Effect of pH on the Adsorption and Activity of Creatine Phosphokinase

Jan 17, 2006 - The combination of in situ ellipsometry with atomic force microscopy in the liquid for the study of adsorption of creatine phosphokinas...
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J. Phys. Chem. B 2006, 110, 2674-2680

Effect of pH on the Adsorption and Activity of Creatine Phosphokinase Sabrina M. Pancera,†,‡ Hartmut Gliemann,‡ Thomas Schimmel,‡,§ and Denise F. S. Petri*,† Instituto de Quı´mica, UniVersidade de Sa˜ o Paulo, P.O. Box 26077, Sa˜ o Paulo, SP, 05513-970, Brazil, Institut fu¨r Nanotechnologie, Forschungszentrum Karlsruhe GmbH, D-76021 Karlsruhe, Germany, and Institut fu¨r Angewandte Physik, UniVersita¨t Karlsruhe (TH), D-76128 Karlsruhe, Germany ReceiVed: June 15, 2005; In Final Form: NoVember 3, 2005

The combination of in situ ellipsometry with atomic force microscopy in the liquid for the study of adsorption of creatine phosphokinase (CPK) onto silicon wafers was shown for the first time. The thickness, adsorbed amount, and topographic information of the adsorbed CPK layers were obtained under different pH conditions. The thickness values of adsorbed CPK layer determined by both techniques were in excellent agreement. At pH 4, CPK monomers present in solution adsorb, forming a very thin (∼0.8 nm) layer, indicating CPK unfolding. Upon increasing the pH to 6.8, the adsorbed layer is composed of a mixture of CPK dimers (native structure) and intermediates, increasing the film thickness (∼2.4 nm). At pH 9, CPK dimers form monolayers with the highest thickness (∼4.0 nm). The nature of interactions between CPK and Si wafers associated with the hydration force seems to control the degree of CPK unfolding upon adsorbing. The enzymatic activity of free CPK and of adsorbed CPK at pH 4, pH 6.8, and pH 9 was measured as a function of pH. In comparison to free CPK in solution, adsorbed CPK presented a shift of the optimal pH from 6.8 toward alkaline pH.

Introduction Adsorption of proteins onto solid surfaces plays a key role in a number of industrial1 and biomedical2-4 applications. Not only the adsorbed amount of protein or enzyme but also the biomolecule orientation upon adsorption affects the activity and stability of the immobilized entities.5-7 The advance of instrumental techniques has allowed one to go deeper into the study of adsorption behavior of protein in liquid. Combined in situ measurements provide reliable information about the thickness, adsorbed amount, and adsorption kinetics with the corresponding three-dimensional structural arrangement,8 without any artifact due to drying process.9-11 Creatine phosphokinase (CPK) has been largely used in clinical assays for the diagnosis of myocardial infarction and muscular disease.12-14 CPK plays an important role in the rapid regeneration of ATP in cells where such demands are high, reversibly catalyzing the transfer of phosphate from phosphocreatine to creatine.15 Ingestion of phosphocreatine has become extremely popular with many athletes for muscle building and performance enhancement. The elevated level of CPK in blood is an important diagnostic indicator for diseases of the nervous system and heart.15 Moreover, some data suggest that creatine and creatinine (dehydrated creatine) may act as precursors of food mutagens and uremic toxins.15 These facts evidence the relevance of developing creatine or creatinine assays.13-14 The activity of CPK in solution has been determined over a broad pH range.16-17 At the pH range of 6-9.6, the native activity is fully retained, while at pH lower than 4.8, the dissociation of dimers (native structure) into monomers causes dramatic loss of activity.17 However, the adsorption of CPK onto solid surfaces over a broad pH range and the determination of CPK activity * To whom correspondence should be addressed. Telephone: 0055 11 3091 3831. Fax: 0055 11 3815 5579. E-mail: [email protected]. † Universidade de Sa ˜ o Paulo. ‡ Forschungszentrum Karlsruhe. § Universita ¨ t Karlsruhe.

