The Effect of Solution pH on the Structure of Lysozyme Layers

Physical and Theoretical Chemistry Laboratory, South Parks Road, Oxford OX1 ... When the Label Matters: Adsorption of Labeled and Unlabeled Proteins o...
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Langmuir 1998, 14, 438-445

The Effect of Solution pH on the Structure of Lysozyme Layers Adsorbed at the Silica-Water Interface Studied by Neutron Reflection T. J. Su and J. R. Lu* 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.

Z. F. Cui Department of Engineering Science, Parks Road, Oxford OX1 3PJ, U.K.

J. Penfold ISIS, CCLRC, Chilton, Didcot OX11 0QX, U.K. Received June 11, 1997. In Final Form: October 29, 1997 We have studied the structure of lysozyme layers adsorbed at the silica-water interface using specular neutron reflection. The effect of pH on the adsorbed lysozyme layer was examined by manipulating the pH in two different cycles at two constant lysozyme concentrations of 0.03 and 1.0 g dm-3; the first cycle was started at pH ) 4 followed by pH ) 7 and then 8, before returning to 4; the second cycle was started at pH ) 7 followed by a decrease to 4 and then back to 7. The neutron reflectivity profiles showed no hysteresis in either adsorbed amount or structure. There was less adsorption at pH ) 4 than at pH ) 7 for both lysozyme bulk concentrations. No variation of the reflectivity with time was found at the experimental resolution of about 5 min per measurement. The lysozyme structure at the interface at pH ) 4 and pH ) 7 was determined from reflectivity profiles at different isotopic compositions of the water. The thickness of the adsorbed layer at the lower concentration of 0.03 g dm-3 was found to be 30 ( 2 Å, suggesting sideways-on adsorption of the ellipsoidally shaped protein. At the higher concentration of 1.0 g dm-3 the thickness of the layer was found to be 60 ( 2 Å, suggesting bilayer adsorption with side-on orientation in each layer. These observations disagree with literature results from surface force and ellipsometric measurements which suggest that a side-on monolayer of 30 Å thickness is formed at dilute bulk concentrations, which switches to end-on adsorption of 45 Å thickness as the bulk concentration increases, eventually reaching a bilayer of 90 Å thickness when the bulk lysozyme concentration is further increased. The neutron measurements indicate that the adsorbed amount and the orientation of the globular protein are determined by the electrostatic repulsion between the lysozyme molecules within the layer.

Introduction Protein adsorption is involved in a wide range of surface and interfacial phenomena; undesirable effects are the deposition of blood proteins onto medical devices and their subsequent biological responses, fouling of bacteria on ships’ hulls, and blockage of filtration membranes in bioseparation; desirable effects are the stabilization of food emulsions and the fabrication of biosensors.1 Many studies have so far demonstrated that weak adsorption of proteins, that is, where there is no denaturation, can be manipulated by adjustment of the balance of surface-protein interaction utilizing variation in the electrostatic forces, hydrogen bonding, van der Waals forces, conformational entropy, and hydrophobic interactions. Thus, surface adsorption can be increased by the gain of conformational entropy upon unfolding at the surface, the shift of pH close to the isoelectric point, or by specific ion binding.2 Although hydrophobic and ionic * Please address all communications to Dr. J. R. Lu. (1) Horbett, T. A.; Brash, T. A. Protein at Interfaces II. ACS Symp. Ser. 1995, 602.

interactions are often regarded as the most important driving forces for surface adsorption, the relative significance of these interactions in a given system depends on the details of the protein structure and the particular surface involved. The variety of the interactions and the variety of differences in size, shape, and flexibility of protein molecules have made it difficult to rationalize the behavior of proteins at interfaces. Strong interaction between protein molecules and the solid surface often leads to irreversible adsorption marked by the denaturation of the protein. The nature of the solid surface, including its hydrophobicity and charge density, has a strong effect on the structure and conformation of the protein layer. For example, the hydrophobic surface often induces exposure of hydrophobic fragments within the protein which may destroy any structural assembly of the protein molecules.3-5 Even at hydrophilic surfaces where the interaction is largely electrostatic, the (2) Haynes, C. A.; Norde, W. Colloid Surf., B 1994, 2, 517. (3) Prime, K.; Whitesides, G. M. Science 1991, 252, 1164. (4) Iwasaki, Y.; Fujike, A.; Kurita, K.; Ishihara, K.; Nakabayashi, N. J. Biomater. Sci., Polym. Ed. 1996, 8, 91. (5) Feng, L.; Andrade, J. D. J. Biomed. Mater. Res. 1994, 28, 735.

