Adsorption of Glucose Oxidase at Organic− Aqueous and Air

Langmuir 2003, 19, 4977-4984

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Adsorption of Glucose Oxidase at Organic-Aqueous and Air-Aqueous Interfaces Dimitra G. Georganopoulou* and David E. Williams Department of Chemistry, University College London, 20 Gordon Street, London WC1H 0AJ, U.K.

Carlos M. Pereira and Fernando Silva Departamento de Quı´mica, Faculdade de Cieˆ ncias, Universidade do Porto, P-4169-007 Porto, Portugal

Tsueu-Ju Su and Jian R. Lu Department of Physics, UMIST, P.O. Box 88, Manchester M60 1QD, U.K. Received June 5, 2002. In Final Form: January 6, 2003 The adsorption of glucose oxidase (GOx) was studied at the interface between two immiscible electrolyte solutions (ITIES) by interfacial capacitance and surface tension measurements and at the air/water (phosphate buffer) interface by surface tension and neutron reflection measurements. The adsorption at both interfaces was found to be time, enzyme concentration, and ionic strength dependent. There was a switch from one interfacial adsorption state to another, as the enzyme concentration was increased. At the ITIES, there was evidence of an interaction between the adsorbed enzyme and the hydrophobic cation in the organic phase (1,2-dichloroethane). The enzyme adsorbed at the air/water interface was found to dissociate into monomers at the lower buffer total concentration of 2 mM while, at the higher buffer concentration of 0.2 M, the adsorbed enzyme retained its dimer structure. The adsorption mostly formed monolayers and the layer thickness varied with bulk concentration, indicating deformation related to the packing of the enzyme at the interface. For enzyme concentrations above 1 µM, in high ionic strength medium, bilayers of enzyme started to form, and the interlayer interactions resulted in a less densely packed second layer forming on the aqueous side of the first one. The switch in properties of the adsorbed layer observed in interfacial tension and capacitance measurements at the ITIES occurred over the same enzyme concentration range as the formation of a more densely packed layer detected from neutron reflection at the air/water interface.

Introduction Glucose oxidase (GOx) is an enzyme that has attracted immense interest, due to its applicability in biosensors for the determination of glucose in body fluids, as well as for removing glucose and oxygen from beverages and food products. Because it is easily obtainable and robust, it is convenient to use as an initial model for exploring the reactivity of redox enzymes at interfaces.1 As part of such a study, we report here an exploration of the adsorption of glucose oxidase at both an organic solvent/water interface and the air/water interface, comparing the results of electrochemical adsorption measurements at the organic/ aqueous interface with those of structural studies using neutron reflection at the air/water interface. Interfacial tension measurements at both interfaces are used to provide a link. GOx is a dimeric globular glycoprotein2 of dimensions 60 × 52 × 77 Å3, made up from two identical subunits, each of molecular weight ∼75 kDa, that are bound with * To whom correspondence should be addressed: Currently at University of North Carolina. Phone: 919-9620458; Fax: 9199621381; E-mail: [email protected]. (1) Georganopoulou, D. G.; Caruana, D. J.; Strutwolf, J.; Williams, D. E. Faraday Discuss. 2000, 116, 109. (2) Kriechbaum, M.; Heilmann, H. J.; Wientjes, F. J.; Hahn, M.; Jany, K.-D.; Gassen, H. G.; Sharif, F.; Alaeddinoglu, G. F. FEBS Lett. 1989, 255, 63.

disulfide bridges, salt linkages, and hydrogen bonds.3 GOx has one redox coenzyme, flavin adenine dinucleotide (FAD), per monomer. The FAD is not covalently bound to the protein and can be released under denaturing conditions. The enzyme has a diffusion coefficient of 4.94 × 10-7 cm2 s-1 in 0.1 M NaCl and a considerable part of hydrophobic side chains located near the surface.4 The mean diameter of the native enzyme in solution, according to photon correlation spectroscopy data, is 76 Å at pH 7.4.5 The same group gives the Stokes radius for the molecule to be 43 Å with a frictional ratio of 1.21, from which they conclude that the enzyme in solution is an elongated protein with rigid structure. Hecht et al.3 state that each monomer is a compact spheroid of dimensions 60 × 52 × 37 Å3. The native protein is acidic with an isoelectric point of 4.44. At pH 7 it is negatively charged with 11 charges. Duinhoven et al.6 summarized four categories of interaction between proteins and surfaces: (i) Electrostatic and Van de Waals interactions, which are dependent on the net charge on both protein and (3) Hecht, H. J.; Kalizs, H. M.; Hendle, J.; Schmid, R. D.; Schomburg, D. J. Mol. Biol. 1993, 229, 153. (4) Hecht, H. J.; Schomburg, D.; Kalizs, H. M.; Schmid, R. D. Biosens. Bioelectron. 1993, 8, 197. (5) Baszkin, A.; Boissonnade, M. M.; Rosilio, V.; Kamyshny, A.; Magdassi, S. J. Colloid Interface Sci. 1999, 209, 302. (6) Duinhoven, S.; Poort, R.; Vandervoet, G.; Agterof, W. G. M.; Norde, W.; Lyklema, J. J. Colloid Interface Sci. 1995, 170, 340.

