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Adsorption of Serum Albumins at the Air/Water Interface J. R. Lu* and T. J. Su Department of Chemistry, University of Surrey, Guildford GU2 5XH, U.K.
J. Penfold ISIS Neutron Facilities, Rutherford Appleton Laboratory, Chilton, Didcot OX11 0QX, U.K. Received February 9, 1999. In Final Form: May 17, 1999 The adsorption of bovine serum albumin and human serum albumin (HSA) at the air/water interface has been studied by neutron specular reflection. All the neutron measurements were performed in null reflecting water (D2O:H2O = 1:11), and the specular reflectivity at this water contrast is entirely from the adsorbed protein layer at the air/water interface. Accurate measurement of surface excesses and layer thicknesses has allowed us to infer the possible structural conformation of the two protein molecules on the surface of water. The effect of bulk protein concentration on the adsorbed amount and the total thickness of the adsorbed layer was examined at pH 5, close to the isoelectric point of 4.8 for the two albumins. The surface excess (Γ) of both proteins increased sharply over the concentration range of 5 × 10-4 to 5 × 10-2 g dm-3, beyond which Γ tended to the respective saturation limits. The saturation value of surface excess of BSA at 1 g dm-3 was found to be 2.8 ( 0.3 mg m-2 as compared with 2.1 ( 0.3 mg m-2 for HSA. The thicknesses of the protein layers at concentrations below 0.1 g dm-3 were found to be close to the short axial length of 40 Å of the globular protein solution structure, suggesting that albumin molecules adsorb with their long axes parallel to the surface of water. The effect of solution pH on the adsorption of the two proteins was examined over the pH range between 3 and 7 and at the two fixed protein concentrations of 5 × 10-3 and 1 g dm-3. At the low protein concentration the thicknesses of the protein layers were found to be constant at 30 ( 3 Å and the corresponding surface excesses to be peaked at pH 5. At the high protein concentration the reduction in surface excess at the pH values away from the IP was more substantiated for both proteins, as evident from the variation of both surface excess and layer thickness with solution pH.
Introduction Serum albumins have been widely used as model proteins for studying the interaction between proteins and different surface substrates. These studies provide useful information not only for the attachment of protein molecules onto solid substrates through physiosorption or chemical bonding as in the case of sensor development but also for the removal of protein molecules from solid substrates as in the process of medical cleaning. Some recent work on the adsorption of bovine serum albumin (BSA) and human serum albumin (HSA) tends to suggest that the surface activities of the two proteins are different.1-6 BSA and HSA have almost identical molecular weight and isoelectric point (IP), but their amino acid sequence differs by some 25%.7 Since both proteins are globular, their surface affinity is strongly affected by the distributions of the polar or charged groups on the outer surface of the globular assemblies. The distribution of these functional groups on the hydrophilic outer shell is usually uneven, with the consequence that some parts of the surface are more hydrophilic than others. Adsorption at the air/water interface is driven by the tendency for the * To whom correspondence may be addressed. (1) Kurrat, R.; Prenosil, J. E.; Ramsden, J. J. J. Colloid Interface Sci. 1997, 185, 1. (2) Liebmann-Vinson; Lander, L. M.; Foster, M. D.; Brittain, W. J.; Vogler, E. A.; Majkrzak, C. F.; Satija, S. Langmuir 1996, 12, 2256. (3) Su, T. J.; Lu, J. R.; Cui, Z. F.; Thomas, R. K.; Penfold, J. J. Phys. Chem. B 1998, 102, 8100. (4) Haynes, C. A.; Norde, W. Colloid Surf. 1994, 2, 517. (5) Blomberg, E.; Claesson, P. M.; Tilton, R. D. J. Colloid Interface Sci. 1994, 166, 427. (6) Fitzpatrick, H.; Luckham, P. F.; Eriksen, S.; Hammond, K. Colloid Surf. 1992, 65, 43. (7) Peters, T. Adv. Protein Chem. 1985, 37, 161.
more hydrophobic regions on the outer surface to minimize their exposure to the aqueous environment. It is expected that when adsorbed at the air/water interface the hydrophobic portion of the protein is exposed to air while the hydrophilic portion is submerged in the aqueous subphase. The structural detail of the adsorbed layer is then determined by the preferential orientation and packing of protein molecules in the interface which is, in turn, associated with electrostatic, hydrophobic, and entropic interactions. It is very likely that the difference in the amino acid sequences between the two albumins may result in some difference in the distribution of polar or charged groups on the surfaces of the two globular proteins, leading to a measurable difference in their surface excesses. The globular structures of the two protein molecules in aqueous solution have been determined by different techniques.7-9 Although different versions of globular structure have been proposed over last four decades, the consensus has been that both albumins are approximately prolate ellipsoid and have the same globular dimension. In normal solution condition their major and minor axes are respectively equal to 140 and 40 Å. The globular structure is approximately composed of three main domains which are loosely joined together through physical forces. In comparison, the six subdomains within the globular framework are wrapped by disulfide bonds. It is thus expected that upon adsorption some possible structural deformation may occur as a result of either the interaction between the protein molecules and the substrate, the steric or electrostatic effects within the adsorbed (8) Bendedouch, D.; Chen, S. H. J. Phys. Chem. B. 1983, 87, 1473. (9) He, X. M. Adv. Protein Chem. 1994, 45, 153.
