Enolase Adsorption onto Hydrophobic and Hydrophilic Solid

The understanding of the adsorption process of biomolecules is very important for biological and engineering applications. Enolase is an enzyme of gly...
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Enolase Adsorption onto Hydrophobic and Hydrophilic Solid Substrates A. T. Almeida,† M. C. Salvadori,‡ and D. F. S. Petri*,† Instituto de Quı´mica, Universidade de Sa˜ o Paulo, P.O. Box 26077, Sa˜ o Paulo, SP 05513-970, Brazil, and Instituto de Fı´sica, Universidade de Sa˜ o Paulo, P.O. Box 66318, Sa˜ o Paulo, SP 05315-970, Brazil Received March 28, 2002. In Final Form: June 7, 2002 The understanding of the adsorption process of biomolecules is very important for biological and engineering applications. Enolase is an enzyme of glycolytic pathway that catalyses a reversible conversion of 2-phosphoglycerate to phosphoenolpyruvate. In this work the adsorption behavior of enolase (2-phosphoD-glycerayte hydrolyase) onto hydrophilic silicon wafers and amino-terminated surfaces (APS) and onto hydrophobic polymer polystyrene (PS) was studied by means of null-ellipsometry. The adsorption kinetics of enolase onto these substrates presented three distinct regions: (i) a diffusion-controlled one; (ii) an adsorption plateau; (iii) continuous, irreversible, and asymptotic increase of the adsorbed amount with time. Atomic force microscopy (AFM) showed that well-packed entities formed an enolase biofilm, which might correspond to the monolayer formation. With increase of the adsorption time, aggregates appeared on the surface, suggesting multilayer formation. The early stages might be predicted by the random sequential adsorption model (RSA), while the cooperative sequential adsorption (CSA) model seems to describe regions ii and iii. No significant influence of ionic strength was observed on the adsorption behavior of enolase onto the present substrates. The adsorption isotherms show that enolase has no preferential adhesion onto hydrophilic or hydrophobic substrates. Contact angle measurements showed that PS surfaces became hydrophilic and silicon surfaces turned hydrophobic after the formation of the enolase biofilm. The study of the influence of pH on the enolase adsorption on silicon and APS surfaces showed that the higher adsorbed amount occurs when pH is close to enolase pI. Far from pI the enzyme solubility decreases and some repulsive forces come out, leading to a decrease in the adsorbed amount.

Introduction Proteins in aqueous solution present a low stability, and the presence of a solid surface can lead in almost all cases to adsorption of the protein to the interface between the two phases. The understanding of the process of protein adsorption onto solid surfaces is very important for industrial1-3 and medical applications.4-6 Many efforts have been made to understand the mechanism of protein adsorption, and many conflicting results have been published. This great difficulty stems from the complex physicochemical characteristics of these biomolecules and the equally complex interactions that arise during the adsorption process.7 Many models developed to describe the adsorption process of proteins are based on reversible thermodynamic, where isotherm fitting following the Langmuir model allows the determination of Gibbs adsorption energy (∆adsG). Lundstro¨m8 developed a competitive adsorption model on the basis of multiple conformational states and * Corresponding author. E-mail: [email protected]. † Instituto de Quı´mica, Universidade de Sa ˜ o Paulo. Telephone: 0055 11 3091 3831. Fax: 0055 11 3815 5579. ‡ Instituto de Fı´sica, Universidade de Sa ˜o Paulo. Telephone: 0055 11 3091 6857. Fax: 0055 11 3091 6749. (1) Fernandez-Lafuente, R.; Armise´n, P.; Sabuquillo, P.; Ferna´ndezLorente, G.; Guisa´n, J. M. Chem. Phys. Lipids 1998, 93, 185. (2) Mattedi, A.; Filho, R. M. Comput. Chem. Eng. 2000, 24, 1111. (3) Alonso, J.; Barredo, J. L.; Armise´n, P.; Dı´ez, B.; Salto, F.; Guisa´n, J. M.; Garcia, J. L.; Corte´s, E. Enzyme Microb. Technol. 1999, 25, 88. (4) Werner, C.; Ko¨nig, U.; Augsburg, A.; Arnhold, C.; Ko¨rber, H.; Ralf Zimmermann, H. J. Colloids Surf., A 1999, 159, 519. (5) Dahint, R.; Seigel, R. R.; Harder, P.; Grunze, M.; Josse, F. Sens. Actuators, B 1996, 36, 497. (6) Slomkowski, S. Prog. Polym. Sci. 1998, 23, 815. (7) Yuan, Y.; Oberholzer, M. R.; Lenhoff, A. M. Colloids Surf., A 2000, 165, 125. (8) Lundstro¨m, I. Prog. Colloid Polym. Sci. 1985, 70, 76.

