlgren, M.; Arnebrant, T. J. Colloid Interface Sci. 1995, 170, 182. (5) Elwing, H.; Askendal, A.; Lundstrom I. J. Colloid Interface Sci. 1989, 128, 296. (6) Wahlgren, M.; Arnebrant, T. Langmuir 1997, 13, 8. (7) Lu, J. R.; Su, T. J.; Thomas, R. K.; Rennie, A. R. J. Colloid Interface Sci. 1998, in press. (8) Rapola, R. J.; Horbett, T. A. J. Colloid Interface Sci. 1990, 136, 480. (9) Yada, R. Y.; Jackman, R. L. Protein Structure Function Relationships in Foods; Blackie Academic & Professional: Glasgow, 1994. (10) Tanford, C. J. Mol. Biol. 1972, 67, 59.

S0743-7463(98)00258-3 CCC: $15.00 © 1998 American Chemical Society Published on Web 09/22/1998

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changes in conformation, leading to the breakdown of the globular structure of the protein.11-16 Among many systems studied, protein complexes with sodium dodecyl sulfate (SDS) have been widely studied because the interfacial and aggregation properties of SDS itself are well-established. The binding of SDS onto a number of model proteins has been examined, and the results suggest that the amount of associated SDS depends primarily on the mass of protein rather than its type, its molecular weight, or its physical state (e.g., whether it is native or denatured).10,17 The extent of SDS binding to protein molecules is affected by the monomer concentration of SDS but not its aggregate concentration. Reynolds et al.17,18 have also demonstrated that the binding of SDS to the highest affinity sites of native bovine serum albumin (BSA) is independent of solution pH, suggesting that the interaction between BSA and SDS is primarily hydrophobic rather than electrostatic. In bulk solution SDS/ BSA complexes have been studied by viscosity, circular dichroism, and light scattering,17,19-21 and the results are broadly consistent with BSA retaining its globular structure at low SDS concentrations but becoming elongated as the SDS concentration increases.17,19 In the present work we study the interaction of SDS and BSA at the hydrophilic silica/water interface by determining the structure and composition of the adsorbed SDS/BSA complex using specular neutron reflection. One of the important features of the neutron reflection experiment is that the complexity caused by the presence of the surfactant and protein in the layer can easily be resolved by deuterium labeling of the surfactant and/or the solvent. The precision of the determination of the structural dimensions in neutron reflection can also reliably reveal any denaturation of the protein molecules before and after the interaction with the surfactant. The latter information has previously been inaccessible but is a vital part of the interpretation of the mode of interaction of proteins and surfactants at the solid/water interface. Experimental Section Neutron reflection experiments were performed on the white beam reflectometer CRISP at the Rutherford-Appleton Laboratory, ISIS, Didcot, U.K.22 using neutron wavelengths from 1 to 6 Å. The sample cell was almost identical to that used by Fragneto et al.23 with the aqueous solution contained in a Teflon trough clamped against a silicon block of dimensions 12.5 × 5 × 2.5 cm3. The collimated beam enters the end of the silicon block at a fixed angle, is reflected at a glancing angle from the solid-water interface, and exits from the opposite end of the silicon block. Each reflectivity profile was measured at three different glancing (11) Anson, M. L. J. Gen. Physiol. 1939, 23, 239. (12) Yang, J. T.; Foster, J. F. J. Am. Chem. Soc. 1953, 75, 5560. (13) Reynolds, J. A.; Herbert, S.; Polet, H.; Steinhardt, J. Biochemistry 1967, 6, 937. (14) Steinhardt, J.; Scott, J. R.; Birdi, K. S. Biochemistry 1977, 16, 718. (15) Nelson, C. J. Biol. Chem. 1971, 246, 3895. (16) Pitt-Rivers, R.; Impiombato, F. S. A. Biochem. J. 1968, 109, 825. (17) Reynolds, J. A.; Gallagher, J. P.; Steinhardt, J. Biochemistry 1970, 9, 1232. (18) Reynolds, J. A.; Tanford, C. J. Biol. Chem. 1970, 245, 5161. (19) Polet, H.; Steinhardt, J. Biochemistry 1968, 7, 1349. (20) Mattice, W.; Riser, J. M.; Clark, D. S. Biochemistry 1976, 15, 4264. (21) Tanner, R. E.; Herpigny, B.; Chen, S. H.; Rha, C. K. J. Chem. Phys. 1982, 76, 3866. (22) Penfold, J.; Richardson, R. M.; Zarbakhsh, A.; Webster, J. R. P.; Bucknall, D. G.; Rennie, A. R.; Jones, R. A. L.; Cosgrove, T.; Thomas, R. K.; Higgins, J. S.; Fletcher, P. D. I.; Dickinson, E.; Roser, S. J.; McLure, I. A.; Hillman, R. A.; Richards, R. W.; Staples, E. J.; Burgess, A. N.; Simister, E. A.; White, J. W. J. Chem. Soc., Faraday Trans. 1997, 93, 3899. (23) Fragneto, G.; Lu, J. R.; McDermott, D. C.; Thomas, R. K.; Rennie, A. R.; Gallagher, P. D.; Satija, S. K. Langmuir 1996, 12, 477.