10.1021/la0205248 CCC: $25.00 © 2003 American Chemical Society Published on Web 05/10/2003

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surface and on the local dipole moment determined by the charge distribution across the protein surface. These interactions are also affected by ions in the layer between the protein and the surface and have been considered in detail by Roth and Lenhoff.7 (ii) Dehydration of interfaces on the outside of the protein and on the surface upon adsorption, which results in the release of a large number of water molecules. (iii) Hydrophobic interactions, leading to structural changes in the protein upon adsorption. A hydrophobic surface may promote unfolding of the protein, such that hydrophobic groups in the interior of the protein can interact with the surface. (iv) Surface-coverage dependent lateral interactions between charges and dipoles. Proteins are commonly classified as “soft” or “hard”, on the basis of assumed conformational rigidity. Soft proteins unfold partially upon adsorption, an effect that increases with increasing net charge on the protein-interface complex. Structural alterations are less important for hard proteins, for which dehydration of hydrophobic surfaces and electrostatic interactions are key. Hard proteins typically adsorb on hydrophilic surfaces only under conditions where the electrostatic interactions are favorable, but they adsorb on all hydrophobic surfaces regardless of the charge. Tripp et al.8 have given an extensive literature review of surface tension studies of adsorption of globular proteins at the air/water interface. Soft, easily foamable proteins with hydrophobic surfaces showed the highest adsorption rate. They noted that many proteins lose their biological activity after adsorption at the air/ water interface and concluded that, after an initial stage determined by diffusion from solution, the protein adsorption rate reflects the chemistry at the interface as the protein unfolds. Rather detailed models, which confirm the general ideas presented above, have been obtained recently by neutron reflection measurements. At the hydrophilic silica/water interface, lysozyme (a hard protein) shows adsorption that is reversible with respect to change of pH, suggesting an electrostatically-driven adsorption process which does not necessarily lead to irreversible denaturation.9 On a silica surface rendered hydrophobic by treatment with octadecyltrichlorosilane, lysozyme, in contrast, shows irreversible denaturation.10 The protein formed a densely packed thin layer next to the surface, with a diffuse thicker layer extending into the bulk solution. The authors suggested that the adsorption was driven by a hydrophobic attraction to the surface of hydrophobic fragments in lysozyme and by electrostatic repulsions within the adsorbed layer. They suggested that the protein unravelled to some extent into peptide chains with the hydrophobic amino acid side chains attached to the hydrophobic surface. This behavior may be contrasted with that of the soft protein bovine serum albumin (BSA) at both air/water11 and hydrophilic silica/ water12 interfaces. At both interfaces, BSA on adsorption showed structural deformation but no denaturation. The adsorption was in the form of a sideways-on monolayer over a wide pH and concentration range. At the air/water (7) Roth, C.; Lenhoff, A. M. Langmuir 1995, 11, 3500. (8) Tripp, B. C.; Magda, J. J.; Andrade, J. D. J. Colloid Interface Sci. 1995, 173, 16. (9) Su, T. S.; Lu, J. R.; Thomas, R. K.; Cui, Z. F.; Penfold, J. Langmuir 1998, 14, 438. (10) Lu, J. R.; Su, T. J.; Thirtle, P. N.; Thomas, R. K.; Rennie, A. R.; Cubitt, R. J. Colloid Interface Sci. 1998, 206, 212. (11) Lu, J. R.; Su, T. J.; Thomas, R. K. J. Colloid Interface Sci. 1999, 213, 426. (12) Su, T. J.; Lu, J. R.; Cui, Z. F.; Thomas, R. K.; Penfold, J. J. Phys. Chem. B 1998, 102, 8100.

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interface, adsorption continued beyond a single monolayer. At high protein concentration, the adsorbed material could be modeled as two layers: a layer at the interface of thickness 4 nm, with volume fraction 0.4, and, adsorbed upon that, a second layer of thickness 3 nm with volume fraction 0.12. Increase of ionic strength from 0.02 to 1 M resulted in more compact layers, indicating that the electrolyte screens ionic repulsions within the adsorbed layer. At the silica/water interface, the apparent thickness of the adsorbed protein layer varied from 3 nm at low BSA concentration to 40 nm at high, indicating that structural deformation is dominated by lateral interactions within the adsorbed layer. The liquid/liquid interface is interesting because, while it is not rigid, it can carry a charge. Hence, a strong electrostatic interaction of protein with the surface may be present, but the surface is deformable. Gajraj and Ofoli13 demonstrated that, with increasing protein concentration, BSA showed a strong and continuing adsorption beyond interfacial saturation at an oil/water interface, until a critical micelle concentration was reached. Conversely lysozyme showed a simple adsorption isotherm approximating Langmuir-type behavior. In preliminary measurements illustrating the applicability of the ac impedance method to study protein adsorption at a polarized organic (nitrobenzene)/aqueous interface, Vanysek et al.14,15 also showed continuing adsorption of BSA beyond initial saturation of the interface, the rather complex variation of interfacial capacitance with concentration implying some reorganization of the interfacial layer with increasing protein concentration. Adsorption of GOx at the air/water interface, through measurement of interfacial tension, has been thoroughly studied by Rosilio et al.16 and Sun et al.17 Rosilio et al., using the crystal structure dimensions, estimated an area for the dimer of 3900 ( 800 Å2/molecule.17 They showed a concentration and time dependent adsorption of GOx if the concentration in the aqueous phase was greater than 4 µg cm-3 (0.026 µM). No adsorption at all was detected at lower concentration. The data were consistent with adsorption initially limited by diffusion toward the interface from the interior of the aqueous phase, followed by reorganization at the interface accompanied by further adsorption when the surface coverage increased above a critical level. The aqueous phase apparently contained no salts in this case. If a lipid was first spread on the interface, then injection of GOx below the interface resulted in an increase of surface pressure at constant area. Even 1 µg cm-3 (0.006 µΜ) GOx caused a significant effect. The effect was most noticeable at low surface pressures. Penetration of the enzyme into the lipid layer, favored by interaction of the lipid chains with hydrophobic groups on the exterior of the protein, was inferred. The enzyme was excluded from the interfacial layer at high surface pressure. A dependence on the orientation of lipid molecules in the interface was deduced. Zhang et al.18 extended this work, demonstrating an effect of lipid chain length and of the presence of double bonds in the lipid chain. Sun et al.17 showed that the observed surface area per enzyme molecule at the air/water interface varied from 700 to 2200 Å2, depending on the conditions of spreading (13) Gajraj, A.; Ofoli, R. Y. Langmuir 2000, 16, 4279. (14) Vanysek, P.; Reid, J. D.; Craven, M. A.; Buck, R. P. J. Electrochem. Soc. 1984, 131, 1788. (15) Vanysek, P.; Sun, Z. Bioelectrochem. Bioenerg. 1990, 23, 177. (16) Rosilio, V.; Boissonnade, M. M.; Zhang, J.; Jiang, L.; Bazskin, A. Langmuir 1997, 13, 4669. (17) Sun, S.; Ho-Si, P. H.; Harrison, D. J. Langmuir 1991, 7, 727 (18) Zhang, J.; Rosilio, V.; Goldmann, M.; Boissonnade, M. M.; Bazskin, A. Langmuir 2000, 16, 1226.