different interaction forces between the substrate and protein. This model considers the protein adsorption as being a reversible process and desorption of the adsorbed molecules might occur. On the other hand, theoretical9,10 and experimental11-13 works show that the protein adsorption is an irreversible process and in this case the Langmuir model can no longer be applied. On the basis of thermodynamic aspects, Haynes and Norde14 showed the several interactions (Coulombic forces, van der Waals forces, Lewis acid-base forces, hydrophobic interactions) that might contribute to protein adsorption. Thus, the adsorption process depends on intramolecular forces within the protein that can lead to a modification of protein conformation. Lyklema and Norde15 also considered these conformational changes due to adsorption process and divided the proteins in soft and hard ones. While the hard ones keep their native structure, the soft ones spread completely after the adsorption on the substrate surface. The driving force for the adsorption of soft proteins is the entropic gain caused by the breakdown of secondary and/ or tertiary structures. The random sequential adsorption (RSA) model is based on the irreversible deposition of colloidal particles for situations in which adsorption does not exceed a monolayer. In this model, the protein molecule is considered as a hard particle (hard disks), which cannot overlap on the (9) Schaaf, P.; Talbot, J. J. Chem. Phys. 1989, 91, 3301. (10) van Tassel, P. R.; Viot, P. Europhys. Lett. 1997, 40, 293. (11) Pancera, S. M.; Alvarez, E. B.; Politi, M. J.; Gliemann, H.; Schimmel, Th.; Petri, D. F. S. Langmuir 2002, 18, 3517. (12) Garrett, Q.; Griesser, H. J.; Milthorpe, B. K.; Garrett, R. W. Biomaterials 1999, 20, 1345. (13) Cho, D.; Franses, E.; Narsimhan, G. Colloids Surf., A 1996, 117, 45. (14) Haynes, C. A.; Norde, W. Colloid Surf., B 1994, 2, 517. (15) Lyklema, J.; Norde, W. Prog. Colloid Polym. Sci. 1996, 101, 9.

10.1021/la0202982 CCC: $22.00 © 2002 American Chemical Society Published on Web 08/08/2002

Enolase Adsorption onto Solid Substrates

surface where another one was previously inserted. The RSA model may be generalized to account for several experimental features such as bulk diffusive transport, surface-induced conformational change, desorption, and multilayer formation.16 The cooperative sequential adsorption (CSA) is an extension of the RSA model, where molecules can adsorb near previously filled sites leading to clusters or island formation and protein multilayer formation.16,17 The aim of this work was to study the large time adsorption behavior of enolase onto hydrophilic silicon wafers and amino-terminated surfaces (APS) and onto hydrophobic polymer polystyrene (PS) by means of nullellipsometry, contact angle measurements, and atomic force microscopy (AFM). Enolase (2-phospho-D-glycerayte hydrolyase), an enzyme of the glycolytic pathway that catalyses a reversible conversion of 2-phosphoglycerate to phosphoenolpyruvate,18 was chosen for this study. It belongs to a novel class of surface proteins that do not possess classical machinery for surface transport yet through an unknown mechanism are transported on the cell surface. Enolase serves as a plasminogen receptor on the surface of a variety of hematopoetic, epithelial, and endothelial cells. Its role in systematic and autoimmune disorders was recognized only very recently.19 Enolase has been recognized as a fungal allergen20 and has been also identified as an allergen by the IgE immunoblot technique.21 Understanding the adsorption behavior of enolase onto common substrates such as hydrophilic silicon wafers and hydrophobic PS films might be of great relevance for the design of sensors. Materials Enolase from Saccharomyces cerevisiae (EC 4.2.1.11, molecular weight of 46.7 kDa, Sigma, St. Louis, MO) was dissolved in the concentration range of 0.002-1.0 g/L in 0.001, 0.01, and 0.1 mol/L NaCl. Silicon (100) wafers (Crystec, Berlin, Germany) with native oxide layer were used as substrates. The Si wafers (cut in a typical dimension of 1 cm2) were rinsed as described elsewhere.22 After this, the surfaces were functionalized with (aminopropyl)trimethoxysilane, APS (Fluka, Switzerland), following a method described elsewhere.22 This method yields a flat and homogeneous amino-terminated monolayer covalent bound on silicon wafers. Polystyrene (PS, Mw ∼ 200 000 g/mol), kindly supplied by BASF Aktiengesellschaft, Ludwigshafen, Germany, was spin-coated (3000 rpm, 30 s) on silicon wafers from solutions prepared in toluene at the concentration of 10 g/L. Toluene, hydrogen peroxide, ammonium hydroxide, and sodium chloride were purchased from Nuclear (Sa˜o Paulo, Brazil) and utilized without previous purification.