S0743-7463(97)00623-9 CCC: $15.00 © 1998 American Chemical Society Published on Web 01/01/1998

pH Effect on the Structure of Lysozyme Layers

adsorption may be irreversible because the accumulated number of direct contacts between protein fragments and the surface may be too large to allow desorption. Irreversibility of this type will generally be sensitive to protein concentration, pH, or salt concentration. Many authors have so far applied the relatively crude Langmuir model to describe this type of adsorption for proteins, for example, see refs 6 and 7, although reversibility is an assumption in the Langmuir treatment. An important step toward understanding protein adsorption is to determine the in situ structure of the adsorbed protein layer because structural information can be related to the interactions in the interfacial region. Many techniques including infrared spectroscopy (IR), radiolabeling, circular dichroism (CD), electrophoresis, NMR, and ellipsometry have all been used to investigate protein adsorption onto either flat solid surfaces or onto solid particles in colloidal dispersions. None of these techniques is able to address the structure of the layer except in a peripheral way.1 Thus, IR has been used to probe the conformational response of amide groups to changes in their environment and this information is then used to infer possible changes within the whole protein layer.8 Several of these techniques do not even give accurate measurements of the adsorbed amount. Radiolabeling and fluorescence can determine the adsorbed amount9,10 but give no information about the structure of the adsorbed layer and fluorescent probes may even alter the surface behavior. Ellipsometry is very sensitive to the adsorbed amount but requires uncertain assumptions in the derivation of structural information.11 In contrast to all these methods neutron reflection offers a powerful and direct means for determining both the amount of adsorbed protein and its low-resolution structure at buried interfaces.12,13 In this paper we report the variation of lysozyme adsorption with pH at the hydrophilic silicawater interface. Lysozyme is a model globular protein and is resistant to denaturation. Its adsorption at different interfaces, including silica and mica, has been extensively examined by other experimental methods, which creates the opportunity to make a systematic comparison. Experimenal Section The neutron reflection measurements were made on the white beam reflectometer SURF at the Rutherford-Appleton Laboratory, ISIS, Didcot, U.K.14 using neutrons of wavelengths 0.5-6.5 Å. The sample cell used was almost identical to that depicted by Fragneto et al. in ref 15 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 angles, 0.35°, 0.8°, and 1.8°, and the results combined. The beam intensity was calibrated with respect to (6) Mizutani, T.; Brash, J. L. Chem. Pharm. Bull. 1988, 36, 2711. (7) Moreno, E. C.; Kresak, M.; Hay, D. I. Biofouling 1991, 4, 3. (8) Ball, A.; Jones, R. A. L. Langmuir 1995, 11, 3542. (9) Rapola, R. J.; Horbett, T. A. J. Colloid Interface Sci. 1990, 136, 480. (10) Baszkin, A.; Boissonnade, M. M. J. Biomed. Mater. Sci. 1993, 27, 145. (11) Malmsten, C. J. Colloid Interface Sci. 1994, 166, 333. (12) Fragneto, G.; Thomas, R. K.; Rennie, A. R.; Penfold, J. Science 1995, 267, 657. (13) Liebmann-Vinson, A.; Lander, L. M.; Foster, M. D.; Brittain, W. J.; Vogler, E. A.; Majkrzak, C. F.; Satija, S. Langmuir 1996, 12, 2256. (14) Bucknall, D.; Penfold, J.; Webster, J. R. P.; Zarbakhsh, A.; Richardson, R. M.; Rennie, A. R.; Higgins, J. S.; Jones, R.; Fletcher, P. D.; Thomas, R. K.; Roser, S. J.; Dickinson, E. International Conference on Advanced Neutron Sources XIII, in press. (15) Fragneto, G.; Lu, J. R.; McDermott, D. C.; Thomas, R. K.; Rennie, A. R.; Gallagher, P. D.; Satija, S. K. Langmuir 1996, 12, 477.

Langmuir, Vol. 14, No. 2, 1998 439 the intensity below the critical angle for total reflection at the silicon-D2O interface. 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 was dependent on the H2O/D2O ratio. The typical background for D2O runs was found to be 2 × 10-6 and H2O to be 3.5 × 10-6 (measured in terms of the reflectivity). Lysozyme (from chicken egg white) was purchased from Sigma (Catalog No. L6876) and used as supplied. D2O (99.9% D) was from Fluorochem. 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 phosphate buffer and the pH varied by changing the ratio of Na2HPO4, NaH2PO4, and H3PO4, keeping the total ionic strength fixed at 0.02 M. There were small differences in pH between H2O and D2O, but this was controlled to within 0.2 pH units. All the experiments were performed at 298 K. The glassware and Teflon troughs for the reflection measurements were cleaned using alkaline detergent (Decon 90) followed by repeated washing in UHQ water. The large (111) face of each silicon block was polished using an Engis polishing machine. The blocks were lapped on a copper plate with a 3-µm diamond polishing fluid and on a pad with a 1 µm diamond followed by 0.1-µm alumina fluids. The freshly polished surfaces were immersed in neutral Decon solution (5%), ultrasonically cleaned for 30 min, and 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, 98% H2SO4 in 100 mL, 25% H2O2) for 6 min at 120 °C.16 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.17 They were then left to soak in UHQ water for at least 24 h. This procedure was found to produce surfaces with reproducible thickness and roughness of the oxide layer, which are completely wetted by water. The reproducibility in surface hydrophilicity between different blocks was examined by measuring reflectivity profiles from the lysozyme adsorbed from a solution of 0.03 g dm-3 of lysozyme in D2O, which always gave a good fit to a thickness of 30 ( 3 Å and a coverage of 1.8 ( 0.2 m gm-2. Neutron Reflection. The reflectivity of neutrons R(κ) is approximately determined by the variation of scattering length density F(z) along the surface normal direction:18