10.1021/la990131h CCC: $15.00 © 1999 American Chemical Society Published on Web 07/30/1999
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layer, or a combination of both processes. Strong interaction can deform the globular framework, leading to a complete breakdown of the globular integrity. It is of interest to examine if any difference in surface adsorption can be detected as a result of the 25% difference in amino acid sequence. However, as many physical properties of the two albumins are similar, any difference in their surface adsorption is not expected to be great. The proposed work thus requires high sensitivity and resolution from the technique to be used for the characterization. Accurate measurement of the adsorbed amount and layer thickness together with the known dimension of the globular structure allows us to deduce some information about the physical state of protein molecules within the adsorbed layer. Techniques such as ellipsometry, surface tension measurement, and radiolabeling have been used to study protein adsorption, but these methods can at most offer estimate about the level of adsorption. They provide no reliable information about the density distribution within the protein layer. Thus, no information is obtained about the possible conformation of the protein molecules at the interfaces. Furthermore, although techniques such as infrared spectroscopy (IR) and circular dichroism (CD) provide useful information about the content of R-helices and β-sheets they provide no indication about the dimension of the protein layer. Neutron reflection has recently been used to study the adsorption of a number of proteins at the air/water and solid/water interfaces.3,10-12 The higher resolution of neutron reflectivity allows a reliable determination of protein layer density distribution along the surface normal direction. The thickness of the protein layer can usually be obtained with a resolution at the level of a few angstroms. The use of isotopic substitution, in the form of deuterium labeling to the self-assembled monolayers on the solid substrate and the change in the ratio of H2O to D2O in the solvent, has enabled us to determine not only the density distribution of the protein layer but also the extent of water mixing across the interface. The recent advances of neutron reflection have allowed us to study the adsorption of a number of globular proteins under different surface and solution conditions. In this paper we present the results of a neutron reflection study on the adsorption of BSA and HSA at the air/water interface. We have made all the measurements in a nonreflecting aqueous medium so that all the specular reflectivities resulted only from the adsorbed protein layers. Experimental Section Neutron reflection measurements were done on the reflectometer SURF at the ISIS pulsed neutron source, RAL, U.K.13 The protein solutions were held in Teflon troughs with a positive meniscus to prevent obstruction of the incoming and reflected beams by the Teflon edges. The troughs were mounted on an active antivibration table to prevent surface vibrations. Samples were accurately aligned using a laser beam which follows the same path as the neutron beam. The neutron beam intensity was calibrated with respect to the reflectivity of clean D2O. A flat background was subtracted from the measured reflectivity (10) Lu, J. R.; Thomas, R. K. J. Chem. Soc., Faraday Trans. 1998, 94, 995. (11) Dickinson, E.; Horne, D. S.; Phipps, J. S.; Richardson, R. M. Langmuir 1993, 9, 242. (12) Lu, J. R.; Su, T. J.; Thomas, R. K.; Penfold, J.; Webster, J. J. Chem. Soc., Faraday Trans. 1998, 94, 3279. (13) . Penfold, J.; Richardson, R. M.; Zarbakhsh, A.; Webster, J. W. 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, A. R.; Richards, R. W.; Staples, E. J.; Burgess, A. N.; Simister E. A.; White, J. W. J. Chem. Soc., Faraday Trans. 1997, 93, 3899.
Lu et al. profiles, and the exact value of the background for a given reflectivity profile 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). The reflectivity was measured in the wavelength range 0.5-6.5 Å and at three different angles of incidence, 0.5°, 0.8° and 1.8°, to ensure a range of κ sufficient to determine the thickness of the protein layer. Fatty acid free BSA (Sigma, catalog no. A0281, lot no. 107H7600) and HSA (Sigma, catalog no. A3782, lot no. 97H7604) were used as supplied. The molecular weight of BSA is 66 267 and that for HSA is 66 439, and their isoelectric point is 4.74.8.7 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. D2O was obtained from Fluorochem (99.9% D), and its surface tension was typically over 71 mN m-1 at 25 °C, indicating the absence of any surface active impurity. H2O was processed through an Elgastat ultrapure water system (UHQ) and its surface tension at 25 °C was constant at 71.5 mN m-1. The glassware and Teflon troughs for the reflection measurements were cleaned using alkaline Decon 90 solution (Decon Laboratories Ltd., U.K.) followed by repeated washing in UHQ water. All the experiments were performed at 25 °C.