Lu et al. angles, 0.35°, 0.8°, and 1.8°, and the results were combined. The beam intensity was calibrated by taking the intensity below the critical angle for total reflection at the silicon oxide/D2O interface to be unity. A flat background 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), was subtracted. For all the measurements the reflectivity profiles were essentially flat at κ > 0.2 Å-1, although the limiting signal at this point depends on the H2O/D2O ratio. The typical background for D2O runs was 2 × 10-6 and that for H2O was 3.5 × 10-6 (measured in terms of the reflectivity). Deuterated SDS was made by reacting deuterated dodecanol with chlorosulfonic acid in dry ethyl ether below 5 °C. Deuterated dodecanol was first dissolved in ether, and the sample flask was then placed on an ice bath. While the solution was stirred, an equivalent molar amount of concentrated chlorosulfonic acid was added slowly into the ethereal solution with the temperature inside the flask maintained below 5 °C. The solution was left for another hour. The acid solution was neutralized by the addition of concentrated NaOH solution. The ether was then rotary evaporated, the remaining solution mixed with propanol, and the hot solution filtered. The cycle of extraction and filtration was repeated until all the SDS was extracted. After the propanol and water evaporated, the solid was dissolved in pure water and the aqueous solution extracted with n-hexane in a continuous liquid/liquid extraction system to remove unreacted dodecanol. Water was subsequently removed by freeze-drying and the solid sample was recrystallized from a water/ethanol mixture several times before a surface tension measurement was made to check the purity of the sample. The absence of a minimum around the critical micelle concentration (CMC ) 8.1 × 10-3 M) in the surface tension plot indicated a high purity of the deuterated sample. The extent of deuteration among the 25 hydrogen atoms in the dodecyl chain was found to be 96 ( 2% by NMR. The deuterated dodecanol used was made by reducing deuterated dodecanoic acid with LiAlD4 (Aldrich, 98%+ D) and was separated from residual fatty acid by chromatographic purification. Hydrogenated SDS was purchased from Polysciences (99%+) and was recrystallized several times before use. Its surface tension was found to be the same as that of the deuterated sample and the tension curves were also in good agreement with previous measurements.24 BSA free of fatty acid was purchased from Sigma and used as supplied (catalogue no. A0281, lot no. 10H9304). The molecular weight of BSA is 66 700 ( 400. D2O (99.9% D) was obtained from Fluorochem and its surface tension was typically over 71 mN m-1 at 298 K, indicating the absence of any surface-active impurity. H2O was processed through an Elgastat ultrapure water system (UHQ) and its surface tension at 298 K was constant at 71.5 mN m-1. The solution pH was controlled by using a phosphate buffer at pH 5.1, keeping the total ionic strength fixed at 0.02 M. There were small differences in the pH between H2O and D2O but this was controlled to within 0.3 pH units. The glassware and Teflon troughs for the reflection measurements were cleaned using alkaline detergent (Decon 90) followed by repeated washing in UHQ water. All the experiments were performed at 298 K. The large (111) face of each silicon block was polished using an Engis polishing machine. The blocks were lapped on a copper plate with 3-µm diamond polishing fluid and on a pad with a 1-µm diamond followed by 0.1-µm alumina suspension. The freshly polished surfaces were immersed in neutral Decon solution (5%) and ultrasonically cleaned for 30 min and this was followed by a further 30 min of ultrasonic cleaning in water. The blocks were then copiously rinsed and soaked in acid peroxide solution (600 mL of 98% H2SO4 in 100 mL of 25% H2O2) for 6 min at 120 °C.25 The blocks were then thoroughly rinsed with UHQ water to remove acid and exposed to UV/ozone for 30 min to remove any traces of organic impurities.26 They were then left to soak in UHQ water for at least 24 h. This procedure was (24) Lu, J. R.; Morocco, A.; Su, T. J.; Thomas, R. K.; Penfold, J. J. Colloid Interface Sci. 1993, 158, 303. (25) Brzoska, J. B.; Shahidzadeh, N.; Rondelez, F. Nature 1992, 360, 719. (26) Vig, J. R. J. Vac. Sci. Technol. 1985, A3, 1027.