Methods Ellipsometry. Ellipsometric measurements were performed in a vertical computer-controlled DRE-EL02 Ellipsometer (Ratzeburg, Germany). The angle of incidence φ was set to 70.0°, and the wavelength λ of the laser was 632.8 nm. This equipment works as a null-ellipsometer.23,24 (16) Talbot, J.; Tarjus, G.; Van Tassel, P. R.; Viot, P. Colloids Surf., A 2000, 165, 287. (17) Bartelt, M. C.; Evans, J. W. J. Stat. Phys. 1994, 76, 867. (18) Kustrzeba-Wo´jcicka, I.; Golczak, M. Comp. Biochem. Physiol., B 2000, 126, 109. (19) Pnacholi, V. Cell. Mol. Life Sci. 2001, 58, 902. (20) Breitenbach, M.; Simon, B.; Probst, G.; Oberkofler, H.; Ferreira, F.; Briza, P.; Achatz, G.; Unger, A.; Ebner, C.; Kraft, D.; Hirschwehr, R. Int. Arch. Allergy Immunol. 1997, 113, 114. (21) Baldo, B. A. Clin. Exp. Allergy 1995, 25, 488. (22) Petri, D. F. S.; Wenz, G.; Schunk, P.; Schimmel, Th. Langmuir 1999, 15, 4520. (23) Fujimoto, J.; Petri, D. F. S. Langmuir 2001, 17, 56. (24) Azzam, R. M.; Bashara, N. M. Ellipsometry and Polarized Light; North-Holland Publication: Amsterdam, 1979.

Langmuir, Vol. 18, No. 18, 2002 6915 For the data interpretation, a multilayer model composed of the substrate, the unknown layer, and the surrounding medium should be used. Then the thickness (dx) and the refractive index (nx) of the unknown layer can be calculated from the ellipsometric angles, ∆ and Ψ, using the fundamental ellipsometric equation and iterative calculations with Jones matrixes, as described elsewhere.24 The thickness of the native SiO2 layers was determined in air, considering the index of refraction for Si as n˜ ) 3.88 - i0.01825 and its thickness as infinite; for the surrounding medium (air) the index of refraction was considered as 1.00. Because the native SiO2 layer is very thin, its index of refraction was set as 1.46225 and just the thickness was calculated. The mean SiO2 thickness measured for 20 samples amounted to 1.9 ( 0.2 nm. After the characterization, the Si wafers were functionalized by the silanization reaction with APS.22 The thickness of the aminoterminated monolayer was determined in air, considering the nominal index of refraction for silane as 1.424. The mean thickness value calculated for the amino-terminated layer was 1.1 ( 0.2 nm. The mean thickness and index of refraction of the PS films were determined as 84 ( 1 nm and 1.583 ( 0.003, respectively.11 The adsorption from solution was monitored in situ with the help of a cell made of poly(methyl methacrylate). This cell has two quartz windows, one for the incident beam and the other for the reflected beam, both with the inclination angle of 70.0°. The measurements were done in an air conditioned room at the temperature of 23° C ( 1° C. The adsorbed amount Γ is determined from11,23,24,26

Γ)

denolase(nenolase - n0) ) denolasecenolase dn/dc

(1)