Adsorption of Glucose Oxidase at Interfaces

Langmuir, Vol. 19, No. 12, 2003 4979 Chart 1

the enzyme on the subphase. In their work they studied films of GOx spread at the air/water interface and transferred to Si or Pt substrates by the LangmuirBlodgett (LB) method. The subphase had a pH ) 6 and consisted of 5 mM BaCl2. Native enzyme gave LB films with very low activity and apparent thickness 30 Å/layer. Given the enzyme dimensions, the implication is that the protein had largely dissociated into monomers at the interface and that these were adsorbed with the shortest axis parallel to the interface. If, however, the enzyme was treated with glutaraldehyde to cross-link together the monomers before spreading on the interface, then the LB films prepared retained a high activity for the oxidation of glucose, which increased linearly with number of layers deposited. The apparent thickness in this case was 48 Å/layer. The implication was that cross-linking with glutaraldehyde prevented dissociation of the enzyme upon adsorption. Rinuy et al.19 used the method of second-harmonic generation to study GOx adsorption at the air/water interface, using aqueous solutions of high ionic strength (I ) 0.1 M at pH ) 7). The signal associated with GOx adsorbed at the air/water interface exhibited a shoulder at 0.10 µM enzyme concentration and a maximum at 0.40 µM, subsequently decreasing for higher enzyme concentrations. They commented that the behavior could reflect the effect of protein-protein interactions, involving rearrangement and reorientation of the enzyme molecules at the interface. At a solid/water interface, in LB films also containing behenic acid, Fiol et al.20 concluded that GOx was a long ovoid with dimensions of about 60 Å × 130 Å and therefore apparently undissociated. Szucs et al.21 showed that the adsorption of GOx on a gold electrode was time and concentration dependent, with the electrode potential having a significant effect on the time dependent behavior of the adsorption. Three different stages of adsorption were distinguished, with one being the adsorption normal to the long axis, (140 Å, “standing”) the second along the short axis (50 Å, “lying”), and the third a thin film, consisting of denatured enzyme, with FAD exposed. The authors attributed these effects to electrostatic forces and molecular interactions and suggested that the transition from one stage to the other is dependent on the surface coverage. The picture that emerges is that GOx behaves to some degree like BSA but that it may dissociate at the interface, that multilayers may form, and that the behavior may be (19) Rinuy, J.; Brevet, P. F.; Girault, H. H. Biophys. J. 1999, 77, 3350. (20) Fiol, C.; Valleton, J. M.; Delphire, N.; Barbey, D.; Barraud, A.; Ruaudel-Teixier, A. Thin Solid Films 1992, 215, 88. (21) Szucs, A.; Hitchens, G. D.; Bockris, J. O. J. Electrochem. Soc. 1989, 136, 3748.