Neutron Reflection Neutron reflection measures the specular reflectivity, which is defined as a ratio of the reflected beam intensity to that of the incident beam. The neutron reflectivity is related to the scattering length density F(z), which is determined by the structural distribution profiles of the interfacial components and their scattering lengths. As scattering length varies from element to element, isotopic substitution can be used to label a given component and the resultant reflectivity arising from the interface can be subsequently altered. Thus, isotopic substitution allows measurements of more than one reflectivity profile for the same chemical system. This aids substantially in offering a structural profile from model fitting. The scattering length density is related to the chemical composition across the surface by14,15
F(z) )
∑ ni(z)bi
(1)
where ni(z) is the number density of element i and bi its scattering length. Since the scattering length of hydrogen is -3.7 × 10 -5 Å, that of deuterium 6.7 × 10-5 Å, and that of oxygen 5.8 × 10-5 Å, the scattering lengths for H2O and D2O are of opposite sign. An important composition of the mixture is the one with the approximate ratio of D2O to H2O of 0.088 that gives a zero scattering length density. This water mixture does not contribute to the specular reflectivity and is usually called null reflecting water (NRW). When a protein layer is adsorbed on the surface of NRW the specular neutron reflectivity is then only from the adsorbed protein layer. Under such circumstance, and if the adsorbed layer is uniform, the area per molecule, A, is given by
A)
∑mibi/Fτ
(2)
where mi is the number of element i with scattering length bi and τ is the thickness of the layer. The surface excess Γ is related to A by (14) Lu, J. R.; Lee, E. M.; Thomas, R. K. Acta Crystallogr. 1996, A52, 11. (15) Crowley, T. L. Physica 1993, A195, 354.
Adsorption of Serum Albumins
Γ ) 1/NaA
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(3)
where Na is Avogadro’s constant. Neutron reflectivity profiles are usually analyzed by means of the optical matrix formalism, described by Born and Wolf16 and Lekner.17 The modeling procedure usually starts by comparing the calculated reflectivity based on an assumed structural model with the experimentally measured profile. The model is then modified until the calculated reflectivity profile is within error identical to the measured one. Although in many situations more than one structural profile may fit the measured one, other information such as the preferred geometry of the layer and the packing constraint in the total volume fraction across the interface will offer sufficient constraint so that the right structural profile can be easily chosen. The parameters used in the calculation are the thicknesses of the layers, τ, and the corresponding scattering length densities, F, which are related to the composition of the layers by eq 1. We have explained previously18 that in fitting the reflectivity profiles with a uniform layer model, τ and F can be varied over a limited range, but that their variations cancel in their contribution to A in such a way that A is not affected by the initial assumption of the layer being uniform. As the composition of amino acids for BSA and HSA is known,7 their total scattering lengths can be calculated and the values are equal to the sum of each of the elements times its corresponding scattering length. The scattering length for each element is listed in ref 19. The total scattering length for BSA was calculated to be 0.1462 Å and that for HSA to be 0.1473 Å. Each albumin molecule contains about 1120 labile hydrogens that exchange with bulk water. In this work the measurements were made in NRW containing 8% D2O. The scattering lengths of the proteins will be affected as a result of the exchange. If complete exchange occurs, the scattering length for BSA is 0.155 Å and that for HSA is 0.147 Å, leading to an increase of some 5%. We have discussed in a previous work3 that under the surface adsorption conditions almost all of the labile hydrogens will exchange to completion. We have therefore used the scattering lengths corresponding to complete exchange. Incomplete exchange will result in an experimental error well within 5% in surface excess and have no effect on the thickness. Results and Discussion (A) Variation of the Layer Structure with Bulk Concentration. The variation of the amount of protein adsorbed on the surface of water was examined by fixing the solution pH at 5, close to its isoelectric point. As outlined in the previous section, information about the adsorbed protein layer can be obtained directly from the reflection measurements on the surface of null reflecting water, as under this condition the specular reflectivity results entirely from the adsorbed protein layer. For each protein we have made reflectivity measurements in NRW at different protein concentrations. The adsorption of both BSA and HSA at the air/water interface was found to be time dependent. At the high protein concentrations around 1 g dm-3 it usually took about 1 h for the adsorption to reach equilibrium, as indicated by the variation of reflectivity with time. However, at the low concentrations (16) Born, M.; Wolf, E. Principles of Optics; Pergamon: Oxford, 1970. (17) Lekner, J. Thoery of Reflection; Nijhoff, 1987. (18) Lu, J. R.; Su, T. J.; Thomas, R. K.; Penfold, J.; Richards, R. W. Polymer 1996, 37, 109. (19) Sears, V. F. Neutron News 1992, 3, 26.