where nenolase and denolase are the index of refraction and thickness of the adsorbed enzyme, n0 is the index of refraction of the solution measured with an Abbe´ refractometer, dn/dc is the increment of refractive index determined with a differential refractometer, and cenolase is the average enzyme concentration within the layer.23,24,26 For our system, n0 was measured for each concentration and dn/dc amounted to 0.16 mL/g at the temperature of 23 °C. From the ellipsometric angles ∆ and Ψ and a multilayer model composed by silicon, silicon dioxide, amino-terminated monolayer or polymeric film, enzyme layer, and solution it is possible to determine the thickness of the adsorbed enzyme (denolase). However, the very low value of denolase and the small differences between the indices of refraction of the substrate, enzyme, and solution made an independent determination of nenolase and denolase impossible. Therefore, nenolase was kept constant as 1.50 and denolase was calculated. Nevertheless, it is important to remember here that, if the index of refraction assumed for the adsorbing layer lies in a reasonable range (between 1.40 and 1.60), the product nenolasedenolase should be a constant value and does not depend on the adopted model.23,24,26 This product is the parameter necessary to calculate the adsorbed amount Γ from eq 1. Contact angle measurements were performed at 23 ( 1 °C in a home-built apparatus equipped with a Casio QV-10 digital camera, which is connected to a computer. Sessile water drops of 8 µL were used for the advancing contact angle (θA), and then the volume was reduced to 4 µL to measure the receding contact angle (θR). The hysteresis in the contact angle measurements (∆θ) was calculated from the difference between (θA) and (θR). At least three spots were done in each sample before and after the adsorption. All the surfaces measured correspond to long periods (16 h) of adsorption. The measurements were performed in a closed quartz cell saturated with water vapor. To verify aging effects on the surface wettability, advancing contact angles were measured for the same drop in time intervals of 10 min during 1 h. The results are mean values of triplicates. Atomic Force Microscopy (AFM). AFM analysis of enolase covered surfaces were carried out in the air at room temperature (25) Edward, D. P., Ed. Handbook of Optical Constants of Solids; Academic Press: London, 1985. (26) Motschmann, H.; Stamm, M.; Toprakcioglu, Ch. Macromolecules 1991, 24, 3229.

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Figure 1. Adsorption kinetics measured for enolase (c ) 0.01 g/L in 0.001 mol/L NaCl) onto silicon wafers (9), aminoterminated surfaces (O), and PS films (+) at 23 ( 1 °C. with a Nanoscope IIIA (Digital Instruments). The microscope operated in the tapping mode using silicon cantilevers with oscillating amplitude of 50-100 nm and resonance frequency close to 300 kHz. Scan areas of 20 × 20, 5 × 5, and 1 × 1 µm2 with a resolution of 512 × 512 measured points (pixels) were obtained. Image processing was performed by using the Nanoscope III software.

Results and Discussion Adsorption Kinetics. The adsorption kinetics of enolase onto bare, amino-terminated (APS), and PScovered silicon wafers in the dilute regime (cenolase ) 0.01 g/L) are shown in Figure 1. They presented similar features and can be divided into three distinct regions: (i) an initial period with a linear increase of adsorbed amount of enolase (Γ) with t0.5; (ii) an adsorption plateau; (iii) a third stage with a continuous increase of Γ with t0.5. The kinetic curves in Figure 1 also show that the first step is very fast (10 min, approximately) whatever is the substrate and that enolase has a higher affinity by silicon surface than PS or amino-terminated surfaces in the dilute range. According to Norde and Giacomelli,27 protein adsorption process involves various stages: transport of the protein from the bulk solution into the interfacial region; attachment of the protein at the substrate; relaxation of the protein on the surface. Many experimental11,27-30 and theoretical8-10,31,32 works show that for the very dilute range the first step of the adsorption is a purely diffusive process. The apparent diffusion coefficient D can be calculated from the relation11,23,26

Γ(t) )

2 cbulkxDt xπ

(2)

To study the influence of ionic strength on the adsorption behavior of enolase onto hydrophilic silicon wafers and hydrophobic PS films, dynamic experiments were carried out in 0.001, 0.01, and 0.1 mol/L NaCl with bulk enolase concentration, cenolase, fixed at 0.01 g/L. Figure 2 shows the adsorbed amount of enolase, Γ, as a function of t0.5 obtained for silicon wafers and PS films, at different ionic strengths. From the initial slopes the apparent diffusion coefficient, D, values were calculated, as presented in Table 1. The D values obtained for enolase adsorbing onto silicon wafers and PS films were on the order of 10-6 and 10-7 (27) Norde, W.; Giacomelli, C. E. J. Biotechnol. 2000, 79, 259. (28) Shibata, A.; Iizuka, Y.; Ueno, S.; Yamashita, T. Thin Solid Films 1996, 284-285, 549. (29) Gage, R. A.; Norde, W. Colloids Surf., B 1997, 9, 139. (30) Oscarsson, S. J. Chromatogr., B 1997, 669, 117. (31) Ramsden, J. J. Colloids Surf., A 1998, 141, 287. (32) Bartelt, M. C.; Privman, V. Int. J. Mod. Phys., B 1991, 18, 2883.