dependent upon the surface charge and electrolyte composition. The present work compares the behavior of GOx at the polarized interface between two immiscible electrolyte solutions (ITIES) and at the air/water interface, using interfacial capacitance measurement at the ITIES, neutron reflection at the air/water interface, and interfacial tension at both. At the liquid/liquid interface, we find that the protein desorbs at potentials positive of the potential of zero charge. At both interfaces, charge screening by the electrolyte is important and leads to an accumulation of protein at the interface. Depending on the electrolyte and enzyme concentration, the adsorbed protein may reorganize and multilayers can form. The dimeric protein can also dissociate to monomers at the interface but does not denature. Experimental Section The organic solvent 1,2-dichloroethane (DCE) (99.9% Sigma, ACS reagent), lithium chloride, (LiCl) (Aldrich, 99%), dipotassium hydrogen phosphate (K2HPO4), and potassium dihydrogen phosphate trihydrate (KH2PO4‚3H2O) (Fluka reagent grade) were used as received. Millipore Q-plus water (18 MΩ cm) was used for the preparation of the aqueous solutions and for washing; D2O (99.9%, D) was obtained from Fluorochem. The pH of the aqueous phase was adjusted to 7 with the phosphate buffer. The organic electrolytes, bis(triphenyl)phosphoranylidene tetraphenylborate, BTPPATPB, and tetrabutylammonium tetraphenylborate, TBuATPB, were prepared by precipitation from aqueous solutions of sodium tetraphenylborate (NaTPB) (Fluka, Chemica) with bis(triphenyl)phosphoranylidene chloride, BTPPACl (Fluka, Chemica), and tetrabutylammonium chloride, TBuACl (Fluka, Chemica), respectively. Glucose oxidase (ex Aspergillus Niger) was purchased from Fluka and was stored at -20 °C. All glassware and Teflon troughs were thoroughly washed with an alkaline detergent, “Decon-90”, followed by thorough rinsing with Millipore Q-plus water and acetone. The enzyme was fully dissolved in the aqueous phase that formed a sharp liquid/liquid interface on top of the organic layer. The enzyme was then allowed to diffuse and adsorb at the interface for different periods of time, as described in each set of experiments in the discussion. All experiments were carried out at room temperature (25 °C). In the current study, two different ITIES cells were used (See Chart 1), and two different air/aqueous interfaces (See Chart 2). The reference electrodes used for the four electrode capacitance measurements were Ag/AgCl, and the counter electrodes were Pt wires (d ) 0.5 mm). The cyclic voltammetry and impedance measurements were carried out using two different instruments: a Solartron 1250 frequency response analyzer with potential control by a Solartron 1287 potentiostat (Solartron, U.K.) and the FRA (frequency response analyzer) module of an Autolab PGStat100 (Ecochemie, The Netherlands). A small amplitude (10 mV) sinusoidal voltage was superimposed on each constant dc-potential, with the range of frequencies used (f ) 0.1-10 Hz) chosen in order to avoid highfrequency artifacts.22 The surface tension measurements were (22) Samec, Z.; Langmaier, J.; Trojanec, A. J. Chem. Soc., Faraday Trans. 1996, 92, 3843.

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Georganopoulou et al. Chart 2

carried out with a Kruss K10 digital tensiometer, and the Pt ring used (d ) 0.5 and 20.5 mm in diameter) was flamed before each set of measurements in an oxidative flame and rinsed in H2O. The neutron reflection measurements were made on the white beam reflectometer SURF at the Rutherford-Appleton Laboratory, ISIS, Didcot, U.K., using neutron wavelengths from 1 to 6 Å. The sample cell described elsewhere13 contained the aqueous solution in a Teflon trough of approximate dimensions 15 × 6 × 3 cm3. The collimated beam entered the water phase from one end of the trough at a fixed angle, was reflected at a glancing angle from the air/water interface, and exited from the opposite end of the trough. Each reflectivity profile was measured at three different glancing angles, 0.35°, 0.8°, and 1.5°, thus covering the full range of momentum transfer with overlapping data sets. The beam intensity was calibrated with respect to the intensity below the critical angle for total reflection at the air/D2O interface. A flat background determined by extrapolation to high values of momentum transfer (Q ) (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 Q > 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 that for H2O, to be 3.5 × 10-6 (measured in terms of the reflectivity).

Theory For the surface tension measurements, the data prior to a break of the surface tension γ versus ln(c) can be fitted to a quadratic equation from which the surface excess Γ (mol m-2) can be calculated using the Gibbs equation:

Γ)-

1 dγ RT d ln(c)

(1)

The area per molecule A of the adsorbed species can then be calculated by

A)

1 NAΓ

(2)

with NA the Avogadro number. The specular reflectivity R(Q) is determined by the variation in scattering length density F(z) along the surface normal direction, z:

R(Q) )

16π2 |Fˆ (Q)|2 2 Q

(3)

with Fˆ (Q) the one-dimensional Fourier transform of F(z):

Fˆ (Q) )

∫-∞∞ exp(-Qz)F(z) dz

(4)

The scattering length density F(z) is related to chemical composition through F(z) ) ∑nibi, where ni is the number density of element i and bi is the scattering length

(amplitude). Since different isotopes (e.g. H, D) have different bi values, the use of isotopic substitution may result in a variety of neutron reflection profiles for a given layer, resulting in a technique called “contrast variation”. The most useful combination is often achieved by exchanging deuterium (bD ) 6.671 fm) for hydrogen (bH ) -3.739 fm), as they have opposite signs. For the reflection at the air/water interface, the contribution to the reflection from bulk solvent can be removed by using null reflecting water (NRW), which contains 8.1% (v/v) D2O, with a resulting zero scattering length density. The profiles obtained are usually analyzed by means of the optical matrix formalism on the basis of the following procedure. An assumption for a structural mode is made for the adsorbed layer, followed by calculation of the reflectivity, on the basis of the optical matrix formula. The calculated reflectivity is then compared with the measured data, and the structural parameters (number of layers, thickness τ, and density F of each layer) are varied in a least-squares iteration until a best fit is found. Assuming a model of a uniformly distributed protein layer, the area per molecule (A) of the adsorbed protein can be calculated directly from the derived scattering length density F and the thickness of the layer τ, using