R(κ) )

16π2 |F(κ)|2 κ2

(1)

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

Fˆ (κ) )

∫

∞

-∞

exp(-iκz) F(z) dz

(2)

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

F ) Σnibi

(3)

where ni is the number density of element i and bi its scattering amplitude (scattering length). Because values of bi vary from isotope to isotope, isotopic substitution can be used to produce different reflectivity from a given chemical structure and this can be a great help in revealing the structural details of an interface. This is particularly the case for systems containing hydrogen atoms. For example, the scattering lengths of D and H are of opposite sign, and hence the scattering length density of water can be varied over a wide range, which can be used to (16) Brzoska, J. B.; Shahidzadeh, N.; Rondelez, F. Nature 1992, 360, 719. (17) Vig, J. R. J. Vac. Sci. Technol. 1985, A3, 1027. (18) Lu, J. R.; Lee, E. M.; Thomas, R. K. Acta Crystallogr. 1996, A52, 42.

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Su et al.

highlight an adsorbed protein layer in different ways. This technique is commonly called contrast variation. Thus, when the solution is made from water consisting of approximately 1 mol of D2O and 2 mol of H2O, which has a scattering length density close to that of silica, the specular reflectivity is largely only caused by the adsorbed protein layer. Under these circumstances, neglecting the small contribution from the oxide layer on the silicon and assuming a model of a uniform adsorbed layer, the area per molecule, A, can be deduced directly from the derived scattering length density F and thickness of the layer τ using

A)

Σnibi Fτ

(4)

where ni and bi depend on the known formula of the protein. The surface excess Γ is related to A by

Γ)

Figure 1. Ellipticity (real part) at the air/silica/silicon interface as a function of wavelength at three different incident angles. The continuous lines were calculated using the optical matrix formula assuming an oxide layer thickness of 20 ( 3 Å.

1 NaA

Although in principle Fourier transformation could be used to determine the interfacial scattering length density profile, in practice, the procedure for extracting structural information from experimental profiles is usually done by fitting models to the data. A structural model is assumed and the corresponding reflectivity is calculated using the optical matrix formula,19 the calculated reflectivity compared with the measured one, and the structural parameters modified in a least-squares iteration. The parameters used in the calculation are the thicknesses of the layers, τi, and the corresponding scattering length densities, Fi. Since the scattering 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. Although a single uniform layer model may not be appropriate for protein adsorption, it is usually sufficiently accurate for the determination of the adsorbed amount, Γ or A.

Results and Discussion Characterization of the Oxide Layer. Polished silicon normally has a thin oxide layer present on its surface and, although this only contributes weakly to the reflected signal, it is essential to characterize it to ensure correct fitting of the structure and composition of any subsequently adsorbed layers. The thickness of the oxide layer was initially determined using spectroscopic ellipsometry on the silicon/air interface. The sensitivity of the ellipticity to the variation of the incident angle was first determined at a fixed wavelength of 400 nm. Measurements were then made at angles selected for large variations in ellipticity. A typical measurement is shown in Figure 1, the results being plotted in terms of ellipticity and wavelength at three different angles. The measured ellipticity profiles were fitted simultaneously using a single uniform layer with a thickness of 20 ( 3 Å, giving the solid lines in Figure 1. Six different positions on the silicon block were examined and the values of the thickness were found to be identical within the 3 Å error, demonstrating the uniformity of the surface. The same surface was subsequently characterized in contact with water using neutron reflection. The oxide layer is expected to be defective in the sense that the roughness resulting from polishing and chemical treatment will probably generate pores in contact with the water. The presence of water in the layer is easily detected and measured by using contrast variation. Thus, measurements were usually made at four different contrasts: (19) Born, M.; Wolf, E. Principles of Optics; Pergamon Press: Oxford, U.K.; 1970.

Figure 2. Neutron reflectivities as a function of momentum transfer κ at the silica-water interface using different water contrasts: (]) D2O; (4) CM4 (scattering length density of water ) 4.0 × 10-6 Å-2); (+) CMSi (scattering length density 2.07 × 10-6 Å-2 ). The continuous lines were calculated using the optical matrix method and an oxide layer thickness of 17 ( 3 Å. Table 1. Physical Constants Used in the Calculation species

scattering lengtha × 105/Å

molecular volume/Å3

scattering length density × 106/Å-2

D2O H2O SiO2 lysozyme

19.2 -1.7 15.9 3377.4c

30 30 47 16717b

6.4 -0.6 3.4 2.0

a

See ref 35. b See refs 33 and 34. c Value in H2O.