around 5 × 10-4 g dm-3 some 10 h was required for equilibration to be reached. Similar time effects have been reported by other authors in the study of protein adsorption at the air/water interface, using surface tension measurements, ellipsometry, and radiolabeling,20-24 where the equilibrium state was indicated by the attainment of constant values of surface tension, ellipticity, or radiation level from the labeled species. The time required for reaching constant reflectivity at a given albumin concentration is broadly comparable to that reported in the literature.20-24 As the variation of adsorption with time offers information about the kinetic process of adsorption, many attempts have been made to characterize the timedependent adsorption processes. One of the most systematic studies has been done by Graham and Phillips20 who have shown that protein adsorption is characterized by a two-stage process, the transport of protein molecules from bulk solution to the interface and the rearrangement of conformational structure within the adsorbed layer. For globular proteins such as lysozyme and BSA the surface excess shows a steady increase with time until a saturation limit is reached. The surface pressure starts to rise after a characteristic period of induction. Over the region where the surface excess has reached the saturation limit, the surface pressure retains its trend of increase, showing the distinct process of structural rearrangement of the adsorbed protein molecules at the constant surface excess. Similar observations have been reported in the recent work of Damodaran et al. and others,21-24 although it should be made clear that these measurements only agree with each other in trend and the reproducibility is generally poor. For example, Graham and Phillips20 have shown that for the adsorption of 0.76 mg dm-3 chicken egg white lysozyme at the air-water interface, the induction time for surface pressure to rise is about 1 h, while Damodaran et al.23 have obtained a value of some 7 h at an even higher lysozyme concentration of 1.5 mg dm-3. In a separate work, Damodaran et al.24 showed that the variation of surface excess for lysozyme with time also has an induction period of some 2 h, which was not observed in their later work23 and which was not shown in the work by Graham and Phillips either.20 It is worthwhile to note that all these studies were done at pH 7 using phosphate buffer with the total ionic strength of 0.1 M. In this work we restrict our discussions to the equilibrium situation. We show in Figure 1 the variation of neutron reflectivity with bulk HSA concentration in the form of log(reflectivity) against κ. These results were recorded after allowing sufficient time for equilibrium to be attained. It can be seen from Figure 1 that reflectivity is only measurable up to 0.15 Å -1 in κ and above this all the reflectivity profiles fall to the level of the background. The basic feature of the adsorbed layers can be deduced from the reflectivity profiles shown in Figure 1 without attempting any model fitting. At the two lower concentrations, increase in bulk protein concentration results in an increase in the level of the reflectivity profiles but no change in the shape. Because the level of reflectivity is an indicator of the surface coverage and its slope is related to the thickness of the adsorbed layer, the close proximity of the two lower (20) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1979, 70, 403. (21) Paulsson, M.; Dejmek, P. J. Colloid Interface Sci. 1992, 150, 394. (22) Tripp, B. C.; Magda, J. J.; Andrade, J. J. Colloid Interface Sci. 1995, 173, 16. (23) Razumovsky, L.; Damodaran, S. Langmuir 1999, 15, 1392. (24) Anand, K.; Damodaran, S. J. Colloid Interface Sci. 1995, 176, 63.
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Figure 1. Plots of log(reflectivity) against κ for the adsorption of HSA on the surface of null reflecting water at pH 5 under the bulk concentrations of 5 × 10-3 (O), 5 × 10-2 (4) and 1 g cm-3 (+). The continuous lines were calculated assuming uniform layer distributions with surface excesses given in Figure 2 and layer thicknesses in Figure 3.
reflectivity profiles shows that an increase in concentration only results in the increase of the adsorbed amount. At the highest concentration of 1 g dm-3, both the level and shape of the reflectivity profile have been changed. At the higher concentrations, the reflectivity profiles decay faster, suggesting that the thickness of the layers has increased. Quantitative structural information about the adsorbed layers was obtained by fitting layer models to the measured reflectivity profiles. For all HSA concentrations studied, the measured reflectivity profiles could be fitted using a model of a single uniform layer. The fits obtained with this model, shown as continuous lines in Figure 1, show that the adsorbed layers are reasonably uniform and therefore that the adsorbed HSA molecules probably retain their globular structure. Denaturation of the protein usually results in a more complex structure at the interface and layers with different densities are usually required to fit the data. For example, in studying the adsorption of lysozyme onto the hydrophobic silicon oxide/water interface formed by coating a monolayer of octadecyl trichlorosilane (OTS) onto the freshly polished oxide surface, we found that the adsorbed protein layer was characterized by two regions, an inner layer of 10-15 Å with some 90% of protein, and an outer layer of 40-80 Å with some 5-20% of protein.25 The breakdown of the globular framework of lysozyme was in this case clearly indicated by the high density of the inner layer and its thickness being much shorter than the short axial length of 30 Å for lysozyme. The formation of the diffuse outer layer is consistent with the distribution of the hydrophilic fragments from the broken globular assemblies. In the current work the physical state of the albumin molecules can again be inferred by comparing the layer structure with their globular dimension. The fits of the single uniform layer model give the layer thickness and its scattering length density. The volume fraction of protein can be derived from the latter. At the two lowest concentrations the thickness of the HSA layer was found (25) Lu, J. R.; Su, T. J.; Thirtle, P. N.; Thomas, R. K.; Rennie, A. R.; Cubitt, R. J. Colloid Interface, Sci. 1998, 206, 212.
