© Copyright 1997 by the American Chemical Society
VOLUME 101, NUMBER 28, JULY 10, 1997
LETTERS Absence of Surface Exclusion in the First Stage of Lysozyme Adsorption Is Driven through Electrostatic Self-Assembly V. Ball and J. J. Ramsden* Department of Biophysical Chemistry, Biozentrum of the UniVersity, Klingelbergstrasse 70, 4056 Basel, Switzerland ReceiVed: NoVember 15, 1996; In Final Form: February 19, 1997X
Adsorption kinetics of hen egg white lysozyme at pH 7.4 onto a Si(Ti)O2 surface were measured at two ionic strengths and under different shear rates by means of optical waveguide lightmode spectroscopy. At low ionic strength, increases of the adsorbed amount linear with time suggest that the adsorption proceeds without surface exclusion. Within the concentration range investigated, the duration of this linear regime was inversely proportional to the bulk concentration. The linear regime always stopped at a threshold value of 0.18 ( 0.02 µg/cm2 and was followed by a second regime with surface exclusion. At high ionic strength the linear regime was not observed, suggesting an electrostatically driven self-assembly process at low ionic strength. This conclusion is supported by the linear arrays of lysozyme at graphite surfaces observed with STM (Haggerty, L.; Lenhoff, A. M. Biophys. J. 1993, 64, 886). Moreover, the adsorption rate at low ionic strength is faster than that predicted by diffusion across the diffusion boundary layer, suggesting that the transport is accelerated by electrostatic attraction. This was confirmed by an estimation of the electrostatic free energy contribution.
Introduction Hen egg white lysozyme was the first protein whose atomic coordinates were determined at atomic resolution (0.2 nm).1 It is a very well-characterized single-domain protein, which has made it an attractive model for investigating its adsorption at solid/liquid interfaces from both a thermodynamical 2-5 and a kinetic point of view.4,6-10 Despite the great number of studies in this field, only a few qualitative tendencies have emerged, in particular that electrostatic interactions constitute a strong driving force for adsorption:2,5 the protein hardly adsorbs under conditions of electrostatic repulsion at hydrophilic surfaces, and hence structural rearrangements play only a minor role as might be expected from its strong structural stability.11 On silicon oxide surfaces8,10 or on silanized quartz9 there is evidence for adsorption in multilayers, but not on polystyrene latex particles.5 * To whom all correspondence should be addressed. Tel: + 41 61 267 21 93. Fax: + 41 61 267 21 89. X Abstract published in AdVance ACS Abstracts, July 1, 1997.
S1089-5647(96)03812-6 CCC: $14.00
Norde and Anusiem4a and Norde et al.4b observed a linear increase of the adsorbed amount with time on a silica surface4a and an indium tin oxide chip covered with multilayers of polymerized 10-12 pentacosadiyonic acid4b in the presence of 10 mM phosphate buffer at pH 7.0, already suggesting the absence of surface exclusion effects, although this inference was not made by the authors. It is the purpose of this paper to analyze precisely the initial buildup of an adsorbed lysozyme layer at the Si(Ti)O2-water interface as a function of both the hydrodynamic conditions and the electrostatic interactions between the protein and the surface. Our experiments were performed with optical waveguide lightmode spectroscopy (OWLS), which allows the adsorbed amount per unit surface area, Γ, of an adsorbed protein layer with a mass resolution of (1 ng/cm2 to be determined.12,13 Experimental Section Protein Solutions. We used hen egg white lysozyme (Worthington, purity > 99%) dissolved either in 10 mM Hepes© 1997 American Chemical Society
5466 J. Phys. Chem. B, Vol. 101, No. 28, 1997