Binding of SDS to BSA Layers

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found to give surfaces with reproducible thickness and roughness of the oxide layer and which were completely wetted by water.

Neutron Reflection Neutron reflectivity R(κ) is primarily determined by the variation of scattering length density F(z) along the surface-normal direction:27,28

R(κ) )

16π2 |Fˆ (κ)|2 2 κ

(1)

where Fˆ (κ) is the one-dimensional Fourier transform of F(z):

tering length density of a given layer varies with isotopic composition, the fitting of a set of isotopic compositions to a single structural model greatly reduces the possibility of ambiguity in the interpretation, although it adds to the complexity of the fitting procedure. The choice of the number of sublayers depends on the complexity of the system, but the general procedure is to use the minimum number that will fit the whole set of data. The volume fractions of each component in a mixed sublayer can be expressed as

F ) φpFp + φsFs + φwFw

(4)

The scattering length density depends on the chemical composition through the following equation:

where F is the total layer scattering length density, Fp, Fs, and Fw are the scattering length densities of BSA, SDS, and water and φp, φs, and φw are their respective volume fractions in the layer. Thus, φp + φs + φw ) 1. If deuterated SDS is adsorbed onto BSA from D2O where Fs is adjusted to be equal to Fw, eq 4 can be written as

F ) Σnibi

F ) φpFp + (1 - φp)Fw

Fˆ (κ) )

∫-∞∞exp(-iκz)F(z)dz

(2)

(3)

where ni is the number density of element i and bi is its scattering length (scattering amplitude). Because different isotopes have different values of bi, isotopic substitution can be used to change the reflectivity for a given chemical structure and this is helpful in determining the composition at a mixed interface. For systems containing hydrogen atoms, isotopic substitution can be achieved by exchanging H with D. Although it is not easy to obtain protein samples with a high deuterium content, it is relatively easy to vary the isotopic labeling of surfactant molecules. Because the scattering lengths of D and H are of opposite signs, the scattering length densities of the surfactant and water can be varied over a wide range, which can be used to highlight an adsorbed protein layer in different ways. This technique is commonly called contrast variation. A simple example of the benefit of contrast variation is as follows. When a surfactant is bound onto the preadsorbed protein layer at the solid/D2O interface, the mixed interfacial layer has three components, protein, surfactant, and water, and it is not easy to obtain the volume fraction distribution of each component across the interface. However, if the surfactant is deuterated and its scattering length density is adjusted to be close to that of D2O, the surfactant is approximately invisible. In these circumstances the reflectivity will give the volume fraction of the protein directly. If a similar measurement is then made using the fully hydrogenated surfactant, the reflectivity gives the combined volume fraction of the protein and surfactant and hence that of the surfactant can be obtained by subtraction. The principle for extracting structural information from the data is straightforward. A structural model is assumed, in which the interface is divided into a suitable number of uniform sublayers, and the corresponding reflectivity is calculated using the optical matrix formulism.29 The calculated reflectivity is then compared with the measured one and the structural parameters subsequently modified in a least-squares iteration to obtain a good fit. The parameters used in the calculation are the thicknesses of the sublayers, τi, and their corresponding scattering length densities, Fi. Since the scat(27) Lu, J. R.; Lee, E. M.; Thomas, R. K. Acta Crystallogr. 1996, A52, 42. (28) Crowley, T. L. Physica A 1993, 195, 354. (29) Born, M.; Wolf, E. Principles of Optics; Pergamon Press: Oxford, 1970.