Lu et al.
to be 32 ( 3 Å and the area per molecule to decrease from 8500 ( 200 to 6900 ( 200 Å2. This suggests that the slight change in reflectivity results entirely from the increase in surface concentration as the layer thickness remains constant within the error. Since the globular structure of HSA in aqueous solution is usually taken to be ellipsoidal with the short axial length of 40 Å, the thickness obtained here suggests that HSA molecules adopt a sideways-on orientation in this low surface concentration range;7 that is, the molecules adsorb with their long axes parallel to the surface. That the thickness of the layer is less than the dimension of the short axis of the ellipsoidal structure of HSA suggests that there might be some flattening of the molecules on adsorption. Alternatively, it could be attributed to the simplified treatment of the cylindrical shape of the protein. When a layer of ellipsoids (or cylinders to a good approximation) is represented by a uniform layer, the effective thickness will be less than the diameter of the ellipsoid. Such an effect is difficult to quantify when the molecules are not true ellipsoids. However, in studying lysozyme layers at the air/water interface we found that the uniform layer thickness exactly matched the short and long axial lengths where sideways-on and longwayson adsorption occurred, respectively.12 Thus, it is unlikely that the difference between the layer thickness of 32 Å and the short axial length of 40 Å can be accounted for by the simplification of the model. When the HSA concentration is increased to 1 g dm-3, the area per molecule decreases to 5500 ( 200 Å2, which is close to the estimated limiting area per molecule of 5600 Å2 for sideways-on adsorption. The thickness is now 48 ( 3 Å as compared with the constant thickness of 32 Å at the two lower concentrations. This result tends to suggest that at this concentration the HSA molecules within the adsorbed layer are starting to repel each other as a result of close packing. Although the net charge at this pH is almost zero, local charges are present on the surface of the globular framework and some weak electrostatic repulsion may still exist within the adsorbed layer. At high concentration the layer thickness is much greater than the short axial length, and this suggests that the globular framework of HSA is not at all rigid. However, it should be remembered that the exact extent of deformation hinges on the assumption about the solution structure of the two proteins, in particular, its short axial length. We will return to this point after the effect of pH on the structure of globular framework has been discussed. Similar measurements have been done for BSA adsorption at the air/water interface. The results are summarized in Figure 2 for the variation of surface excess with bulk concentration and in Figure 3 for the variation of the total layer thickness with bulk concentration. To emphasize the changes in the low concentration range, plots of surface excess and layer thickness versus log(concentration) are included as inserts in the two figures. The results for HSA are also shown for comparison. It can be seen from Figures 2 and 3 that over the low concentration region the thickness and surface excess for the two albumins are identical within the experimental error. At 5 × 10-2 g dm-3 the surface excess for BSA adsorption is about 0.5 mg m-2 greater than that of HSA. The layer thickness for the BSA layer is 37 ( 3 Å, as compared with 32 ( 3 Å for HSA. A further increase in BSA concentration to 1 g dm-3 produces a layer distribution which cannot be modeled well by uniform layer distribution. The simplest model that is consistent with the measured reflectivity profile at this concentration is a two-layer model. The best model fit gave a dense top layer of 48 Å on the air side with the area per molecule of 5500 Å 2 and an inner layer of 32 Å
Adsorption of Serum Albumins
Figure 2. Variation of surface excess versus bulk concentration for the adsorption of BSA (2) and HSA (b) on the surface of null reflecting water. Plots of surface excess versus log(concentration) are also included as inserts in the diagram to highlight the variation over the low concentration range. The solid lines are drawn for a guide.
Figure 3. Variation of total layer thickness versus bulk concentration for the adsorption of BSA (2) and HSA (b) on the surface of null reflecting water. Plots of layer thickness versus log(concentration) are also included as inserts in the diagram to highlight the variation over the low concentration range. The solid lines are drawn for a guide.
with the area per molecule of 12 000 Å2. The structure of the top layer was hence the same as that for the HSA at the same concentration, as already discussed earlier. There are three possible conformational structures corresponding to the total density distribution profile. First, the loose inner layer is composed of sideways-on BSA molecules with their globular structure being retained. The dimension of both layers is then equivalent to two sideways-on adsorption. Second, the interface is composed of the sideways-on monolayer intermixed with a small fraction of tilted conformation. Third, the majority of the BSA
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molecules retain their globular structure, but a small fraction of them has been denatured. The neutron measurement itself cannot distinguish between these different models, but in light of the high degree of deformation at 5 × 10-2 g dm-3 either of the first two models is more likely. It can be seen from Figure 2 that the surface excess for BSA is systematically higher than that of HSA, showing that BSA is more surface active than HSA over this concentration region. The relative surface activity of different albumin proteins has long been of interest, and several studies have been made to examine the adsorption of albumins at the air/water interface. It has however been difficult to compare the exact surface excesses as the differences are relatively small and conventional techniques such as surface tension measurements, ellipsometry, and radiolabeling are not sufficiently accurate to yield reliable values for comparison. For example, Graham et al.20 have used both ellipsometry and 125I radiolabeling to determine the surface excess and layer thickness of BSA. Over the concentration region comparable to our work, their surface excesses are greater than our values by a factor of 2 and their layer thicknesses also well exceed our neutron data at least by a factor of 2-3. The discrepancy in the thickness must be caused by the insensitivity