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NaOH (Sigma) buffer, pH 7.4 (buffer 1) or in the same buffer to which 0.15 M NaCl (pro analysi, Merck) was added (buffer 2). The Debye screening lengths LD were 3.04 and 0.76 nm respectively for buffers 1 and 2. The refractive indices of the cover medium (nc) were 1.331 689 and 1.333 299 at a temperature of 25 °C for buffers 1 and 2 as determined using an LI3 interferometer (Carl Zeiss, Iena). The buffer solutions were freshly prepared before each experiment and were injected in the cell through an Acrodisc 0.2 µm membrane. The pH values were checked by means of a calibrated digital E-532 pH meter (Metrohm, Herisau, CH), and the buffers were again filtered through 0.2 µm membranes (Sartorius) before and after addition of the lyophilized protein powder. Relevant physicochemical properties of the lysozyme molecule are well-known.4,5 Of particular importance here are the diffusion constant (1.04 × 10-10 m2/s) and the isoelectric point (11.1), which determine respectively the rate of arrival at the interface (at a given bulk concentration) and the electrostatic interaction with the Si(Ti)O2 substrate. Surface Parametrization. Optical waveguides consisted of Si0.6Ti0.4O2 sputtered onto a 48 × 16 × 0.5 mm glass chip into which a grating coupler (grating constant Λ ) 833 nm) has been photolithographically etched and were obtained from Artificial Sensing Instruments, Zu¨rich (type 1400). The Si(Ti)O2 layer was 200 ( 2 nm thick as measured during equilibration with buffer alone and allowed only the zeroth order transverse electric (TE) and transverse magnetic (TM) modes to propagate.14 The density Ns of silanol groups on silica15 is about 5 nm-2 and the point of zero-charge pH0 approximately 2.5.16 Ns should be relatively uniform from one surface to another when the silica is in aqueous solution where the siloxane bounds are disrupted after water chemisorption.17 The pK difference between the two acid/base couples SiOH2+/SiOH and SiOH/SiO- is ∆pK ) 5.5.16 These values allow the surface potential ΨS to be estimated using the ionizable surface group model.16 At 298 K and for pH 7.4 considering the 10 mM hepes buffer as a 1:1 electrolyte (even though it is mainly in a zwitterionic form at this pH), a graphical resolution of the basic equation of the Healy and White model16 leads to ΨS ) -167 mV. For TiO2 the same calculation, with values 5.8, 2.0, and 5 nm-2 for pH0, ∆pK, and Ns respectively, yields ΨS ) -87 mV. Thus the surface potential of the mixture is -135 mV. For buffer 2 the same calculations lead to ΨS ) -80 mV. The pH value at the surface (pHS) is different from that in the bulk solution according to eq 116
pHs ) pH + with ys )
ys 2.303
(1)
eψs kT
where e is the value of an elementary charge, k the Boltzmann constant, and T the absolute temperature. Hence pHS ) 5.12 for buffer 1 and 6.05 for buffer 2. Both at the surface and in the bulk, the lysozyme is positively charged and takes between 7 and 10 elementary positive charges (a precise value would require measurement or calculation of the titration curve, which are both difficult and inaccurate at a low ionic strength of 10 mM) and the overall electrostatic interaction between the protein and the surface is attractive, the more so as the protein approches closer to the surface. Mode Spectrum Measurements. The technique and physical principle of optical waveguide lightmode spectroscopy are fully described elsewhere.13,18 Briefly, it consists of measuring
Figure 1. Schematic diagram of the chip with glass support (S), Si(Ti)O2 waveguiding film (F), proteinaceous adlayer (A), and cover medium (C). n and d refer to refractive indices and the thickneses respectively. G is the grating coupler (grating constant Λ ) 833 nm), which is a distance s (0.25 cm) from the inlet. F is the injection flow rate, and P is one of the two photodiodes measuring the light L incoupled into the waveguide from a He-Ne laser source at an angle of incidence R. The other photodiode (not represented) is placed on the left-hand side of the waveguide and measures the intensity of the guided modes in the left direction. The laser is fixed and R continuously varied by rotating the waveguide holder.