A)

∑mibi Fτ

(5)

with mi the number of ith atoms and ∑mibi the total scattering length of the protein. The surface excess, Γ, can be estimated then using eq 2. The MW (∼75 kD per monomer) and total scattering length (∼0.16 Å per monomer in null reflecting water) of GOx were obtained on the basis of the amino acid sequence and crystal structure available. The area per molecule and surface excess are then obtained from the layer thickness, τ, and scattering length density, F, obtained from the fit of simulated reflectivity profiles to the experimental data. Although the equations presented here apply under the condition of a uniform layer distribution, yet they are directly applicable to each of the sublayers that may be present in the model. The total adsorbed amount is obtained by summing over the sublayers used in the fitting procedure. Results Impedance Measurements. Cyclic voltammetry demonstrated that addition of 1 µM GOx in the aqueous phase did not cause any shift of the edges of the potential window in both cells 1 and 2. There was no sign of visible precipitation at the interface, such as might have followed from gross denaturation of the enzyme. It is inferred that the enzyme did not induce or participate in any ion transfer

Adsorption of Glucose Oxidase at Interfaces

Figure 1. (a) Complex plane impedance plot of -Z′′ vs Z′ for f ) 10-0.1 Hz, cell 2, E ) 360 mV, 0.40 µM enzyme. Experimental values are represented as points, and the solid line is the fit using the equivalent circuit shown in the inset, with Rs the solution resistance, CD the double-layer capacitance, WCT the Warburg diffusion impedance, and RCT the chargetransfer resistance. (b) Capacitance, CD, derived from fitting the equivalent circuit as a function of potential difference between aqueous and organic reference electrodes, Ew - Eo, and various GOx concentrations, using cell 2 (see text). From top to bottom at less positive potentials: 0, 0.05, 0.10, 0.15, 0.20, 0.30, 0.40, 0.50 µM GOx. The lines are polynomial fits.

of the supporting electrolytes. Impedance diagrams in the presence of different GOx concentrations were obtained at different constant dc potentials, within the potential window established by cyclic voltammetry. Figure 1a shows a typical complex plane impedance plot. All the impedance data obtained at each potential, for all the GOx concentrations and for both cells, were fitted well by the equivalent circuit shown in the inset of Figure 1a (fitting errors were less than 2%). There was no need to include an adsorption impedance element in the equivalent circuit. The potential range E ) 0.25-0.45 mV was scanned with both increasing and decreasing potential, with 50 mV steps, to obtain data at 25 mV intervals across the entire range, after equilibration of 1 h, with the experiments lasting up to 4 h. The resulting impedance responses were found to be essentially time-invariant, especially for high enzyme concentrations. However, further investigation of interfacial capacitance variation with time showed that, while for high enzyme concentrations the double-layer capacitance reached a steady value within 3 h, for the lower concentrations small changes continued over a period of at least 10 h. The effect of these small changes was not discernible on the impedance diagrams. Values for the double-layer capacitance obtained from fitting the data to the equivalent circuit are plotted as a function of interfacial potential difference and bulk enzyme

Langmuir, Vol. 19, No. 12, 2003 4981

Figure 2. (a) Effect of bulk enzyme concentration on the interfacial capacitance at the the pzc with cell 2. (b) Effect of bulk enzyme concentration on the interfacial capacitance at the pzc with cell 1.

concentration in Figure 1b. With increasing enzyme concentration, the minimum of the curves, assumed to be the potential of zero charge (pzc) on the interface, was displaced to increasingly more negative potentials. For every enzyme concentration studied, the double-layer capacitance, CD, was lowered for potentials negative of the pzc in the base electrolyte, the effect increasing with increasing enzyme concentration. For potentials positive of the pzc, for all enzyme concentrations including zero, the capacitance was either coincident with or slightly higher than the capacitance curve in the absence of the enzyme. Figure 1b does not include the responses obtained for high enzyme concentrations (c > 0.6 µM), as these could not be followed reproducibly, showing occasional shifts of the pzc to more positive potentials and capacitance values higher than those observed for lower enzyme concentrations. Figure 2 shows the capacitance values at the pzc plotted against enzyme concentration for the two different cells. With neither cell is there a monotonic decrease of capacitance with increasing enzyme concentration, such as would be obtained with a simple adsorption isotherm. Instead, it appears that the adsorption of the enzyme in both cells is governed by at least two different dynamic processes and that the system switches from one adsorbed state to another with increasing concentration. The effect was much more marked for the lower concentration of organic electrolyte, cell 1. This cell also had a more bulky, slightly more hydrophobic organic cation. Surface Tension Measurements. Figure 3 shows the variation of surface tension γ with concentration c for the liquid/liquid interface of cell 1. No significant changes for each GOx bulk concentration measurement were observed after 2 h of establishing the interface. The surface tension decreased almost 15 mN m-1 within the range of enzyme concentrations studied. Again, no monotonic decrease of surface tension was observed with increasing enzyme

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Figure 3. Interfacial tension, γ, vs bulk enzyme concentration, c, after 4 h of adsorption of GOx at the organic/aqueous interface using cell 1. Figure 5. Experimental reflectivity profiles for increasing enzyme concentration in NRW with 0.002 M, pH ) 7 phosphate buffer. From bottom to top, bulk enzyme concentrations: 0.01 (O), 0.10 (+), 0.40 (4), and 1 µM (×) of GOx. The solid lines represent fits using the uniform layer model (see text). The inset shows the increase in reflection for 0.10 µM and θ0 ) 1.5°, after 1 h (O) and 12 h (+).