(5)

The surface excess of the protein in each sublayer can be expressed as

Γ)

τ(1 - φp) n′wVwNa

(6)

where τ is the thickness of the protein layer, n′w is the number of water molecules associated with each protein molecule, and Vw is the water molecular volume. n′w contains a contribution from the deuterated SDS and can be evaluated from the following equation:

n′w )

(1 - φp)bp FVw - bw(1 - φp)

(7)

where bw and bp are the scattering lengths for water and protein. Because Fp is known, Fs and Fw can be obtained by applying eq 4 to a reflectivity measurement at different contrasts. Similarly, the exact value for the number of water molecules associated with each protein molecule can also be obtained. The set of equations above applies to a single sublayer, and the total adsorbed amounts, and so forth, are obtained by summing over the sublayers used in the fitting procedure. Results and Discussion The thickness and composition of the oxide layer present on the freshly polished silicon (111) surface may vary from block to block, and since this layer makes a small contribution to the reflectivity, it is necessary to measure its thickness and composition accurately before the main adsorption experiment. The structure of the oxide layer is most reliably determined by measuring its neutron reflectivity profile with the block in contact with water using the sample cell described in the Experimental Section. The water helps to highlight the oxide layer especially when it is defective. Measurements were made at two different water contrasts, D2O and water whose scattering length density is matched to silicon (CMSi, F ) 2.07 × 10-6 Å-2). Figure 1 shows these reflectivity profiles. The reflectivity is weak for CMSi because at this contrast the signal is only from the oxide layer. If there are any defects in the oxide layer, water may penetrate the layer and this would result in the total scattering length density for the oxide layer being lower than that of pure silica (3.41 × 10-6 Å-2) in CMSi, but in D2O the

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Figure 1. Characterization of the structure of the oxide layer at the silica-water interface using neutron reflectivity with (b) D2O and (+) water contrast matched to silicon (CMSi with scattering length density ) 2.07 × 10-6 Å-2). The continuous lines were calculated using the optical matrix method with an oxide layer thickness of 8 ( 3 Å and F ) 3.4 × 10-6 Å-2. No roughness was used in the fitting.

total scattering length density of the oxide layer would be higher than that of pure silica. However, all the reflectivity profiles were fitted using a thickness of 8 ( 3 Å and F ) 3.41 × 10-6 Å-2 for the oxide layer, suggesting that, for the block used, there is no penetration of water into the layer, since the scattering length density of the layer is exactly as expected for amorphous oxide. The thickness of the oxide layer used in the previous work was 12 ( 3 Å, slightly thicker that that of the oxide layer on the present block. Such a difference should not affect the protein adsorption. Furthermore, no roughness was necessary to fit the reflectivity, suggesting that the oxide surface is quite smooth. Although differences in the thickness of the oxide layer are not expected to affect protein adsorption, the latter may be sensitive to variations in the hydrophilicity of the oxide surface and this could change during the course of the experiment. We therefore monitored the reflectivity profiles of BSA adsorbed at the silica/D2O interface at different times. After each SDS binding experiment, the solid surface was cleaned by rinsing with SDS solution at its critical micelle concentration (CMC) and this was followed by copious rinsing with UHQ water and a final rinsing with D2O. The standard 0.15 g dm-3 solution in D2O was then introduced into the sample cell. Figure 2 shows representative reflectivity profiles for BSA adsorption at different stages of the experiment. That the reflectivities from the BSA/D2O interface remain identical throughout the experiment suggests that the affinity of BSA for the surface did not change. The amount of protein adsorbed (surface excess) was obtained by fitting a uniform layer model to the reflectivity profiles using the optical matrix formalism already outlined. The continuous line in Figure 2 was calculated for a uniform layer of protein adsorbed on silicon oxide, assuming the structure of the oxide layer to be the same as that already determined in pure water. The volume fraction and surface excess of the adsorbed protein in the surface layer can be calculated using eqs 4-7 with the values of the scattering length density of the pure protein of the appropriate isotopic composition that are given in Table 1. The good fit of a single uniform layer to the whole set of measured reflectivity profiles indicates the

Lu et al.

Figure 2. Neutron reflectivity profiles showing the adsorption of BSA at the silica/D2O interface from a 0.15 g dm-3 phosphate buffered BSA solution. The solution pH was 5.1 and the ionic strength was 0.02 M. The measurements were made at the beginning (b), the middle (+), and the end (]) of the experiments over a period of 20 h. The consistency of the results suggests that the hydrophilicity of the solid surface remained unchanged. The continuous line was calculated for a layer thickness of 35 ( 4 Å and F ) 5 × 10-6 Å-2. The reflectivity from the bare silicon oxide-D2O interface is also shown for comparison (dashed line). Table 1. Scattering Length Densities of BSA in Different Water Contrasts at pH 5.1a contrast

F × 106/-2

D2O CMSi zero scattering H2O

3.34 2.41 1.85

a The total molecular volume was taken to be 79 111 Å 3.35 The degree of ionization of different amino acid groups was taken from ref 36.