the effective refractive indices of transverse electric (TE or “s” waves) and transverse magnetic (TM or “p” waves) modes incoupled under total internal reflection conditions into the Si(Ti)O2 oxide layer via the grating coupler G (Figure 1). By varying the angle of incidence, the mode spectrum is detected as a series of narrow peaks (fwhm ∼ 3 × 10-6 radian) by photodiodes P positioned at the two ends of the waveguide (Figure 1, only one photodiode is represented for reasons of simplicity). The photodiode monitors the intensity of the incident wavelength (linearly polarized He-Ne line reflected on a mirror to obtain the TE and TM fields before incoupling), and different diffraction orders are allowed depending on the line density of the the grating coupler. The intensity due to Stokes and anti-Stokes light scattering from the TiO2 microcrystallites in the waveguide film is negligible in comparison to the Rayleigh line. The angle of incidence R is varied in a (7° angular range by means of an IOS-1 goniometer (Artificial Sensing Instruments, Zu¨rich). Light is incoupled into the waveguide film only when the following condition is fulfilled13,14
n sin R +
lλ )N Λ
(2)
where n, l, and N are the refractive index of air, the diffraction order (always 2), and the effective refractive index of the guided mode respectively. The simultaneous solution of the linearized mode equations for the waveguiding film allow both the thickness dA and refractive index of the proteinaceous adlayer nA to be calculated.13,14 The adsorbed amount per unit surface area, Γ, can then be obtained according to13,14,19
Γ)
(nA - nC)dA dn/dc
(3)
where dn/dc is the refractive index increment (0.182 cm3/g for lysozyme).19 All the adsorption experiments were performed at (25 ( 1) °C. Temperature was followed by means of a Pt100 resistance embedded in the aluminium waveguide holder; the temperature variations in an individual experiment were lower than 0.2 °C. The protein solution was injected (flux F) by means of a syringe pusher connected to the inlet of an anodized aluminium
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J. Phys. Chem. B, Vol. 101, No. 28, 1997 5467
cuvette sealed to the surface by means of an O-ring. The cuvette was constituted by two parallel disks 3.14 × 10-2 cm apart and had a cross-sectional area A of 2.83 × 10-2 cm2 at the center of the diffraction grating where our optical measurements are performed. It thus mimics the situation where the fluid is moving over a plane surface. Then the thickness of the diffusion boundary layer (through which the proteins can reach the surface only by diffusion) is given by20
δ)3
(Dκ ) (κsu) 1/3
1/2
(4)
where D is the translational diffusion coefficient for the lysozyme molecule, κ is the kinematic viscosity of the fluid (taken equal to that of pure water, i.e, 9 × 10-3 cm2/s at 298 K), s is the coordinate along the plate counting from the inlet (0.25 cm to the center of the grating), and U is the average velocity of the fluid. Thus
U)
F A
(5)
Since A is a function of the position along the cuvette, the value of U is minimal at the position of measurement where the crosssectional area is maximal. The wall shear rate γ is a function of the flux, the area A, and the height h of the cuvette according to eq 6:
γ)
6F Ah
(6)
In our study, proteins were injected at flow rates F of either 8.9 or 29.0 cm3/h; these values were checked by weighing. The corresponding shear rates γ were equal to 16.6 and 54.2 s-1 respectively. The diffusion boundary layer thicknesses, calculated with eqs 5 and 4, are then equal to 234 and 130 µm for the low and high flow rates respectively. Before each experiment, the chips were cleaned with ethanol (10 min), Nanopure water, 0.2 M HCl, and again water for at least 10 min. After this treatment the Si(Ti)O2 surface was hydrophilic as qualitatively judged by the good spreading of water. Although no attempts were made to quantify precisely the contact angle for water, this contact angle was smaller than 10° in all the experiments performed here. Results and Discussion A typical experiment, shown in Figure 2, starts with the injection of Nanopure water followed by buffer 1, protein solution at 2.3 µg/cm3, buffer 1, a 0.2 M HCl solution, and finally water again. We can see that the HCl rinsing removes all the adsorbed lysozyme (as expected from electrostatic considerations since both the protein and the surface are strongly positively charged at pH ∼ 1) and that subsequent water rinsing regenerates the waveguide. In Figure 3a we represent experiments performed in buffers 1 and 2 at a lower protein concentration of 0.3 µg/cm3. The experiment in Figure 3a was stopped before saturation was reached in order to check for reversibility against buffer rinse in the linear regime, which is also seen but for a shorter time in Figure 2. Marked differences can be seen between the two experiments in Figure 3: first of all, in buffer 1 the rate of lysozyme adsorption is constant for at least 1000 s, a sign that the preadsorbed molecules do not exclude any subsequently reaching the interface, whereas in the presence of 150 mM NaCl (buffer 2, see also Figure 3b which is an enlargement of the lower curve in Figure 3a) adsorption progressively decelerates. In other experiments with buffer 1, in which adsorption was pursued for longer times before rinsing
Figure 2. Adsorption kinetics of lysozyme at a concentration of 2.3 µg/cm3 from 10 mM Hepes buffer, pH 7.4. The flow rate F was constant during the whole experiment and equal to 8.9 cm3/h. Arrow a, injection of buffer; arrow b, beginning of protein injection; arrow c, new injection of buffer; arrow d, injection of 0.2 M HCl solution; arrow e, injection of Nanopure water.