Figure 4. Interfacial tension, γ, vs bulk enzyme concentration, c, after 14 h of adsorption of GOx at the air/water interface, at low ionic strength (open symbols) and high ionic strength (filled symbols).

concentration. Instead a switch from one adsorbed state to another with increase of the concentration of GOx is again suggested. As with the impedance measurements, the surface tension at high enzyme concentrations, c > 1 µM, was not reproducible. For the air/water interface, the approach to the final state of adsorption was significantly slower than that at the ITIES. For both cells 3 and 4, after 2 h of adsorption, the interfacial tension had barely changed from the enzyme-free value, throughout the bulk enzyme concentration range studied. Figure 4 shows surface tension measurements with bulk GOx concentration after 14 h of adsorption, after which time the overall change in interfacial tension was similar in magnitude to that observed at the ITIES after 2 h. These results are consistent with those reported by Rosilio et al.17 As with the behavior shown at the ITIES, the system could be represented as switching from one state to another with increase of enzyme concentration, with the effect being somewhat dependent on the ionic strength of the aqueous phase. Neutron Reflection Measurements. Figure 5 shows the variation of neutron reflectivity log(R) versus momentum transfer Q, for selected enzyme concentrations at the air/NRW (null reflecting water) interface, in the presence of a low concentration of phosphate buffer (cell 3, 0.002 M total buffer), and Figure 6 shows the variation for the same concentrations in the presence of a high concentration of phosphate buffer (cell 4, 0.2 M buffer). The specular reflectivity obtained due to contrast with NRW was attributed solely to the adsorbed layer. At values of Q above 0.1 Å-1, all the curves fell to the level of the background. The magnitude of the reflectivity at a given Q is an indication of the surface coverage, and the slope, d(log R)/dQ, is related to the thickness of the adsorbed

Figure 6. Experimental reflectivity profiles for increasing enzyme concentration in NRW, for 0.20 M phosphate buffer. Bulk enzyme concentrations from bottom to top: 0.01 (O), 0.10 (+), 1 (4), and 6 (×) µΜ. Solid lines represent fits for a uniform layer model for c < 0.40 µΜ and a two-layer model for c > 1 µΜ.

layer. The adsorption was found in both cases to be time dependent, with typical time scales needed to reach equilibrium being for high enzyme concentrations ∼6 h and for low enzyme concentrations ∼14 h. The period needed for the system to reach pseudoequilibrium was based on the variation of the reflectivity at θ ) 1.5° with time, as shown in the inset of Figure 5. Figure 5 shows that in the presence of a low ionic strength aqueous phase (0.002 M buffer) there was a steady increase of the magnitude of reflectivity with increasing enzyme concentration, while the slopes of the curves remained essentially the same. This suggests a simple behavior in which, with increasing enzyme concentration, the surface coverage increased while the thickness of the adsorbed layer remained essentially unchanged. Figure 6 shows that with the higher ionic strength aqueous solution (0.2 M buffer) the behavior was very different. First, increase of enzyme concentration through the lower range studied (0.01 to 0.40 µM) caused a significant increase in d(log R)/dQ, with increasing concentration, implying an increase in the thickness of the interfacial layer as well as its surface coverage. Second, further increase in enzyme concentration beyond this range resulted in a lower magnitude of reflectivity and a

Adsorption of Glucose Oxidase at Interfaces

change of slope. Thus, without attempting any model fitting, we can deduce the existence of different adsorbed states of different thickness and layer structure, dependent on the enzyme concentration and the ionic strength of the aqueous solution. Discussion The impedance results showed that the enzyme was strongly adsorbed at the ITIES. The shift of pzc observed in Figure 1b is attributed to increased adsorption of GOx as the organic phase becomes more positive with respect to the aqueous phase. When the potential of the aqueous phase relative to the organic phase was negative of the pzc, then the capacitance was decreased in the presence of the enzyme, indicating that the enzyme was behaving as a simple organic dielectric layer, blocking the interface. The variation of interfacial capacitance with time and with enzyme concentration demonstrated that with increasing surface coverage of enzyme the adsorption state of the enzyme changed. This switch in state was slow. The high enzyme concentration state had the higher capacitance. Furthermore, the impedance results showed that the nature and concentration of the supporting organic electrolytes affected the adsorption of the enzyme at the ITIES, in that the presence of a slightly less bulky organic cation (TBuA rather than BTPPA), in higher concentration (10 mM rather than 1 mM), caused the switch of adsorption state with increasing enzyme concentration to be much less pronounced. One speculative interpretation is that the hydrophobic parts of the enzyme surface might penetrate somewhat into the organic phase. Then there could be some interaction between the enzyme and the organic ions. Incorporation of the organic cation into the adsorbed layer of negatively charged enzyme might reduce the conformational reorientations of the adsorbed enzyme. There is some analogy with the work of Rosilio et al.,17 where adsorption of GOx at the air/ water interface was promoted by the coadsorption of lipid. When the potential of the aqueous phase was positive of the pzc, the capacitance was somewhat increased in the presence of the enzyme. Kakiuchi et al23 note that a common point of intersection of Cdl versus E curves corresponds to a point of inflection in the dependence of adsorbed amount on potential, for a system at equilibrium. It is not clear whether our data indeed show a common intersection point, and the time variation of capacitance as the adsorbed state of the enzyme changes is a confusing factor. The measured capacitance is C ) ∂q/∂(Ew - Eo), where q denotes the charge stored at the interface. Any phenomenon which increased the dependence of charge storage on interfacial potential difference would cause an increased capacitance. An interpretation of our data is simply that the adsorbed enzyme, with increasing coverage, switches from one adsorbed state to a different one and that these two states have a different dependence of charge storage on interfacial potential difference. One element of the charge stored in the interfacial enzyme layer would be that associated with ions from both the aqueous phase and the organic phase which were coadsorbed with the enzyme. Potential dependent coadsorption of ions might be the reason for the increased capacitance of the high enzyme concentration state, shown in Figure 2. The breaks observed in the surface tension measurements at the ITIES (Figure 3) can also be ascribed to the presence of the two different adsorption states. A time and concentration dependence was demonstrated by surface tension measurement, as well as a dependence on (23) Kakiuchi, T.; Kondon, T.; Kotani, M.; Senda, M. Langmuir 1992, 8, 169.