of ellipsometry to the protein layer distribution on the surface of water. The heavy mixing of protein layer with water resulted in a total refractive index close to that of pure water. It is of relevance to mention that using the optical technique Kurrant et al.1 have recently examined the adsorption of BSA and HSA at the hydrophilic optical guide/water interface. The optical guide was made of a mixture of silicon oxide and titanium oxide. Kurrant et al. found that at pH 7 the surface excess for BSA was 1.2 mg m-2 and that for HSA was 2.2 mg m-2, showing that HSA was clearly more surface active than BSA. This trend is somewhat opposite to what was observed in this work at the air/water interface. LiebmannVinson et al.2 have studied HSA adsorption at the hydrophilic silicon oxide/water interface using neutron reflection and found that the amount of HSA adsorbed at 0.05% and at pH 7 was negligible. In a similar experiment Su et al.3 have studied the adsorption of BSA at the silicon oxide/water interface under almost the same solution condition as that of Liebmann-Vinson et al. and found that the BSA surface excess was about 0.8 ( 0.2 mg m-2. These results thus show that at the silicon oxide/water interface BSA is more surface active than HSA, a trend consistent with that observed at the air/water interface. It would be of interest to compare the surface excess of albumins at the air-water interface with that at the hydrophobic solid/water interface, but such results are not available. We have discussed elsewhere26 that the thicknesses of layers measured at the air/water interface always contain a contribution from the thermal motion of the surface (capillary waves). The relationship between the total measured thickness τ and capillary roughness w is given approximately by14,26
τ2 ) σ2 + w2
(4)
where σ is the intrinsic thickness of the layer in the absence of capillary waves. According to Pershan et al.27 the roughness from the thermal motion on the surface of a (26) Lu, J. R.; Simister, E. A.; Thomas, R. K.; Penfold, J. J. Phys. Condens. Matter 1994, 6, A403. (27) Schwartz, D. K.; Schlossman, M. L.; Kawmoto, G. H.; Kellogg, G. J.; Pershan, P. S.; Ocko, B. M. Phys. Rev. A 1990, 41, 5687.
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Figure 4. Plots of log(reflectivity) against κ for the adsorption of HSA on the surface of null reflecting water at the pH values of 3 (+), 5 (O), and 7 (4). The bulk concentration of HSA was fixed at 5 × 10-3 g dm-3. The continuous lines were calculated assuming uniform layer distributions with the surface excesses given in Figure 5a and layer thicknesses given in Figure 5b.
liquid is inversely proportional to the square root of the surface tension. At 25 °C, the surface tension of pure water is about 72 × 10-3 N m-1 and the surface roughness is 2.8 Å. This value is equivalent to a value of w of 7 Å in our model, as there is a factor of 2.3 in difference arising from the different definition of the roughness.26 Since the surface tension of the two albumins at 1 g dm-3 is about 53 mN m-1,22 the thermal roughness of the solution at this concentration is estimated to be 9 Å. Taking the thickness of HSA at 1 g dm-3 to be 48 Å gives an intrinsic thickness of 47.5 Å. As this correction is well within the experimental error, it is clear that thermal roughness does not make any measurable contribution to the thickness of protein layers. This situation is rather different to what was observed in the surfactant monolayers where roughness is an important structural feature.14,26 (B) The Effect of Solution pH. A change in pH will affect the distribution of charges within protein molecules. This in return affects the electrostatic interaction within an adsorbed layer and the degree of hydration of protein molecules at the interface. Hence it is of interest to determine how the structure of protein layers is affected by pH. Furthermore, we have mentioned previously that the two albumins have a difference in amino acid sequence by 25%. It is worthwhile to examine if such a difference affects their response to solution pH variation. The effect of pH on protein adsorption has been examined at two fixed protein concentrations: 5 × 10-3 g dm-3 and 1 g dm-3. Figure 4 shows the reflectivity profiles measured in NRW for HSA adsorption at pH 3, 5, and 7 and at 5 × 10-3 g dm-3. The two reflectivity profiles at pH 3 and 7 are close, suggesting a similar structural profile for the adsorbed layers under these conditions. The reflectivity profile at pH 5 is higher but otherwise parallel in shape to the other two at pH 3 and 7, and this suggests that despite the increase in surface excess the layer thickness does not change. The subsequent fitting of these reflectivity profiles to a uniform layer model gave a good consistency between the measured reflectivities and the calculated ones (continuous lines in Figure 4), showing that the actual
Figure 5. Variation of surface excess (a) and total layer thickness (b) with solution pH at the BSA (2) and HSA (b) concentration of 5 × 10-3 g dm-3. The solid lines are drawn for a guide.
distributions under different pH conditions are well described by the uniform layer model. Figure 5 summarizes the fitted structural parameters, with the variation of surface excess versus pH shown in Figure 5a and that of the layer thickness versus pH in Figure 5b. For comparison, the corresponding values for BSA in terms of surface excess and layer thickness are also shown in Figure 5. It can be seen that for both proteins, while the surface excesses show clear maximum at pH 5, the thicknesses are virtually constant at 32 ( 3 Å. We have further examined the effect of pH on adsorption by measuring the neutron reflectivity at the high protein concentration of 1 g dm-3, and Figure 6 shows the corresponding reflectivity profiles. The main feature here is that with the increase of bulk concentration, both the level and the shape of the reflectivity profiles vary, suggesting that at the high protein concentration, a change in pH affects both the adsorbed amount and the structure of the adsorbed layer. Figure 7 summarizes the variation of surface excesses (Figure 7a) and the variation of
Adsorption of Serum Albumins
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Figure 6. Plots of log(reflectivity) against κ for the adsorption of HSA on the surface of null reflecting water at the pH values of 3 (+), 5 (O) and 7 (4). The bulk concentration of HSA was fixed at 1 g dm-3. The continuous lines were calculated assuming uniform layer distributions with the surface excesses given in Figure 7a and layer thicknesses given in Figure 7b.