Figure 2. Adsorption kinetics measured for enolase (c ) 0.01 g/L) onto silicon wafers (9) and PS films (+) at 23 ( 1 °C in (a) 0.001 mol/L, (b) 0.01 mol/L, and (c) 0.1 mol/L NaCl. Table 1. Diffusion Coefficients Calculated for Enolase Adsorbing onto Silicon Wafers and PS Films from Different Ionic Strength Media D (cm2 s-1) substrate 0.001 mol/L NaCl 0.01 mol/L NaCl 0.1 mol/L NaCl Si/SiO2 PS

2 × 10-6 5 × 10-7

3 × 10-6 2 × 10-7

2 × 10-6 3 × 10-7

cm2 s-1, respectively, over the whole ionic strength range. All experiments were performed at pH 6.7, which is close to the theoretical value of the isoeletric point (pI) of enolase (6.4).33 This might explain why no influence of the ionic strength on the diffusive behavior could be observed, since under such conditions the enolase molecules present null net charge and the screening effects are minimized. As stated above, a second stage in the adsorption kinetics is characterized by an adsorption plateau, indicating the formation of an enolase monolayer. Figure 2 shows that, for both substrates upon increasing the ionic strength, the plateau values decreased by 0.9 ( 0.2 mg/m2 over the NaCl concentration range of 0.001-0.1 mol/L. This might have been caused due to the screening effect on the adsorbed enolase, which reduced the electrostatic repulsion among its charged segments, leading to a more contracted conformation. Upon increase of the ionic strength, the plateau duration was reduced for both substrates. During the monolayer formation the adsorbed biomolecules might undergo conformational relaxations.15 The increase in the ionic strength might have caused a reduction in the electrostatic repulsion making these (33) www.expasy.ch (ID ) ENO1).

Enolase Adsorption onto Solid Substrates

structural rearrangements faster. Lateral diffusion of the already adsorbed enolase molecules could be also considered as a possible reason for the observed effects. Brownian dynamics simulations34 showed that at high ionic strength the Coulombic interaction among the proteins is shielded and lateral diffusion of the already adsorbed proteins leads to clustering on the surface and opens up additional room for the arriving molecules. In the third stage, a continuous increase of the adsorbed amount was observed in all systems. After approximately 12 h of adsorption at the ionic strength of NaCl 0.01 or 0.1 mol/L the adsorbed amount of enolase reached the same value (Γ ≈ 4.9 mg cm-2) for hydrophilic silicon surfaces and for hydrophobic PS films. In a comparison of this figure with those obtained for low ionic strength (0.001 mol/L NaCl), Γ ≈ 5.5 mg cm-2 onto silicon wafers and Γ ≈ 4.5 mg cm-2 onto PS; the slight difference lies within the experimental error. Therefore, in the third stage, no effect of ionic strength on the adsorption features could be observed. The continuous adsorption can be explained by considering that, after the monolayer formation, the substrate is essentially invisible for the proteins adsorbing in the second and consecutive layers, since the substrates are no longer silicon wafers or PS films but they are enolase biofilms with similar characteristics. This might have been caused by cooperative binding, which is a common process in biological systems. Models were developed to describe the dynamics of multilayer adsorption.10,16,17,32,34 They are based on the random sequential adsorption (RSA) model, where particles are considered irreversibly adsorbed in a monolayer whenever they reach the substrate or another particle. Due to the exclusion area around each adsorbed particle, the surface coverage ranges from 0.38 to 0.75, depending on the geometrical dimensions chosen. One important feature of this model is its infinite memory: once a particle is placed on the surface, it affects the geometry of all later placements. The extended RSA model for multilayer deposition is the so-called CSA model. The first layer is strongly bound and well packed. If a particle sticks on a free site of the surface, it remains there permanently. If it lands on an already adsorbed particle, an overhang amount is described by two parameters: the adsorption parameter; the rolling parameter. If the former overcomes the latter, adsorption will take place. Otherwise, the particle rolls toward the surface and, if the path is unblocked, the particle adsorbs. If the path is blocked by another particle, it desorbs. By adjusting the values of the adsorption parameter and the rolling parameter, one can try to describe experimental situations. The adsorption of the first layer is a fast diffusive process, while the formation of the subsequent layers is a slower process, which can be asymptotically described. To verify if enolase adsorption follows the RSA or CSA model, desorption experiments were performed in the ellipsometric cell exchanging the enolase solution after 16 h of adsorption by pure solvent and further in situ measurements were performed overnight. Enolase did not desorb from any of the studied substrates, indicating irreversible adsorption as predicted by RSA and CSA models. The molecular packing was confirmed by AFM images (Figure 3) obtained in the tapping mode operation for adsorption experiments with enolase bulk concentration of 0.5 g/L (0.001 mol/L NaCl) on silicon wafers. The AFM images show that after 5 min in contact with the enolase solution (Figure 3a), a homogeneous enolase biofilm was formed on silicon surface. The surface rough(34) Ravichandran, S.; Talbot, J. Biophys. J. 2000, 78, 110.