Langmuir, Vol. 19, No. 12, 2003 4983 Table 1. Structural Parameters for the GOx Layer Adsorbed on the Surface of Water with Total Phosphate Buffer Concentration 0.002 Ma c/µM

τ ( 3/Å

A ( 500/Å2

Γ ( 0.30/(mg dm-3)

0.01 0.1 1

25 28 37

9500 7700 5050

1.5 1.6 2.4

a c ) GOx concentration; τ ) adsorbed layer thickness; A ) area/ molecule; Γ ) surface excess of GOx.

the ionic strength of the aqueous phase. A qualitatively similar behavior was observed for the air/water interface in Figure 4, which suggests that the dominant effects are at the aqueous side of the interface. However, the interfacial reorganization processes are slower at the air/ water interface than at the ITIES. Differences are attributed to, first, the presence of the organic cations at the ITIES and, second, the control of interfacial potential at the ITIES. Assuming that the interfaces saturate at the second break of each γ versus ln(c) curve (∼0.40 µM enzyme), the surface excess obtained from the slope prior to the break can be very approximately estimated using eq 1, to give a value for the area/molecule of 2500-3000 Å2 for both interfaces. The values for the area per molecule correlate well with values given by Sun et al.18 (700-2200 Å2). The expected area per molecule from the crystal structure dimensions varies from 2450 to 3620 Å2 depending on the axis normal to the surface. A structural picture of the adsorption at the air/water interface which is consistent with the results of interfacial tension measurements and which confirms the models reviewed in the Introduction was obtained by neutron reflection. The important result was the difference in adsorption found with the different ionic strengths of the aqueous solution. For high ionic strength there was an increased enzyme adsorption, which could reasonably be attributed to reduced lateral electrostatic repulsion because the coadsorbed ions would screen the charges of the adsorbed charged molecules. The clearly observed effect is that the adsorbed area/molecule decreases. It is suggested that the aqueous electrolyte ions affect the structure of the interface, where the enzyme adsorbs: the size and charge of the electrolyte ions could affect the way in which these ions coadsorb with the enzyme at the interface, thereby altering the lateral interactions between different enzyme molecules. In the case of low ionic strength (0.002 M buffer), the profiles for the whole range of enzyme concentration fitted a simple single uniform adsorbed layer model. The structural parameters from Figure 5 are shown in Table 1. As the surface excess increased with concentration, the layer thickness slightly increased but in all cases remained below 37 Å. The physical state of the adsorbed enzyme can be deduced by comparing the layer structure with the enzyme dimensions from the crystal structure (elongated dimer, 60 × 52 × 77 Å3; each monomer being a compact spheroid, 60 × 52 × 37 Å3). The layer thickness indicates the possible adsorption of monomers, and the uniformity of the layers may suggest that despite the dissociation of the dimers into monomers there was no further breakage into polypeptides. The relatively large limiting area/ molecule (∼5000 Å2) compared with the value expected from the crystal structure (∼3000 Å2) may reflect the effect of strong electrostatic repulsion within the layer under such low ionic strength conditions. The results given in Table 2 show no evidence, therefore, for any significant denaturation of the enzyme at the air/water interface. This finding is broadly consistent with the suggestion of the dissociation deduced by Sun et al.18 The neutron data, however, have provided solid evidence for the first time to support this hypothesis.

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Georganopoulou et al.

Table 2. Structural Parameters for the GOx Layers Adsorbed on the Surface of Water with Total Phosphate Buffer Concentration 0.20 Ma c/µM

τ1 ( 3/Å

A1/Å2

0.01 0.1 1 2 6

41 50 48 48 50

9500 4700 4500 4500 4450

τ2 ( 3/Å

50 50 45

A2/Å2

Γ ( 0.20/(mg dm-3)

33 000 32 000 20 500

1.35 2.7 3.1 3.1 3.3

a Layer 1 is adjacent to the interface; layer 2 is on the aqueous side of layer 1.