correctness of the model. The thickness was found to be 35 ( 3 Å and the protein volume fraction to be 0.45, in good agreement with the values obtained from our previous measurements.30 In calculating the amount of protein adsorbed, complete exchange of labile hydrogens within BSA and the surrounding D2O has been assumed. Because the extent of exchange will affect the calculated surface excess, discussion will be given later to justify this assumption. It can be said at this stage that structural deformation within the globular framework after surface adsorption promotes the exchange process, resulting in a thorough exchange. Since the previous neutron adsorption measurements were made over longer time scales, the comparison of the two sets of data suggests that the adsorption of BSA reaches equilibrium within the first few minutes. This observation is different from many ellipsometric results where the ellipsometric signal changes significantly over the first 30 min. Our results now imply that the changes in the ellipsometric signal are not associated with the changing amounts of protein at the surface, as has generally been assumed.2-6 We do not have an explanation for this discrepancy, but we note that the ellipsometric signal may be sensitive to the presence of counterions and to their type and these may continue to change after the protein adsorption is essentially complete. Neutron reflection is only directly sensitive to the protein. (30) Su, T. J.; Lu, J. R.; Thomas, R. K.; Cui, Z. F.; Penfold, J. J. Phys. Chem. B 1998, in press.

Binding of SDS to BSA Layers

Figure 3. Neutron reflectivity profiles showing the binding of deuterated SDS to the preadsorbed BSA layer in D2O. The continuous line was calculated using a layer thickness of 48 ( 4 Å and values of F corresponding to volume fractions of 0.38 for BSA, 0.17 for SDS, and 0.45 for water. The reflectivity fitted to the preadsorbed BSA in D2O is shown as a dashed line for comparison.

Figure 4. Neutron reflectivity profiles showing the binding of hydrogenated SDS to the preadsorbed BSA layer measured over a period of 20 min (b) and 2 h (+). The overlap of the two profiles indicates that the measurements are reproducible and that there is no time effect. The continuous line was calculated using the parameters similar to those used for the calculated profile in Figure 3, and the long dashed line through the two measured profiles was calculated by allowing the distribution of SDS to be slightly more skewed toward the bulk solution. The reflectivity fitted to the preadsorbed BSA in D2O is shown as a short dashed line for comparison.

The SDS binding was studied with the preadsorbed BSA layer in equilibrium with 0.1 mM buffered SDS solution at the chosen isotopic contrast. The addition of SDS has a large effect on the neutron reflectivity as shown by the variation of the reflectivity with different labeling of SDS. The comparison is given for measurements in D2O in Figures 3 and 4. While the binding of deuterated SDS (d-SDS) does not change the profile much from that of the preadsorbed BSA layer (see Figure 3), the binding of hydrogenated SDS (h-SDS) generates a sharp interference fringe (see Figure 4). The shift of the interference fringe toward lower κ indicates that the layer thickens in the presence of SDS. In the case of h-SDS the bound SDS cannot be distinguished from the protein layer because