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ness (roughness mean square ) RMS) amounted to 0.50 nm. The RSA model estimates a maximum coverage of 55% of surface for spherical particles.9,16 An estimate of the surface coverage θ as a function of time was made by considering the corresponding adsorbed amount of enolase (Figure 4) and the spherical area of one adsorbed enolase molecule as 22.9 × 10-14 cm2. The latter was calculated from the radius of gyration of an enolase molecule assumed as 1.35 nm.35 This value corresponds to the half of the radius of gyration of the enolase dimmer measured in the presence of magnesium ions.35 The silicon wafer presented an area of 1.0 cm2. The number of adsorbed molecules was calculated from the adsorbed amount and from the molecular weight of one enolase chain (46.7 kDa). The surface coverage θ is calculated from

θ ) (NA)/area

(3)

where N is the number of adsorbed molecules, A is the projected area of an adsorbed molecule, and area is the substrate area. Figure 4a shows the adsorption amount as a function of time obtained for enolase at bulk concentration of 0.5 g/L (0.001 mol/L NaCl) on silicon wafers. Figure 4b shows the surface coverage θ calculated for the first 5 min of adsorption. It increased continuously until the formation of a plateau. After 24 s of adsorption a surface coverage of 52%, which is close to the limit of RSA model, was reached. After 200 s the surface was totally covered (θ ) 1.00) and the monolayer formation was evidenced in Figures 3a and 4a. The θ values corresponding to 5 min or longer are higher than 1.00. It can be due an overestimate about the radius of gyration of enolase. A decrease of 10% in this value would yield a surface coverage of 1.00 for adsorption times longer than 5 min. With increase of the adsorption time to 1, 2, 3, and 16 h, the presence of enolase aggregates could be observed on the homogeneous enolase biofilm, as shown in Figure 3b-g. The number of aggregates and the surface roughness increased with the adsorption time. The latter reached the maximum value of 2.32 nm for 16 h of adsorption. This observation corroborates the kinetic behavior illustrated in Figure 4a. A magnification of the homogeneous biofilm obtained after 2 h (Figure 3d) showed the presence of small spherical entities closely packed on the surface. The aggregates were not present in the early stage (Figure 3a); they appeared as the adsorption time increased, indicating that the preexisting biofilm induced the aggregation of the arriving enolase molecules on the surface. After 16 h of adsorption (Figure 3f), the presence of very large aggregates (52 ( 2 nm high) appeared dispersed on a homogeneous film (Figure 3g). The kinetic curve in Figure 4a presents features similar to those obtained in the dilute range (Figure 1), a fast and linear increase of the adsorbed amount as a function of time in the initial stages, an adsorption plateau until 2 h, indicating the monolayer formation, and for adsorption times longer than 1 h an asymptotic increase of the adsorbed amount with the time takes place. The plateau value of 4.2 mg/m2 observed for enolase onto silicon wafers is comparable to those found for blood proteins onto silicon wafer.11,36 The asymptotic behavior was predicted by the theoretical models described above and might be attributed to cooperative binding. Therefore, the CSA model with adsorbing parameter > rolling parameter seems to (35) www.rcsb.org (pdbID ) 1ONE). (36) Ortega-Vinuesa, J. L.; Tengvall, P.; Lundstro¨m, I. Thin Solid Films 1998, 324, 257.

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Figure 3. AFM images corresponding to the dried enolase-covered silicon wafer after (a) 5 min, (b) 1 h, (c, d) 2 h, (e) 3 h, and (f, g) 16 h. The inserted table shows the RMS data obtained for each image.

describe the large time adsorption behavior of enolase onto solid substrates adequately. Adsorption Isotherms. The adsorption isotherms obtained for enolase from NaCl solution (0.001 mol/L) onto hydrophilic substrate (silicon wafer) and hydrophobic polymeric film (PS) during 10 min, 50 min, and 24 h showed a continuous increase of Γ as a function of the bulk concentration, as shown in Figure 5. Enolase adsorbed strongly onto both substrates. However, a preferential

adsorption onto the hydrophilic or hydrophobic substrate could not be observed. According to Tengvall et al.37 proteins are only slightly stable in aqueous solutions and, in the presence of a foreign surface, the adsorbed state is often the energetically favorable; i.e., the adsorption energy of the system is minimized (∆adsG ) ∆adsH - T∆adsS (37) Tengvall, P.; Lundstro¨m, I.; Liedberg, B. Biomaterials 1998, 19, 407.