D2O, H2O, CM4 (F ) 4 × 10-6 Å-2), and CMSi (F ) 2.07 × 10-6 Å-2). The sensitivity of the reflectivity profiles to this contrast variation is demonstrated in Figure 2. In the case of CMSi the reflectivity is weakest because the signal is only from the oxide layer, which contains contributions from the oxide itself and any water in its porous structure. When the water contrast is 4 × 10-6 Å-2, it almost matches the whole oxide layer and the signal is mainly from other parts of the interface. All the reflectivity profiles were fitted using a thickness of 17 ( 3 Å for the oxide layer and this value is consistent with the ellipsometric measurements. The volume fraction of water in the oxide layer was found to be 0.2, and the small value of 3 Å used for the roughness of the oxide-water interface suggests that the surface is reasonably smooth. The scattering lengths and molecular volumes used in the fitting are given in Table 1. Lysozyme Adsorption at Different pH. Although the pattern of lysozyme adsorption with respect to its bulk concentration is well-known, the effects of pH on this adsorption are not well-established. We have found for

pH Effect on the Structure of Lysozyme Layers

Langmuir, Vol. 14, No. 2, 1998 441

Figure 4. Comparison of surface excesses obtained from two independent pH cycles at bulk lysozyme concentrations of 0.03 and 1 g dm-3. Two pH cycles were made at 0.03 g dm-3: the first started at pH ) 4 (]), then raised to 7 (]), and 8 (]), and returned to 4 (×); the second started at pH ) 7 (4), then lowered to 4 (4), and returned to 7 (+). The pH cycle at 1 g dm-3 was started at pH ) 7 (0), then lowered to 4 (0), and returned to 7 (3).

Figure 3. Effect of pH on lysozyme adsorption measured as the variation of reflectivity in D2O at 0.03 g dm-3. (a) The solution pH was initially 4 (]), then raised to 7 (4), followed by 8 (+), and then returned to 4 (×). (b) The pH was initially 7 (]), lowered to 4 (4), and finally returned to 7 (+). The continuous lines are the uniform layer fits to the measured reflectivities at pH ) 4 and 7 and are drawn for guidance. The results suggest completely reversible adsorption.

several protein systems that the amount of protein adsorbed is sensitive to both pH and the pH history of the interface, that is, the route used to establish the final pH.20 The effect of pH on lysozyme adsorption was investigated at a fixed protein concentration of 0.03 g dm-3 in two ways. In the first the pH was started at 4, increased to 7, then 8, and finally back to 4. Figure 3a shows the four reflectivity profiles in D2O measured at these four values of the pH. That the two reflectivity curves at pH ) 4 are identical shows that the adsorption is reversible with respect to this particular pattern of pH variation. Figure 3a also shows quite clearly that the adsorbed amount increases at pH ) 7 and reaches its highest value at pH ) 8. To check the reversibility of the adsorption further, we also carried out the pH cycling by starting at pH ) 7, moving down to 4, and then returning to 7, and these results are shown in Figure 3b. The results again show that adsorption is completely reversible and that the adsorbed amount at pH ) 4 is less than that at pH ) 7. Comparison of the reflectivity profiles between the two independent cycles indicates that the adsorption only depends on the actual pH and not on the route to a given pH. The exact surface excesses were determined by fitting the measured reflectivity profiles using the optical matrix method. We started by using the simplest model for the adsorbed lysozyme layer, a uniform layer distribution. We have shown previously that the value obtained for the (20) Su, T. J.; Lu, J. R.; Thomas, R. K.; Rennie, A. R., to be published.

surface excess is to a good approximation independent of the structural model used to fit the data.21 The measured reflectivity profiles were therefore fitted using a uniform layer for the oxide layer and the protein layer. We further assumed that protein adsorption does not affect the structural composition of the oxide layer, in particular, the percentage of water in the oxide layer. This means that the structure of the oxide layer was taken to be the same as in the absence of protein. The continuous lines in Figure 3a,b were calculated using this model, and the resulting parameters are given in Table 2. The variation of the surface excess with pH from both pH cycles can be seen more clearly in Figure 4 where it is also clear that the two independent sets of measurements produce identical surface excesses. It is interesting to note that the surface excess at pH ) 8 is 1.9 mg m-2 which is almost twice the value at pH ) 4, showing that pH has a strong effect on the lysozyme adsorption. It is also interesting that because the isoelectric point for lysozyme is at about pH ) 11 a decrease in pH will make the protein more positively charged.22,23 Thus the decrease of surface excess with pH is opposite to what one might expect from simple electrostatic considerations, indicating that other interactions may play an important role. We discuss this further below. The lysozyme surface excess at pH ) 8 is equivalent to an area per protein molecule of 1400 Å2. Assuming that a solvated lysozyme molecule has the same dimension as in its crystalline form, the limiting area per molecule if adsorbed sideways-on would be 1350 Å2. The surface coverage at this pH will thus reach saturation if the molecules are adsorbed in the form of side-on monolayer. Similarly, the area per molecule at pH ) 4 is about 2660 Å2, equivalent to a surface coverage of 50% in sidewayson geometry and only 34% if the end-on orientation were adopted. It may be inappropriate to assume a constant limiting area per molecule for lysozyme because its dissolved state is likely to be different from its crystalline structure because of the effects of solvation and pH dependent charge. (21) Lu, J. R.; Su, T. J.; Thomas, R. K.; Penfold, J.; Richards, R. W. Polymer 1996, 37, 109. (22) Tanford, C.; Roxby, R. Biochemistry 1972, 11, 2192. (23) Haynes, C. A.; Sliwinski, E.; Norde, W. J. Colloid Interface Sci. 1994, 164, 394.