thicknesses (Figure 7b) for the two proteins. At the higher protein concentration both surface excess and layer thickness show a clear maximum at pH 5. In comparison with the results shown in Figure 5, the increase in bulk concentration also leads to an increase in both the adsorbed amount and layer thickness. In addition, as the bulk concentration increases, the difference in the amount of adsorption between the two proteins becomes more obvious. BSA shows a consistently greater surface excess than HSA and a much thicker layer at pH 5, further supporting the view that BSA is more surface active. The variation of surface excess with pH correlates well with the change in the extent of electrostatic interaction within the adsorbed albumin layers. At the IP the electrostatic repulsion should be at a minimum since the net charge within the protein is zero. The adsorption peaks at this pH for both albumins. Lateral repulsion starts to increase as the pH is varied away from the IP, and the surface excess for the two proteins decreases accordingly. An increase in the number of net charges within the proteins will also increase their hydrophilicity and reduce their tendency to adsorb onto the surface of water. Bendedouch et al.8 have shwon that albumin molecules tend to elongate when solution pH is lowered. At the higher protein concentration studied, the surface layer is well packed and the extent of elongation of albumin molecules with solution pH may also contribute to the increased steric and electrostatic repulsion within the adsorbed layer, leading to the reduction in surface excess as pH is shifted away from the IP. We have previously studied the adsorption of BSA at the hydrophilic silicon oxide/water interface using neutron reflection and found that the BSA surface excess also attained its maximum value at the protein IP.3 The study was made between pH 3 and 7 over which the oxide surface was weakly negatively charged. The major difference at the air/water interface is that the surface itself does not have an intrinsic charge which otherwise also varies with pH. There is no electrostatic interaction comparable with that between protein and solid substrate, although
Figure 7. Variation of surface excess (a) and total layer thickness (b) with solution pH at the BSA (2) and HSA (b) concentration of 1 g dm-3. The solid lines are drawn for a guide.
electrostatic forces within the adsorbed layer are still important. The hydrophobic effect, gains in entropy arising from protein dehydration, and change in structural conformation are the main factors contributing to surface adsorption at the air/water interface. Adsorption at both air/water and solid/water interfaces shows a maximum at the IP of the protein, indicating that the level of adsorption is dominated by the lateral electrostatic repulsion within the protein layers and is less influenced by the interaction with the solid substrate. It should also be noted that apart from the values of the surface excess the thicknesses of the layers at the two interfaces are also close; that is, they are of the order of 40 Å at all pH and over the entire concentration range, except for BSA adsorbed at the concentrations above 0.5 g dm-3 at pH 5, showing that the main feature of the adsorption at the two interfaces is the formation of sideways-on monolayers. In many studies of adsorption of globular proteins at the air/water interface, the difference in surface activity is usually attributed to the different degree of flexibility in the globular structure. In the case of albumins, it is not
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so obvious to make such correlation because information about the packing of the globular structure and the distributions of amino acid groups on the globular framework is not available. Nevertheless, the composition and amino acid sequence for the two proteins are already known7 and there are a number of differences between them. Some of these differences may be responsible for the variation in surface excess. On the level of the composition of amino acids, one of the obvious differences between the two albumins is the number of prolines. BSA has 28 prolines in the peptide chain while HSA has 24. The presence of proline groups tends to disturb the formation of secondary structures as they interrupt the continuity of hydrogen bonding and hence the secondary structures. In addition, BSA has 14 isoleucines while HSA only has 8. It is however difficult to suggest that BSA is more hydrophobic, because such difference in hydrophobicity is almost completely offset by the variation in other amino acid groups between the two albumins. For example, BSA has 46 alanines while HSA has 62. On the level of amino acid sequence, the main difference between the two proteins is within the third loop; of some 77 amino acids in this loop 34 groups do not match. Although it is generally true that the large extent of variation in the amino acid sequence will affect the stability of globular structure, it is difficult to be precise without knowing how in each case the amino acid groups are packed together. On the level of globular conformation, the main difference is the net charge distributions within the three main domains. At pH 7, the net charges within BSA are -10 on the N-terminal domain, -8 on the middle domain, and 0 on the C-terminal domain, while in HSA, the net charges are -9, -8, and +2, respectively. The variation in the charge distribution within albumins can easily lead to the observed level of difference in surface excess over the high concentration range. Change in pH alters the charge distributions within protein molecules and hence their stability and tendency for surface adsorption. The transition of the globular conformation of serum albumin in bulk solution under different pH conditions has been carefully examined by Foster et al.28 Different forms of serum albumin, denoted as extended (E), fast (F), normal (N), basic (B), and aged (A) were observed and the transitions between them were found to occur at pH values of 3, 4, 8, and 10, respectively. The R -helical content was also found to vary over the whole pH range, again suggesting that the globular structural conformation of serum albumin varies with pH. The direct measurement of the effect of pH on the globular structure of BSA and HSA in aqueous solution has been made using various techniques including sedimentation, viscosity measurements, small-angle X-ray scattering (SAXS), and small angle neutron scattering (SANS).8,29-31 Most of these studies have shown that over the pH range between 5 and 7, the proteins are predominantly ellipsoidal and have the approximate globular dimension of 40 × 40 × 140 Å3. Some authors have shown that at the pH around 5 the shape of serum albumin may also be represented by other geometrical forms, for example, flat and right prisms. In the study of X-ray scattering from albumin solution Anderegg et al.29 showed that the X-ray scattering intensity could be fitted assuming that BSA is (28) Foster, J. F. In Albumin Structure, Function and Uses; Rosenoer, V. M., Oratz, M., Rothschild M. A., Eds.; Pergamon: Oxford, 1977. (29) Anderegg, J. W.; Beeman, W. W.; Shulman, S.; Kaesberg, P. J. Am. Chem. Soc. 1955, 77, 2927. (30) Squire, P. G.; Moster, P.; O’Konski, C. T. Biochemistry, 1968, 7, 4261. (31) Olivieri, J. R.; Craievich, A. F. Euro. Biophys. J. 1995, 24, 77.