(Figure 2), an exclusion effect begins to influence the adsorption kinetics: the linear adsorption regime always stops at Γmax ≈ 0.18 µg/cm2 (see Table 1). The product of the duration of the linear adsorption regime (∆tlinear) and the bulk concentration cb is approximately constant (Table 1) and equal to 500 ( 200 (µg‚s)/cm3. These observations with buffer 1 strongly suggest an adsorption mechanism without surface exclusion; in other words during this regime the available area function21 φ has to be taken as constant and equal to 1, in contrast to previous observations of protein adsorption22 or particle adhesion23 in which φ was found to be a decreasing function of Γ.21 We are thus in the presence of a nonrandom adsorption mechanism, such as the growth of ordered molecular arrays, which have been observed for lysozyme on a graphite surface with scanning tunneling microscopy.24 The mechanism appears to involve favorable electrostatic interactions between the proteins that have an asymmetric electrostatic surface potential:24 when the ionic strength increases one expects a diminution of these intermolecular attractive interactions, abolishing the nonrandom growth pattern and thus inducing a process where exclusion effects play a role. This is indeed observed in our experiments with buffer 2 (Figure 3b). We will analyze the second adsorption regime, where exclusion indeed takes place, in a forthcoming paper25 and focus now on the influence of transport phenomena on lysozyme adsorption in the linear regime. Inspection of the data in Table 1 suggests the following relationship
(dΓdt )
linear
) k*cb
(7)
where (dΓ/dt)linear is the (constant) adsorption rate in the linear regime. The data is plotted in Figure 4, and linear regression yields k* ) 2.2 × 10-4 cm/s. This value has to be compared directly with
kcd )
D δ
(8)
representing the coefficient of convective diffusion toward a surface acting as a perfect sink, which equals 4.4 × 10-5 cm/s with the δ value obtained from eqs 4 and 5. This implies either that the adsorption is accelerated through electrostatic attractions
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Figure 4. Variation of the constant adsorption rates in the linear adsorption regime for a flow rate of 8.9 cm3/h (solid line) and of 29 cm3/h (dashed line). The dotted lines represent the limits of the 95% confidence intervals for the linear regression.
Figure 3. (a, top) Comparison between the behavior of lysozyme adsorbed from a 0.3 µg/cm3 solution at an ionic strength of 10 mM (buffer 1, O) and that of 160 mM (buffer 2, 2). Flow rate during the whole experiment: 8.9 cm3/h. Arrows b and c have the same significance as in Figure 2. (b, bottom) Enlargement of the lower curve in (a); see legend of (a).
TABLE 1: Parameters for Lysozyme Adsorption at the Si(Ti)O2-Water Interface at pH 7.4 in 10 mM Hepes Buffer at Two Different Flow Rates F (Second Column)a cb/ µg‚cm-3
F/ cm3‚h-1
∆tlinear/s
∆tlinear‚cb/ µg‚cm-3‚s
Γmax/ µg‚cm-2
(dΓ/dt)linear/ µg‚cm-2‚s-1
0.13 0.31 0.35 0.92 0.95 2.27 2.75 6.40 1.00 2.60
8.9 8.9 8.9 8.9 8.9 8.9 8.9 8.9 29 29
3100 1200 1020 800 700 332 330 110 490 140
403 372 357 736 665 754 908 704 490 364
0.17 0.18 0.18 0.20 0.17 0.20 0.20 0.17 0.19 0.17
5.65 × 10-5 8.25 × 10-5 1.73 × 10-4 1.97 × 10-4 2.49 × 10-4 5.87 × 10-4 5.61 × 10-4 1.45 × 10-3 4.20 × 10-4 1.15 × 10-3
a Cb: bulk concentration. ∆tlinear: duration of the linear adsorption regime. Γmax: amount of lysozyme adsorbed at the end of the linear regime. (dΓ/dt)linear: constant rate of adsorption in the linear regime.