442 Langmuir, Vol. 14, No. 2, 1998

Figure 5. Effect of pH on the reflectivity from adsorbed lysozyme in D2O at 1 g dm-3. The solution pH was initially 7 (]), then lowered to 4 (4), and finally returned to 7 (+). The continuous lines are the best fits drawn for guidance. The results suggest completely reversible adsorption.

We also examined the pattern of adsorption at a higher protein concentration. Figure 5 shows the reflectivities at a lysozyme concentration of 1.0 g dm-3 when the solution pH is cycled from 7 through 4 and back to 7. Again the two measurements at pH ) 7 are identical within error showing that adsorption and desorption are reversible. The surface excess increases substantially with bulk lysozyme concentration. Thus, at pH ) 4 the surface excess Γ increased from 0.9 to 2.3 mg m-2. The variation of Γ with solution pH is again shown in Figure 4. The pH dependence of the adsorption of globular proteins on different substrates has been discussed by several authors.2,24,25 Electrostatic interaction and surface hydrophilicity are usually considered to be the dominant forces and the adsorbed amount determined by a combination of the interactions between the protein molecules in the layer and between the protein and the solid substrate. The surface excesses are normally found to be bell-shaped with respect to pH variation with the peak located in the region of the isoelectric point of the protein. In studying the adsorption of human serum albumin on charged polystyrene particles and charged halide surfaces, Haynes and Norde2 found that the maximal surface excesses peaked at the isoelectric points of the protein/ substrate complex. This was because on contact the negative charges on the particle surface neutralize some of the positively charged groups on the protein, causing a shift of the bulk solution isoelectric point to a lower pH. Away from the isoelectric point the increased charge on the protein molecules increases the repulsion between protein molecules inside the adsorbed layer, which leads to a decrease in the surface excess. Iler26 has indicated that the negative charge density on the silicon oxide surface is approximately constant between pH ) 4 and 8. On the other hand, Tanford et al.22 and Haynes et al.23 suggest that the net positive charge on lysozyme increases from 8 at pH ) 8 to 10 at pH ) 4. The combination of these two observations suggests that the electrostatic attraction between lysozyme and the surface should increase with decreasing pH, in contrast with the observed decrease of surface excess. This indicates that the effect of electrostatic attraction between the oxide (24) Morrissey, B. W.; Stromberg, R. R. J. Colloid Interface Sci. 1974, 46, 152. (25) Bagchi, P.; Birnbaum, S. M. J. Colloid Interface Sci. 1981, 83, 460. (26) Iler, R. K. The Chemistry of Silica; Wiley: New York, 1979.

Su et al.