Enolase Adsorption onto Solid Substrates

Langmuir, Vol. 18, No. 18, 2002 6919 Table 2. Advancing and Receding Contact Angles Measured for Water Drops on Bare Substrates and on Dried Enolase-Covered Substrates at 23 ( 1 °Ca

Figure 4. (a) Adsorption kinetics measured for enolase (c ) 0.5 g/L in 0.001 mol/L NaCl) onto silicon wafers at 23 ( 1 °C. (b) Surface coverage θ values as a function of time calculated for the first 5 min of adsorption (see details in the text).

Figure 5. Adsorption isotherms measured for enolase onto (a) silicon wafers and (b) PS films at 23 ( 1 °C. The adsorbed amount values correspond to 10 min (9), 50 min (O), and 24 h (2) of adsorption. The lines are guides for the eyes. The error bars correspond to the standard deviations obtained for the mean values of triplicates.

< 0). Enzymes are very complex adsorbates because they possess hydrophobic, hydrophilic, and charged domains. According to PDB data35 and the Raswin Molecular Graphics (Rasmol) program, an enolase chain is composed of 436 amino acid residues, of which 47% are hydrophobic and 53% are hydrophilic.35 Therefore, there is no predominance of one kind of domain. Favorable interactions between the hydrophilic segments and silicon surfaces due to electrostatic interaction and hydrogen bonding, for instance, can contribute to the adsorption process (reduction of ∆adsG). On the other hand, hydrophobic bonding between PS and the hydrophobic enolase segments might lead to small ∆adsH, and in this case, the entropic gain might be the main driving force for the adsorption.15,38 Moreover, depending on the residue distribution on the enolase surface, there may be patches of hydrophobic groups that result in strong adsorption onto hydrophobic substrates. Contact Angle Measurements. Enolase-covered substrates corresponding to those obtained at cenolase ) 0.01 g/L (0.001 mol/L NaCl) and 16 h of adsorption were dried under a stream of N2 prior to the contact angle measurements. Table 2 presents the advancing (θA) and the receding (θR) contact angles measured for bare substrates and for the enolase covered substrates. These findings show that the enolase molecules acquired different conformations when adsorbed onto hydrophobic and hydrophilic substrates. The hydrophilic silicon21 and amino-terminated surfaces21 became hydrophobic after enolase adsorption, indicating that the hydrophobic segments of enolase are exposed to the air, while the hydrophilic segments might be interacting with the surface. PS hydrophobic surface39 turned hydrophilic after the formation of enolase biofilm. In this case, the interac(38) Haynes, C. A.; Norde, W. J. Colloid Interface Sci. 1995, 169, 313. (39) Siqueira-Petri, D. F.; Wenz, G.; Schunk, P.; Schimmel, Th.; Bruns, M.; Dichtl, M. Colloid Polym. Sci. 1999, 277, 673.

substrate

θA (deg)

θR (deg)

∆θ (deg)

Si/SiO2 APS PS Si/SiO2/enolase APS/enolase PS/enolase

5(1 24 ( 1 88 ( 1 72 ( 1 70 ( 1 41 ( 2

19 ( 2 79 ( 2 39 ( 1 32 ( 1 27 ( 1

5 9 33 38 14

a The hysteresis in the contact angle measurements (∆θ) was calculated from the difference between θA and θR. The samples were obtained after16 h of adsorption at cenolase ) 0.01 g/L in 0.001 mol/L NaCl.

Figure 6. Contact angles measured for water drops on dried enolase-covered silicon wafers (9), amino-terminated surfaces (O), and PS films (2) at 23 ( 1 °C as a function of time. The samples were obtained after 16 h of adsorption at cenolase ) 0.01 g/L in 0.001 mol/L NaCl. The lines are guides for the eyes. The error bars correspond to the standard deviations obtained for the mean values of triplicates.

tions between hydrophobic segments of enolase and PS hydrophobic surface lead to the exposure of hydrophilic segments of enolase to the air. The preferential orientation of one kind of enolase segment to the air as a function of the substrate should probably lead to a minimization of the system free energy. The increase in the hysteresis in the contact angle measurements (∆θ) after enolase deposition is in agreement with surface roughness measured by AFM. Figure 6 shows the contact angle values for water drops on enolase biofilm formed on silicon wafer, APS, and PS surfaces as a function of time. The measurements were realized in intervals of 10 min, and the initial time corresponds to the measurement right after the deposition of water drop on the surfaces. During the first 20 min the contact angle values changed up to 10° as a function of time, indicating possible conformational rearrangements on the biofilm in contact with water. According to Lyklema and Norde,15 the proteins can be classified as hard and soft ones. A protein is considered hard when upon adsorption it maintains the structure it has in the dissolved state. On the other hand, a soft protein spread completely upon adsorption, leading to conformational changes in its structure. Thus, enolase might be considered as a soft protein since conformational changes were evidenced by contact angle measurements. pH Effect. Proteins behave like polyelectrolytes, and at moderate ionic strength and variation of pH, they can undergo intramolecular charge interaction, inducing conformational changes due to protonation and deprotonation.40 To understand how pH influences the adsorption behavior of enolase onto silicon wafers and aminoterminated surfaces, adsorption experiments were carried (40) Bagchi, P.; Birnbaum, S. M. J. Colloid Interface Sci. 1981, 83, 460.