For the case of the higher concentration of phosphate buffer (0.2 M), Figure 6 suggests that, depending on the concentration, the enzyme adsorbs in different states that have different thicknesses. The structural parameters obtained are shown in Table 2. The profiles for the first range of concentrations in Table 2 fit a uniform layer with thickness that increases with concentration and then stabilizes, going from 41 (3 Å at 0.01 µM to 50 (3 Å for the concentrations 0.01-0.40 µM. For higher concentrations (c > 0.40 µM), the uniform layer was not an appropriate fit anymore. Instead, a two-layer model, incorporating a second layer within the aqueous phase, adsorbed upon the primary surface layer, was found to better fit the neutron reflectivity profiles. Both layers showed thickness of 50 (3 Å. No fit of the experimental data assuming less ordered fragment distributions was satisfactory. This suggests that the protein did not denature, and its globular framework did not break down to peptide fragments, for all the ranges of concentration studied. Complete denaturation of the protein would result in a more complex structure at the interface, with various layers of different densities. A reasonable proposition is that, at sufficiently high ionic strength, the electrostatic charges on the protein are screened and the intermolecular electrostatic repulsions decreased. This process increased adsorption, thus decreasing the area per molecule while maintaining a reasonably constant layer thickness. Furthermore, comparison with the enzyme dimensions from the crystal structure implies that the enzyme did not dissociate but adsorbed with the short axial axis of the globular dimer structure (52 Å) perpendicular to the interface and the long axes, 60 and 77 Å, parallel to the interface. At very low concentrations (0.01 µM) the enzyme flattened at the interface to give a layer of thickness of 41 ( 3 Å. For higher concentrations (0.10 µM < c < 0.40 µM), the thickness of the closely packed layer (50 ( 3 Å) became comparable to the short axis length and the area/molecule became comparable to the area estimated from the crystal structure dimensions (3620 Å2). The assumption is therefore that, at sufficiently high surface coverage, the electrostatic repulsions within the surface layer counteract the adsorption interaction which tends to flatten the enzyme on the interface. A close-packed layer could possibly be formed. It appears that the second layer of enzyme that adsorbed on top of the first closely packed layer also had thickness close to the dimension of the short axial axis of the protein dimer. The large area per molecule of the second layer implies that, under the experimental conditions, adsorption into this layer was not complete. The enzyme adsorption certainly involved different adsorption states that were concentration and time dependent. Capacitance and surface tension breaks over similar concentration ranges supported the neutron reflection findings. The model implies that at low concentration the initial adsorption of the enzyme at the interface is followed by conformational changes and reorientation of the adsorbed molecules that consequently flatten somewhat. At higher concentrations lateral in-

teractions prevent the denaturation of the enzyme at the interface. A close-packed enzyme layer can be formed. For even higher concentrations a second layer of molecules adsorbs upon the primary adsorbed layer. This interfacial layer of increasing thickness therefore consists of bilayers of adsorbed enzyme that interact with each other. Keeping in mind the size of the enzyme, the interfacial layer could be considered as a third phase at the interface. Variations in the composition of this phase could account for the irreproducible results obtained with the surface tension and capacitance methods at the ITIES. This model could also account for the SHG findings of Rinuy et al.20 and the surface tension findings of Rosilio et al.17 on GOx adsorption at the air/water interface. They also provide a new insight on the findings of Fiol et al.21and Szucs et al.22 for GOx adsorption at the solid/liquid interface. The interfacial capacitance measurements indicate that GOx adsorption appears to be very similar to BSA adsorption at the liquid/liquid interface.15 From the neutron reflection results, it was also evident that GOx adsorption at the air/water interface was very similar to BSA adsorption.12 On the basis of these comparisons, it is argued that GOx appears to be a “soft” protein with limited conformational stability on adsorption, depending on concentration. Conclusions The adsorption of glucose oxidase at both the ITIES and the air/water interface was time, enzyme concentration, and ionic strength dependent. The effects found by interfacial tension measurements were similar at both types of interface. Equilibration was much faster at the ITIES. Both interfacial tension and capacitance measurements showed a switch from one interfacial adsorption state to another, as the enzyme concentration was increased. At the ITIES, there was evidence from capacitance measurements for an interaction between the adsorbed enzyme and the hydrophobic cation in the organic phase. Structural parameters of the interfacial enzyme layer were provided by the neutron reflection technique, at the air/water interface. The enzyme adsorbed at the air/water interface was found to dissociate into monomers in the presence of low salt concentration (2 mM buffer) while, at a high salt concentration (0.2 M buffer), the adsorbed enzyme retained its dimer structure. The adsorption mostly formed monolayers and the layer thickness varied with bulk concentration, indicating some deformation related to the packing of the enzyme at the interface. For concentrations above 1 µM, in high ionic strength medium, bilayers of enzyme started to form, and the interlayer interactions resulted in a less densely packed second layer forming on top of the first one. The switch in properties of the adsorbed layer seen in interfacial tension and capacitance measurements occurred over the same enzyme concentration range at which the neutron reflection showed a more densely packed layer forming. That this switch was associated with both an increase of capacitance and an increase of interfacial layer thickness implies the coadsorption of ions from the electrolyte, which would provide a mechanism for changing the charge stored in the layer in response to changes in electrode potential. Acknowledgment. This work was supported by the European Union program on Training and Mobility of Researchers (Contract FMRX-CT96-0078). We thank the Engineering and Physical Sciences Research Council (EPSRC) for support of this work under Grant GR/L 92778. We thank Drs. Daren Caruana and Jorg Strutwolf for useful discussions. LA0205248