surface and lysozyme becomes less significant with pH increase and that lateral electrostatic repulsion between the charged protein molecules within the layer is more important, that is, the pH dependence of lysozyme adsorption is governed more by protein-protein interaction and less by protein-surface interaction. It is generally accepted that the irreversibility of adsorption is related to the number of contacts between protein and surface. Although the fraction of segments in contact with the surface may be small and typically less than a few percent, the adsorption energy can easily be greater in magnitude than -100 kJ mol-1 because of the large total number of contacts. Since the amount of lysozyme adsorbed on the silica surface is dominated by lysozyme-lysozyme interaction, there must be relatively few lysozyme-surface contacts and hence adsorption on this surface is reversible. This is further reinforced by lysozyme being a robust globular protein with a welldefined equilibrium structure in bulk solution within the pH range studied.2 The stability of lysozyme is also supported by the microcalorimetry work of Privalov et al.27 who have shown that the denaturation of small singledomain proteins like lysozyme is thermodynamically reversible over a wide pH range. Recently, Haynes and Norde28 have also shown that the loss of native secondary structure on the adsorption of globular proteins is sensitive to the nature of the substrate surface. It is interesting to compare our surface excesses with those determined by other techniques. McGuire et al.29 studied lysozyme adsorption onto silica using ellipsometry. At a fixed lysozyme concentration of 1 g dm-3 they observed a surface excess of 2.8 mg m-2 as compared with our value of 3.3 mg m-2. Using the almost identical mode of ellipsometry Claesson et al.30 found a surface excess of 2.5 mg m-2 while Wahlgren et al.31 obtained a higher value of 3 mg m-2 at the same lysozyme concentration. The difference between the three different sets of ellipsometric data may reflect the range of uncertainty in ellipsometry measurements. However, these surface excesses are all too low when compared with the value of 4.8 mg m-2 for the adsorption of lysozyme on mica obtained using the technique of electron spectroscopy for chemical analysis by Blomberg et al.32 It is likely that such a large discrepancy is caused by the difference in the substrate. McGuire et al. also observed a steady increase of surface excess with time over the initial 30 min. Claesson et al.30 have also observed similar time dependence for lysozyme adsorption onto mica using surface force apparatus. They suggested that the cause of the time dependence of the change of force between the two mica plates was both increasing adsorption and the reorientation of lysozyme molecules within the adsorbed layer. We also examined the effect of time by following the change of reflectivity at different time intervals. Although we were not able to obtain information within the first 5 min of the solution being in contact with the surface, we had sufficient (27) Privalov, P. L.; Khechinashvili, N. N. J. Mol. Biol. 1976, 86, 665. (28) Haynes, C. A.; Norde, W. J. Colloid Interface Sci. 1995, 169, 313. (29) McGuire, J.; Wahlgren, M.; Arnebrant, T. J. Colloid Interface Sci. 1995, 170, 182. (30) Claesson, P. M.; Blomberg, E.; Froberg, J. C.; Nylander, T.; Arnebrant, T. Adv. Colloid Interface Sci. 1995, 57, 161. (31) Whalgren, M.; Arnebrant, T.; Lundstrom, L. J. Colloid Interface Sci. 1995, 175, 506. (32) Blomberg, E.; Claesson, P. M.; Froberg, J. C.; Tilton, R. D. Langmuir 1994, 10, 2325. (33) Tanford, C. J. J. Phys. Chem. 1972, 76, 3020. (34) Chalikian, T.; Totrov, M.; Abagyan, R.; Breslauer, K. J. Mol. Biol. 1996, 260, 588. (35) Sears, V. F. Neutron News 1992, 3, 26.

pH Effect on the Structure of Lysozyme Layers

Langmuir, Vol. 14, No. 2, 1998 443 Table 2. Structural Parameters Obtained Using the Uniform Layer Model concentration/ g dm-3

pH

Γ ( 0.2/ mg m-2

τ/Å

φa

0.03 0.03 0.03 1.0 1.0b

4 7 8 4 7

0.9 1.7 1.9 2.3 3.3

30 ( 3 30 ( 3 30 ( 3 35 ( 4 50 ( 5

0.21 ( 0.05 0.38 ( 0.05 0.45 ( 0.05 0.45 ( 0.05 0.46 ( 0.1

a φ denotes the volume fraction of lysozyme in the layer. b Uniform layer model did not fit the reflectivity profile well.

Figure 6. One-layer fits to the reflectivity profiles at pH ) 4 in the presence of 0.03 g dm-3 of lysozyme: (]) D2O; (4) CM4; (+) CMSi. The continuous lines were calculated using Γ ) 0.85 mg m-2 and τ ) 30 Å.

sensitivity to monitor any subsequent changes in the surface excess. We observed no time effects for any of the lysozyme concentrations or pHs studied in this work. Structure of Lysozyme Layers. Upon adsorption onto a solid substrate proteins form a number of direct contacts with the substrate surface. Contact formation may lead to partial breakdown of fragments of the Rhelix or β-sheet, which may generate further contacts. Lysozyme is a globular protein, and its native state in aqueous solution is highly ordered with most of its polypeptide backbone having little or no rotational freedom. Although structural rearrangement may occur upon adsorption, the internal coherence of the globular protein should prevent it from completely unfolding into loose random structures on the surface. This is supported by the work of Haynes and Norde2 who have examined a wide range of globular proteins using data from different sources and techniques. The adsorbed layer dimensions of globular proteins are thus expected to be comparable with their dimensions in aqueous solution. Measurements of the layer thicknesses can then be used to assess the orientation of the protein molecules on the surface. Thus, Claesson et al.30 have made this assumption in their determination of the conformation of lysozyme at the solid-water interface using a surface force apparatus and ellipsometry. The advantage of neutron reflection over most other techniques is that it is very sensitive to the structural distribution normal to the interface and can probe interfacial layer thickness with fewer assumptions to an accuracy better than a few angstroms. This is especially the case if the measurements are made at different contrasts. H2O/D2O mixtures similar to those used for the characterization of the structure of the oxide layer as described above can also be used to highlight the adsorbed lysozyme layer. It should be noted that although measurements in D2O provide reliable information about the amount of lysozyme adsorbed under different pH conditions, they may be ambiguous in revealing structural distributions.21 The use of a range of water contrasts gives a much more certain procedure for reducing the choice of possible structures. Figure 6 shows reflectivity measurements from 0.03 g dm-3 of lysozyme solution at pH ) 4 and different water contrasts. In comparison with the reflectivity profiles for the solid-water interface shown in Figure 2, the adsorption of protein changes the shape and the level of the reflectivity curves at all contrasts. The difference is largest for the measurement in CMSi