between the positively charged protein and the negatively charged surface or that eq 4 is not able to give δ and thus kcd correctly. In order to assess this second possibility we performed adsorption experiments with buffer 1 at a flow rate of 29 cm3/h. We obtain a new straight line (dashed line in
Figure 4) whose slope is equal to 4.4 × 10-4 cm/s. The ratio of the two slopes is 2, in agreement with the square root of the ratio of the flow rates since (29/8.9)0.5 ≈ 1.8 as predicted by eq 4. Thus eq 4 appears to be essentially correct. Hence the adsorption process must be accelerated by attractive electrostatic interactions, implying that their contribution to the Gibbs free energy of adsorption should be significant at low ionic strength. If we assume the lysozyme molecule to be a spherical particle of radius R ) 2 nm, this contribution can be calculated according to26
[
( )]
∆Gelpws(l) ) RΨpΨs ln 1 + exp -
l LD
(9)
where subscript pws means that the interaction takes place between the protein (medium p) and the Si(Ti)O2 surface (medium s) through water (w). Superscript el refers to the electrostatic nature of the interaction, is the dielectric constant of water and l is the separation between both surfaces. The main problem with eq 9 is to know the surface potential Ψp of the lysozyme molecule. Indeed one should rather use the ζ potentials for both surfaces to take account of the nonaccessibility of molecules inside the inner Helmholtz plane.28 This would reduce the value of the Gibbs energy significantly. But, to our knowledge the ζ potential of lysozyme is not known at the ionic strengths used in our experiments. Thus we can only e1 give a crude approximation for ∆Gpws (l), our aim being mainly to evaluate the effect of the ionic strength. We then make a further approximation by considering the lysozyme molecule to be a uniformly charged sphere carrying an average of seven elementary positive charges at a pH close to 7.5 as obtained from proton titration curves in KCl solutions.29 This leads to a surface charge density σp of -2.2 × 10-2 C‚m-2 for the protein from which the surface potential can be estimated according to30
Ψp(σp, 1/R) )
[
LD q - 1 2kT ln(p + q) e R pq
]
(10)
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J. Phys. Chem. B, Vol. 101, No. 28, 1997 5469
with
p)
σpeLD 20kT
Acknowledgment. V. Ball is supported by a Lavoisier fellowship from the French government.
(11)
and
q ) (1 + p2)1/2
(12)
where all the symbols have been previously defined. Clearly this second approximation underestimates the value of Ψp, because the protein is asymmetric with respect to its charge distribution. At this point we cannot argue if the overestimation due to the use of surface potentials instead of ζ potentials cancels the underestimation due the evenly spread charge approximation: we want only to compare electrostatic effects in two different buffers for a given protein-surface separation. For el (l) values is thus ≈8 × buffers 1 and 2, the ratio of the ∆Gpws 4 10 at l ) 10 nm with LD,1 ) 3.04 nm, ΨS,1 ) -135 mV and LD,2 ) 0.76 nm, ΨS,2 ) -80 mV. This very high value makes it easy to understand both qualitative differences of adsorption behavior between buffers 1 and 2 and the electrostatic acceleration of the transport toward the surface in the presence of buffer 1. Our conclusion relative to the adsorption mechanism is then in contrast to the results obtained by Norde and Anusiem 4a and Norde et al.4b who have observed that the ratio between the experimental adsorption rate and the rate predicted for a process only limited by transport toward the surface was 0.6 (conditions: 10 mM phosphate buffer, pH 7.0, and a pure silica surface4a or a Langmuir-Blodgett fatty acid film deposited on indium tin oxide4b). In other words, a lysozyme molecule reaching those interfaces has a probability of only 60% to adsorb. The difference between the behavior observed in our study and that observed in the group of Norde may arise from the different buffering system. Indeed phosphate buffer has been found to dramatically inhibit the adsorption of seralbumin to silica-titania surfaces.29 The effect of the nature of ions upon the adsorption behavior of proteins needs careful additional studies. What little data is available points to dramatic effects possibly due to ion-induced structural modifications in the protein.31,32 The kind of electrostatically driven self-assembly we observed by means of kinetic experiments seems to be a rather general phenomenon in nature and has been also observed in the adsorption of DNA onto cationic phospholipids by means of atomic force microscopy33 and may play a role in important biological processes.