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both species are hydrogenated and therefore this reflectivity profile cannot show whether SDS binding has resulted in the partial removal of BSA from the oxide surface. In contrast, the profile from d-SDS/BSA in D2O contains direct information about the BSA distribution because, since the scattering length density of d-SDS is 6.4 × 10-6 Å-2, which is almost identical to that of D2O, its presence in the complex is masked by D2O. Thus, the change in the shape of the d-SDS/BSA profile from that of BSA alone must be caused entirely by structural changes in the BSA. The best fit is shown as a continuous line in Figure 3 and was calculated with a layer thickness of 48 ( 3 Å and F ) 5.2 × 10-6 Å-2. That the single layer model fits the data well indicates that although the total layer thickness has increased, the distribution of the BSA along the surface normal is still uniform. The volume fraction of the protein in the layer can be calculated using eq 6 and the new value was found to be 0.38 as compared with 0.45 in the original preadsorbed BSA layer. Taken in conjunction with the increased thickness of the protein layer, this change in the volume fraction leads to a surface excess of BSA of 2.5 mg m-2, which is exactly the same as that before the addition of SDS. This result therefore shows that the SDS binding at this bulk concentration of 0.1 mM does not cause the removal of any BSA from the oxide surface. The expansion of the BSA layer indicates that some SDS molecules have penetrated into the BSA layer, causing the disintergration of the compact BSA structure. It is possible that some SDS molecules also adsorb on the outer surface of the BSA layer. Although this cannot be revealed in the presence of d-SDS due to the matching contrast of d-SDS to D2O, the use of h-SDS will highlight this outer SDS layer. Since a typical SDS layer is at least some 15 Å, the total layer thickness is going to be around 60 Å. However, the sharp minimum in Figure 4 indicates that the dimension of the overall layer is only about 45 Å, showing the absence of such an outer SDS layer. Alternatively, the SDS can be uniformly distributed within the expanded BSA layer and the continuous line in Figure 4 was calculated with a layer thickness of 50 ( 4 Å and F ) 4.2 × 10-6 Å-2. If it is assumed that the isotopic substitution of the SDS does not affect the composition of the interface, the volume fractions of SDS and water can be calculated from eq 6 since the volume fraction of BSA is already known. The values are found to be 0.17 for SDS and 0.45 for water, respectively. It can be seen from Figure 4 that the single-layer model fits the data well up to κ ) 0.12 Å-1, indicating that the mixed SDS/BSA layer is reasonably well represented by a uniform layer distribution. The small deviation above 0.12 Å-1 is however systematic and appears to be above the error range. Several models assuming different distributions for BSA or SDS were tested. The one that clearly improves the fitting in the high κ region is to assume some level of unsymmetrical distribution for SDS within the mixed layer. The long dashed line through the measured reflectivity profile in Figure 4 was calculated assuming that the SDS distribution is composed of two regions, an inner layer of 32 Å with F ) 4.3 × 10-6 Å-2 and an outer layer of 13 Å with F ) 3.5 × 10-6 Å-2. This gives a total layer thickness of 45 Å, close to the value of 48 Å from the profile involving d-SDS described above. The corresponding volume fraction of SDS was subsequently found to be 0.16 in the inner layer and 0.26 in the outer layer, equivalent to an average total SDS volume fraction of 0.19, as compared with 0.17 from the direct uniform layer model. This suggests that although BSA is uniformly distributed, the distribution of SDS is slightly skewed

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toward the outer part of the mixed layer, possibly as a result of the steric restriction of the solid substrate. That the total SDS surface excess from the single-layer and two-layer models is within error identically suggests that the total interfacial compositon is less model-dependent, an observation already described in the previous work.27 The validity of the uneven SDS distribution will be further tested in the following when measurements under other contrasts are introduced. The binding of SDS to a variety of proteins has been studied in bulk solution. Reynolds and Tanford31 examined the binding of SDS to several denatured proteins and found that the weight ratio of SDS to protein was almost constant at 0.4 below 0.8 mM SDS and this increased to 1.4 as the SDS concentration was increased. They also found that the binding only depended on the monomer SDS concentration and had little to do with the identity or molecular weight of the proteins. Thus, on breakdown of the native structures by β-mercaptoethanol or a combination of guanidine hydrochloride (GuHCl) and β-mercaptoethanol, proteins are reduced to mixed polypeptide chains in a random coil conformation and the binding is then only determined by the number of peptide groups available. In a parallel experiment Reynolds et al.17,18 studied the binding of SDS onto native BSA and found that at 0.1 mM SDS the weight ratio is below 0.1, which is much lower than that for the denatured proteins (the weight ratio for the binding to native BSA is, incidentally, nearly independent of pH over the pH range 4.8-6.8). Thus, the ratio of the observed volume fraction of 0.43 for the complex at the solid/water interface is close to the weight ratio of 0.4 for the binding of SDS onto denatured BSA. This suggests that binding of SDS to BSA at the solid/water interface has resulted in the complete breakdown of the globular assembly and all the hydrophobic domains are open to bind SDS molecules. The binding of SDS to proteins may be time-dependent and in several previous studies of the binding of SDS to different proteins in bulk solutions the mixed solutions were normally left for several hours to allow equilibrium to be reached before measurements were made.32 We have examined the reproducibility and possible time effects by measuring the binding of h-SDS onto the preadsorbed BSA layer in two independent measurements. In the first measurement the BSA solution was in contact with the freshly cleaned oxide surface for just 5 min, the solution was then drained, and the buffered h-SDS solution was added. The neutron reflectivity measurement of this sample was made within a period of 30 min. In the second measurement, the BSA solution was left in contact with the oxide layer for over 1/2 h before the BSA solution was drained and the preadsorbed BSA layer put in contact with the h-SDS solution. The subsequent neutron measurement was made over a period of 2 h. Figure 4 compares the two reflectivity profiles. It can be seen that there is negligible systematic difference in the shape of the two curves and the position of the minimum, suggesting that there is no time effect in the BSA adsorption and the subsequent SDS binding. The reliability of the structural parameters obtained from the models proposed above can be verified further by performing reflectivity measurements at other isotopic compositions. The most direct examination of the distribution of SDS within the interfacial complex is to use d-SDS in CMSi. At this contrast, the scattering length (31) Reynolds, J. A.; Tanford, C. Proc. Natl. Acad. Sci. U.S.A. 1970, 66, 1002. (32) Mascher, E.; Lundahl, P. J. Chromatogr. 1989, 476, 147.