because the protein layer makes a relatively larger contribution to the signal at this particular contrast. We first attempted to fit all the reflectivity profiles using a single uniform layer model for the protein layer together with the single-layer structure for the oxide layer, just as for the determination of the surface excesses above. The structure of the oxide layer, for example, the extent of water penetration and the thickness, was taken to be unchanged from the oxide surface in direct contact with water. The roughness of 3 Å at the oxide surface was also included, although it has little effect on the final reflectivity. Complete exchange of the labile hydrogens in the lysozyme with the bulk water was assumed when the molecular scattering length was calculated. This will certainly be the case for the labile hydrogens associated with the polar or charged groups, but may be less complete for the labile hydrogens on amide groups. Because lysozyme has 129 amino acid residues, there are 127 labile hydrogens associated just with the amide groups. The barrier preventing the exchange is the hydrophobic encapsulation of the labile hydrogens inside the globular structure. If we assume that half of these hydrogens do not exchange, the effect on the scattering length is slightly less than 10%. Since the area per molecule is proportional to the scattering length, the uncertainty introduced into the determination of the area per molecule is also less than 10% and is the main factor determining the error quoted for the surface excesses in Tables 2 and 3. Since the difference in scattering length for the labile hydrogens is largest in D2O, this effect will be reduced for the other contrasts. However, the rather extreme possibility given above is unlikely because any slight conformational perturbation will allow the free exchange of labile hydrogens within the globular structure with D2O. It should be noted that the uncertainty in scattering length has no effect on the derived thickness. The continuous lines in Figure 6 were the resultant best fits using the set of structural parameters listed in Table 2. The thickness of the layer at pH ) 4 was found to be 30 ( 3 Å, suggesting that the protein molecules are adsorbed sideways-on at this concentration. Similar measurements at the same three different water contrasts were made at pH ) 7 and are shown in Figure 7. Although the amount of protein in the layer increases at pH ) 7, the thickness of the layer remains 30 Å, again suggesting side-on adsorption. Claesson et al.30 have suggested that adsorption changes from side-on at very low lysozyme concentration to endon when the lysozyme concentration is around 0.02 g dm-3, leading to the formation of a close-packed monolayer, arguing that the changeover enables the surface to accommodate more protein molecules. However, an endon monolayer would give a monolayer thickness of 45 Å, and the difference from our results of 15 Å is well-outside the range of error in neutron reflection. We have attempted to fit the three reflectivity profiles at pH ) 7 to a thickness of 45 Å, allowing the surface excess to float

444 Langmuir, Vol. 14, No. 2, 1998

Su et al. Table 3. Structural Parameters Obtained from the Two-Layer Model concentration/ Γ ( 0.3/ τ1 ( 3/ τ2 ( 5/ g dm-3 Å Å pH mg m-2 0.03 0.03 0.03 1.0 1.0 a

4 7 8 4 7

0.98 1.78 2.0 2.4 3.6

30 30 30 30 30

15a 15a 15a 15 30

φ1

φ2

0.21 ( 0.03 0.38 ( 0.03 0.40 ( 0.05 0.47 ( 0.05 0.55 ( 0.05

0.06 ( 0.03 0.06 ( 0.03 0.06 ( 0.03 0.28 ( 0.03 0.28 ( 0.03

Denotes insensitive parameters in the calculation.

Figure 7. One-layer fits to the reflectivity profiles at pH ) 7 in the presence of 0.03 g dm-3 of lysozyme: (]) D2O; (4) CM4; (+) CMSi. The continuous lines were calculated using Γ ) 1.65 mg m-2 and τ ) 30 Å.

Figure 9. One-layer model fits to the reflectivity profiles at pH ) 7 in the presence of 1.0 g dm-3 of lysozyme in D2O. The continuous lines were calculated using Γ ) 3.3 mg m-2 and τ ) 50 Å. The dotted line was calculated using a thickness of 45 Å and the dashed line using a thickness of 60 Å.