Lu et al.
a rectangular parallelepiped having a dimension of 82.5 × 27.5 × 63 Å3. The crystallographic results obtained by He et al.9 have suggested that around pH 5.5 the domains and subdomains are arranged to a heart-shaped triangle. The thickness of the triangle is about 30 Å and its side length is about 80 Å. While these results have clearly shown that the solution structures of albumin molecules are strongly affected by pH, the complexity of the shape has posted limitations on the determination of the precise size and shape by existing techniques. Consequently, the commonly accepted ellipsoidal shape and dimension for the albumins under the normal pH condition are possibly an oversimplification. Although the uncertainty about the precise size and shape of the albumin molecules casts limitations on the estimation of the exact extent of the structural deformation after adsorption, the thicknesses obtained under most of the pH and concentration range are close to the short axial length of the globular framework, suggesting that the molecules are predominantly adsorbed sideways-on under all these conditions. Since the three main domains are joined together through physical interactions it is quite reasonable to expect some deformation as a result of the reorganization of the structure within the globular framework. While the true shape of the globular structure is indeed complex, it is useful to ask what the shortest axial length of the deformed globular structure is. Some estimate of this can be obtained from the measurements in the dilute concentration range. At the concentration below 5 × 10-2 g dm-3, the thickness is always constant at 32 ( 3 Å over the whole pH range. Because few albumin molecules are adsorbed on the surface of water, the lateral repulsion is relatively weak. The measured thickness of 32 Å may then be taken to correspond to the true short axial length for the globular structure. That thickness does not vary much with pH may suggest that the variation of the globular structure with pH observed in bulk solution is caused by change in shape. The commonly accepted value of 40 Å of the short axial length may hence suffer from the possible errors arising from the implementation of rotational ellipsoidal model. Nevertheless, our value of 32 Å is close to the more recent crystallographic thickness of 30 Å by Carter et al.,32 although care should be taken in making a direct comparison between the solution structure and its crystalline form. The thickness of the layer increases with bulk concentration at the concentration above 5 × 10-2 g dm-3, clearly showing that at this point the structure of the layer is dictated by lateral repulsion. Conclusions Neutron reflection combined with the contrast variation of the solvent allows a direct determination of the structure of the two albumin layers at the air/water interface. The use of null reflecting water throughout the experiment means that all the reflectivity profiles measured were almost entirely from the adsorbed protein layers. These measurements have clearly demonstrated that neutron reflection is much more sensitive than most other existing techniques for the characterization of protein layers on the surface of water. The main finding of this work is that the thicknesses of the two albumin layers were all close to the short axial length of the globular solution structure for the two albumins over almost all pH and concentration range studied, suggesting that the layers must be predominantly (32) He, X. M.; Carter, D. C. Nature 1992, 358, 209.
Adsorption of Serum Albumins
adsorbed sideways-on. The formation of uniform monolayers under most solution conditions also suggests that despite possible structural deformation, adsorption has not caused the breakdown of the globular framework, leading to the formation of nonuniform density profiles characteristic of the distribution of peptide fragments. Although BSA adsorbs slightly more than HSA over the high protein concentration range, the overall level of surface excess and the thickness of the layers are similar, showing that the difference in amino acid sequence between the two albumins has not caused any major difference in their surface layer structure. For both
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albumins, the surface excess shows a maximum at their isoelectric point, similar to that observed at the hydrophilic silicon oxide/water interface. These results suggest that while the globular framework is retained, the pattern of adsorption is dominated by the lateral repulsion within the adsorbed layers. The interaction between the solid substrate and protein is of lesser importance. Acknowledgment. We thank the Biotechnology and Biological Sciences Research Council for support. LA990131H