References and Notes (1) Blake, C. C. F.; Koenig, D. F.; Mair, G. A.; North, A. C. T.; Phillips, D. C.; Sarma, V. R. Nature 1965, 206, 757. (2) Barroug, A.; Lemaitre, J.; Rouxhet, P. G. Colloids Surf. 1989, 37, 339. (3) (a) Fraaije, J. G. E. M.; Norde, W.; Lyklema, J. Biophys. Chem. 1991, 40, 303. (b) Fraaije, J. G. E. M.; Norde, W.; Lyklema, J. Biophys. Chem. 1991, 40, 317. (4) (a) Norde, W.; Anusiem, A. C. I. Colloids Surf. 1992, 66, 73. (b) Norde, W.; Giesbers, M.; Pingsheng, H. Colloids Surf. B 1995, 5, 255. (5) (a) Haynes, C. A.; Sliwinsky; Norde, W. J. Colloid Interface. Sci. 1994, 164, 394. (b) Haynes, C. A.; Norde, W. J. Colloid Interface. Sci. 1995, 169, 313. (6) Ivarsson, B. A.; Hegg, P. O.; Lundstro¨m, I.; Jo¨nsson, U. Colloids Surf. 1985, 13, 169. (7) Horsley, D.; Herron, J.; Hlady, V.; Andrade, J. D. ACS Symp. Ser. 1987, 343, (8) Wahlgren, M.; Arnebrant, T.; Lundstro¨m, I. J. Colloid Interface. Sci. 1995, 175, 506. (9) Schmidt, C. F.; Zimmermann, R. M.; Gaub, H. E. Biophys. J. 1990, 57, 577. (10) Malmsten, M. J. Colloid Interface. Sci. 1994, 166, 333. (11) Gekko, K.; Hasegawa, Y. Biochemistry 1986, 25, 6563. (12) Tiefenthaler, K. AdV. Biosensors 1992, 2, 261. (13) Ramsden, J. J. J. Stat. Phys. 1993, 73, 853. (14) Tiefenthaler, K.; Lukosz, W. J. Opt. Soc. Am. B 1989, 6, 209. (15) Zuhravlev, L. T. Langmuir 1987, 3, 316. (16) Healy, T. W.; White, L. R. AdV. Colloid Interface. Sci. 1978, 9, 303. (17) Fripiat, J.; Chaussidon, J.; Jelli, A. Chimie-Physique des phe´ nome` nes de surface. Application aux oxydes et aux silicates; Masson: Paris, 1971. (18) Ramsden, J. J.; Roush, D. J.; Gill, D. S.; Kurrat, R.; Willson, R. C. J. Am. Chem. Soc. 1995, 117, 8511. (19) de Feijter, J. A.; Benjamins, J.; Veer, F. A. Biopolymers 1978, 17, 1759. (20) Levich, B. Discuss. Faraday Soc. 1947, 1, 38. (21) Schaaf, P.; Talbot, J. J. Chem. Phys. 1989, 91, 4401. (22) Ramsden, J. J. Phys. ReV. Lett. 1993, 71, 295. (23) Wojtasczczyk, P.; Schaaf, P.; Senger, B.; Zembala, M.; Voegel, J.-C. J. Chem. Phys. 1993, 99, 7198. (24) Haggerty, L.; Lenhoff, A. M. Biophys. J. 1993, 64, 886. (25) Ball, V.; Ramsden, J. J.; in preparation. (26) Van Oss, C. J.; Wu, W.; Giese, R. F. ACS Symp. Ser. 1995, 602, 80. (27) Kurrat, R.; Prenosil, J. E.; Ramsden, J. J. J. Colloid Interface Sci. 1997, 185, 1. (28) Hiemenz, P. C. Principles of Colloid and Surface Chemistry, 2nd ed.; M. Dekker: New York and Basel, 1986; Chapter 12. (29) Haynes, C. A.; Sliwinsky, E.; Norde, W. J. Colloid Interface Sci. 1994, 164, 394. (30) Winterhalter, M.; Helfrich, W. J. Phys. Chem. 1992, 96, 327. (31) Shibata, C. T.; Lenhoff, A. M. J. Colloid Interface Sci. 1992, 148, 485. (32) Liu, H.-S.; Wang, Y.-C.; Chen, W.-Y. Colloids Surf. B. 1995, 5, 25. (33) Fang, Y.; Yang, J. J. Phys. Chem. B 1997, 101, 441.