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enzyme solubility increases and some repulsive force takes place, leading to a decrease in the adsorbed amount. Conclusions

Figure 7. Adsorbed amount of enolase (cenolase ) 0.01 g/L in 0.001 mol/L NaCl) as a function of pH onto silicon wafers (9) and amino-terminated surfaces (4) at 23 ( 1 °C. The values correspond to 16 h of adsorption. The lines are guides for the eyes. The error bars correspond to the standard deviations obtained for the mean values of triplicates.

out in 0.001 mol/L NaCl during 16 h with the pH values ranging from 3.5 to 9.5. Figure 7 shows the adsorption plateau values Γ as a function of pH. The silicon surface (SiO2) is negatively charged only in pH values above 6.8,41 while the amino-terminated surface is positively charged at pH < 4. In both substrates, the curves are symmetrical with respect to the isoelectric point (pI) of the enolase, where a maximum of the amount adsorbed is observed. This maximum might be explained on the basis of not only the specific interactions, like hydrogen bonding, for instance, with the substrate but also the protein-protein electrostatic repulsion and on the reduction of enzyme solubility at the pI. For pH < pI some enolase segments (carboxylic and amino groups, for instance) might be protonated, increasing the solubility. In this case, the interaction between enolase carboxylic groups and the substrates (SiO2 and amino groups stemming from APS) might be due to hydrogen bonding and electrostatic repulsion between protonated amino groups from enolase and APS surface is expected especially near pH 3.5. For pH > pI, some enolase segments (carboxylic and phosphate groups) might be partially charged, and the favorable interaction with the substrates might be due to hydrogen bonding. On the other hand, electrostatic repulsion between partially charged SiO2 and negatively charged groups from enolase might have reduced the adsorbed amount of enolase. Therefore, far from enolase pI the (41) Loidl-Stahlhofen, A.; Schmitt, J.; No¨ller, J.; Hartmann, T.; Brodowsky, H.; Schmitt, W.; Keldenich, J. Adv. Mater. 2001, 13, 1829.

The long adsorption kinetics of enolase onto hydrophilic silicon wafers, amino-terminated substrates, and hydrophobic PS films presented three distinct regions: (i) a diffusion-controlled one; (ii) an adsorption plateau; (iii) continuous, irreversible, and asymptotic increase of the adsorbed amount with time. Such features are predicted by the RSA and CSA models. At the enolase pI no influence of ionic strength was observed on the enolase diffusion coefficient. However, upon increase of the ionic strength, the plateau values and duration were reduced, probably due to screening effects on the conformational rearrangements. Rearrangements in the adsorbed enolase were also evidenced by means of changes in the contact angle measurements along the time. AFM images of films formed by well-packed entities were attributed to the enolase monolayer formation. Increasing the adsorption time the number of aggregates on the biofilm increased significantly. The adsorption isotherms showed a continuous increase of the adsorbed amount with the bulk concentration, either on hydrophobic or on hydrophilic substrates, which is probably due to the cooperative binding among the enolase biomolecules. No preferential adsorption of enolase onto one kind of the present substrates was observed, even after 24 h of adsorption. It is probably due to the almost equivalent content of hydrophilic and hydrophobic segments in the enolase structure. A maximum of the amount adsorbed was observed at the enolase pI. This maximum might be explained on the basis of not only the favorable interactions between substrate and enolase but also the reduction of its solubility. On the basis of the present findings, enolase could be quickly immobilized either onto hydrophobic or hydrophilic substrates to design sensors for the control of the phosphoenolpyruvate production, an important intermediate in the carbohydrate metabolism, or for the detection of immonuglobulin levels. Acknowledgment. The authors acknowledge the FAPESP (Proc. Nos. 00/08051-3 and 97/13070-2) and CNPq for financial support. We are grateful to the “Laborato´rio de Filmes Finos do IFUSP” of Brazil for the SPM facility (FAPESP Proc. No. 95/5651-0). LA0202982