Figure 8. One-layer fit to the measured reflectivity at pH ) 7 in the presence of 0.03 g dm-3 of lysozyme in D2O. The thickness was fixed at 45 Å assuming end-on adsorption and the surface excess floated to obtain the best fit.

to obtain the best possible match between calculated and observed reflectivities, and Figure 8 shows the result for the measurement in D2O. The disagreement between this calculation and the observed reflectivity shows convincingly that the lysozyme cannot be adsorbed in wholly endon orientations. It may be more realistic to take the layer to consist of a mixture of side-on and end-on adsorption and the question is then whether the proportion of the two orientations at the interface can be determined reliably. To obtain this information, we have fitted the three contrasts at each pH using a two-layer model with a first layer of 30 Å and a second layer of 15 Å, which would correspond to the contribution from the outer fragments of lysozyme molecules adsorbed end-on. It was found that the optimal two-layer fit for the set of reflectivity profiles would not tolerate more than 29 ( 5% of the molecules in the end-on conformation at pH ) 4 and not more than 16 ( 5% at pH ) 7 (see Table 3). The problem of introducing a two-layer model for the protein layer is, of course, that it introduces two more variables which could lead to less unambiguous structural information. However, the results clearly indicate that if any molecules are adsorbed in the end-on orientation, the fraction must be less than the values given in Table 3. The surface force apparatus is always sensitive to the largest dimension of an adsorbed layer. Claesson et al.30 proposed that the side-on-end-on transition is driven by packing re-

Figure 10. Two-layer fits to the reflectivity profiles at pH ) 7 in the presence of 1.0 g dm-3 of lysozyme: (]) D2O; (4) CM4; (+) CM2.9 (F ) 2.9 × 10-6 Å-2). The continuous lines were calculated using Γ ) 3.6 mg m-2, τ1 ) 30 Å, and τ2 ) 30 Å.

quirements. The neutron results show that this cannot be the case because although the surface excess at pH ) 4 is less than that at pH ) 7, there is a larger percentage of end-on adsorption, which indicates that the molecules adopt the end-on conformation to reduce the increasing electrostatic repulsion between protein molecules as the proton concentration increases. Thus, the orientation is not determined by simple packing but rather by electrostatic repulsion within the protein layer. The variation of the structure of the adsorbed lysozyme layer with pH at the higher concentration of 1 g dm-3 was also determined using different water contrasts. At pH ) 7 a single uniform layer model did not fit the reflectivity profiles well in the high κ range. Figure 9 shows calculated reflectivities for 45 and 60-Å thick layers, neither of which

pH Effect on the Structure of Lysozyme Layers

Langmuir, Vol. 14, No. 2, 1998 445

Figure 11. Schematic diagram to illustrate the surface coverage and orientation of lysozyme molecules adsorbed at the silicawater interface: (a) at 0.03 g dm-3 and (b) at 1 g dm-3.

fit the measured reflectivity. This suggests that the layer is neither an all end-on monolayer nor a uniform bilayer. The best uniform layer fit produced a layer thickness of 50 ( 5 Å with a surface excess of 3.3 mg m-2. A fit of a two-layer model was then attempted, taking the amount of lysozyme in the two layers to be different. It was found that a two-layer model consisting of 65% surface excess in the inner 30 Å layer and 35% in the outer 30-Å layer provides the best fit as depicted in Figure 10. This again shows that both protein layers are adsorbed sideways-on, not end-on adsorption as proposed by Claesson et al.30 The data at pH ) 4 gave a thickness of 30 Å for the inner layer and 10 Å for the outer layer, suggesting a combination of end-on and side-on orientations, with a fraction of end-on molecules of 37 ( 5%, the highest percentage observed in all our measurements. It is therefore clear that lateral electrostatic repulsion within the adsorbed protein layer determines not only the level of adsorption but also the structural orientation of the molecules inside the layer. Because the positive charges are mainly located on the C-terminal lobe of the lysozyme molecule, we speculate that C-terminal lobes make direct contacts with the negatively charged surface. The exact location of the charge may also contribute to the tilting of the molecules in the layer. Conclusions The effects of pH on lysozyme adsorption are summarised schematically in Figure 11. Since the roughess of 3 Å on the oxide surface is comparable with the error quoted for the thickness of the lysozyme layer, it is not shown in the diagram. The sketch indicates that the

lysozyme layer itself is rough. However, where the two components which are generating the roughness are so completely mixed as here, the neutrons will effectively “see” a uniform mixed layer, that is, they do not distinguish a perfectly uniform mixture of protein and water in this layer from the nonuniform layer shown. It is only the combination of the coincidence of the thickness of the layer with the dimension of free lysozyme, and the reversibility of the adsorption that suggests that the layer must be as drawn rather than uniform. The lateral electrostatic interaction in the interfacial region determines both the level of the surface excess and the orientation of the molecules. Thus, at low lysozyme concentration, lysozyme molecules adsorb sideways-on to the silica surface. The surface coverage decreases with increasing proton concentration as a result of increased repulsion between the molecules inside the monolayer, and this increasing level of repulsion is also reflected in the increased percentage of end-on orientations when the pH is decreased. At higher bulk concentrations, adsorption produces a side-on bilayer at pH ) 7 and the measurements suggest that there is less lysozyme in the outer layer. Only monolayer adsorption can occur at pH ) 4 because of the strong electrostatic repulsion within the protein layer, but the higher coverage (compared with the lower bulk lysozyme concentration) leads to a higher fraction of molecules adopting the endon orientation. Acknowledgment. We thank the Biotechnology and Biological Sciences Research Council for support. LA970623Z