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Figure 5. The neutron reflectivity from deuterated SDS bound to preadsorbed BSA in CMSi. The continuous line was calculated using a layer thickness of 50 ( 3 Å and F ) 2.6 × 10-6 Å-2. The dashed line was calculated assuming that the SDS follows the same degree of unsymmetrical distribution as that for the BSA/ h-SDS/D2O profile.

density of BSA is 2.4 × 10-6 Å-2, which is close to the value of 2.07 × 10-6 Å-2 for both silicon and water and so the protein is almost invisible. The measured reflectivity is then dominated by the d-SDS layer. If the volume fractions for the three components are taken to be the same as those obtained previously from the uniform layer distribution, the total scattering length density for the layer is calculated to be 2.8 × 10-6 Å-2. The best fit, given as a continuous line in Figure 5, was modeled using F ) 2.6 × 10-6 Å-2 and τ ) 48 Å, in good agreement with the original uniform layer result. If, however, the two-layer model corresponding to the best fit to the BSA/h-SDS in D2O is used, the calculated reflectivity profile under this contrast is systematically higher than the measured one. The discrepancy is likely to be caused by the unreliability of the two-layer model which involves more variables in the fitting. On the other hand, the measured profile shown in Figure 5 is of coarse resolution, arising from the low signal under this contrast. Thus, the quality of the data does not offer any reliable justification for sophisticated modeling. Furthermore, the deviation between the measured and the calculated ones based on the two-layer model appears to be related to the calibration. While this can be easily checked with respect to the total reflection in the case of D2O profiles, there is no alternative information for checking the measurements in CMSi. However, the consistency between the uniform layer fits tends to indicate that it is the two-layer model which requies further refining when better data is available in the future. Two further verifications of the structural model were made using water of zero scattering length density with h-SDS and d-SDS. Figures 6 and 7 show these reflectivity profiles. The former measurement is dominated by the preadsorbed BSA layer. The same volume fractions for the BSA and SDS as obtained above give a combined scattering density of 8 × 10-7 Å-2, which gives an excellent fit to the data of Figure 6 with a layer thickness of 50 Å. The combined scattering length density for the mixed layer with d-SDS was calculated to be 1.7 × 10-6 Å-2, again in good agreement with the value of 1.5 × 10-6 Å-2 used for the calculation of the continuous line in Figure 7 with a layer thickness of 48 Å. The average layer thicknesses used for the different contrasts are all between 45 and 50 Å, a variation within the quoted experimental error. Thus,

Binding of SDS to BSA Layers

Figure 6. The neutron reflectivity from hydrogenated SDS bound to preadsorbed BSA in water of zero scattering length density. The continuous line was calculated using a layer thickness of 50 ( 3 Å and F ) 8 × 10-7 Å-2.

Figure 7. The neutron reflectivity from deuterated SDS bound to preadsorbed BSA in water of zero scattering length density. The continuous line was calculated using a layer thickness of 48 ( 3 Å and F ) 1.5 × 10-6 Å-2.

to a good approximation, the results all support the formation of a uniform mixed layer of SDS and protein. An attempt was also made to fit the two-layer model obtained above to these two measurements. As expected, the profile under the contrast shown in Figure 6 was found to be insensitive to the slightly skewed distribution of h-SDS because of the close scattering length densities. Although the measurement given in Figure 7 could more or less be fitted with the two-layer model, the fact that it does not fit the BSA/d-SDS/CMSi, as already discussed above, cannot guarantee any conclusion to be drawn about the distribution of SDS at this stage. In fitting the reflectivity profiles at different contrasts, it has been assumed that isotopic substitution in the surfactant and water does not affect the surface activity of the surfactant and its subsequent interaction with the preadsorbed protein layer. If it were the case, the interfacial compositions would vary with isotopic substitution and it would not be possible to fit the whole set of reflectivity profiles to a single set of structural parameters. That all the measured reflectivity profiles fit to a single structural model suggests that any isotopic effects in the system fall within the range of experimental error. A

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further assumption made in the calculation of the scattering lengths of BSA given in Table 1 is that the labile hydrogens in BSA exchange completely with the aqueous environment on the time scale of the neutron experiment. This assumption is not easy to justify because BSA is known to have hydrophobic domains which may not be accessible to water. The extent of exchange of the labile hydrogens in globular proteins with D2O has been extensively investigated in the past and has been reviewed by Hvidt and Nielsen.33 More recent work by Dobson et al.34 has extended this work with the aim of revealing the mechanisms relating to the folding and unfolding of protein molecules under different solution conditions. The accessibility of labile hydrogen atoms to the surrounding water has been widely used as a measure of the masking of portions of the polypeptide chain. Of the 1015 potentially labile hydrogen atoms per BSA molecule, about 750 exchange almost instantly at pH 7 and 0 °C. Some further 200 exchange over a period of a few minutes up to 2 h, but the remaining 50-100 appear not to exhange easily within another 24 h. Thus, about 90% of the labile hydrogens are readily exchangeable. Hvidt and Nielson also found that the pH and temperature substantially affect the exchange rate. The most difficult hydrogens to exchange are those on the amide groups in the peptide chain, of which BSA has 582. The barrier preventing the exchange is the hydrophobic encapsulation of any labile hydrogens inside the globular framework but since some of the labile hydrogens in the peptide chain are on the outer surface of the globular structure, they are easily exchanged. The fraction which is buried inside the hydrophobic domains is not expected to be exchanged within the time scale of the experiment. The incomplete exchange will introduce an error in the scattering length and a consequent error in the derived surface excess. The typical accuracy of the neutron reflection experiment is 5% or less. If, at the time of adsorption, exchange is incomplete by more than about 5%, one would expect to obtain a small difference in the BSA surface excess in the presence and absence of SDS. This is because the binding of SDS opens up the protein assembly and, since the binding is of hydrophobic origin, the buried labile hydrogens would be more exposed to the surrounding water. That the surface excess does not change suggests that the exchange within the preadsorbed BSA layer, where the globular structure of BSA is deformed but its main framework is still retained, is already complete. The structure of the SDS/BSA complex in bulk solution has been examined by various techniques.18-21 The binding sites have been a matter of interest and the determination of these has been based on changes in ultraviolet absorption or in optical rotation. Binding of SDS onto BSA shifts several ultraviolet bands in the wavelength range 2000-3000 Å, indicating that tryptophan residues are at or near the strongest binding sites and that additional sites with slightly lower affinity are at or close to tyrosine residues. Results from viscosity studies by Reynolds and Tanford18 were modeled in terms of rodlike aggregates and the data suggested that as the level of SDS binding increases, the long axis of the protein becomes more extended. This model was later supported by dynamic light-scattering measurements by Tanner et al.21 which showed a consistent increase of the hydrody(33) Hvidt, A.; Nielsen, S. O. Adv. Protein Chem. 1966, 21, 287. (34) Radford, S.; Buck, M.; Topping, K. D.; Dobson, C. M.; Evans, P. Proteins 1992, 14, 237. (35) Van Krevelen, D. W. Properties of Polymers, 3rd ed.; Elsevier: New York, 1990. (36) Stryer, L. Biochemistry, 3rd ed.; W. H. Freeman and Co.: New York, 1988.

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namic radius with increased SDS binding. The results of Tanner et al. also showed that when the binding weight ratio was below 1, the short axial radius of the prolate spheroid was 21 Å and the long axial radius was about 80 Å, as compared with the values of 21 and 70 Å for the two corresponding radii for native BSA in solution. Thus, SDS binding apparently elongates the rodlike native structure but does not alter its cross-sectional diameter. This picture does not appear to fit what has been observed at the solid/ water interface. If the SDS/BSA complex retains its structure at the solid/water interface, the thickness of the mixed layer should be the same as the preadsorbed BSA without bound SDS. That the layer thickness has

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increased substantially suggests that the structure of SDS/ BSA is different from that in bulk solution. Thus, the presence of the solid interface must have induced further structural deformation. The high packing density within the mixed protein layer and the geometrical constraint of the wall probably forces the layer to expand when the globular framework is broken. Acknowledgment. We thank the Biotechnology and Biological Sciences Research Council for support. LA980258R