after adsorption are still unexplored. Here, we show the effect of pH on the adsorption behavior of CPK onto silicon wafer, a hydrophilic substrate, by means of in situ ellipsometry and atomic force microscopy (AFM) under water and the role played by the adsorption of CPK on the enzymatic activities under different pH conditions. Experimental Section Materials. Silicon, Si, (100) wafers purchased from Wacker (Germany) with a native oxide layer approximately 2 nm thick were used as substrates. Si wafers cut in typical dimension of 1 cm × 1 cm were rinsed with a mixture of NH3 (28% in volume), H2O2 (30% in volume), and distilled water in the volume ratio of 1:1:5 at the temperature of 75 °C during 20 min. Afterward, the wafers were washed with jets of distilled water and dried under a stream of N2.18 The adsorption of CPK (Sigma C3755) onto silicon substrates was studied at 25 ( 1 °C in the dilute regime (enzyme concentration ) 0.005 g L-1) in a 1 mM NaCl solution at pH 4 (obtained with HCl), pH 6.8, and pH 9 (obtained with NaOH). Creatine phosphate (P-6502), β-Nicotinamide adenine dinucleotide phosphate (NADP+, N0505), adenosine 5′-diphosphate (ADP, A-2754), β-D-(+)glucose (G5250), glucose-6phosphate dehydrogenase Type IX from bakers yeast (G-6-PDH, G4134), and hexokinase (HK, H5000), obtained from Sigma (St. Louis, USA) were used without further purification. Methods. Ellipsometry. Ellipsometric measurements were performed in a vertical computer-controlled DRE-EL02 Ellipsometer (Ratzeburg, Germany), as described elsewhere.19 The angle of incidence φ was set to 70.0°, and the wavelength λ of the laser was 632.8 nm. For the data interpretation, a multilayer model composed by the substrate, the unknown layer, and the surrounding medium should be used. Then the thickness (dx) and index of refraction (nx) of the unknown layer can be calculated from the ellipsometric angles, ∆ and Ψ, using the fundamental ellipsometric equation and iterative calculations

10.1021/jp0532364 CCC: $33.50 © 2006 American Chemical Society Published on Web 01/17/2006

Adsorption and Activity of Creatine Phosphokinase

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with Jones matrixes20

ei∆ tan Ψ ) Rp/Rs ) f(nk,dk,λ,φ)

dimers, monomers, or unfolded monomers reported in the literature,24 by the Stokes-Einstein relation

(1)

where Rp and Rs are the overall reflection coefficients for the parallel and perpendicular waves, respectively. They are a function of the angle of incidence φ, the wavelength λ of the radiation, the indices of refraction, and the thickness of each layer of the model, nk, dk. First of all, the thickness of the SiO2 layers was determined in air, considering the index of refraction for Si as n ) 3.88 i0.01821 and its thickness as an infinite one, for the surrounding medium, the index of refraction was measured at 25 °C with an Abbe´ refractometer. The index of refraction determined for NaCl 0.001 mol/L amounted to 1.3335. Because the native SiO2 layer is very thin, its index of refraction was set as 1.462, and just the thickness was calculated. The mean SiO2 thickness measured for 50 samples amounted to 1.9 ( 0.1 nm. The adsorption from solution was monitored in situ with the help of a cell made of poly(methyl methacrylate), which is filled with 30 mL of enzyme solution. 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 a conditioned room at the temperature of 25 ( 1 °C. The small differences in the indices of refraction of the substrate, CPK and solution make an independent determination of nenzyme and denzyme impossible. Therefore, nenzyme was kept constant as 1.50, and denzye was calculated. The hydrodynamic thickness could be calculated if the optical contrast in the system were higher. 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 nenzymedenzyme should be a constant value.22 This product is the parameter necessary to calculate the adsorbed amount of enzyme Γ23

Γ)

denzyme(nenzyme - n0) dn/dc

(2)

where n0 is the index of refraction of the solution, dn/dc is the increment of refractive index determined with a differential refractometer. For our system, dn/dc amounted to 0.16 mL/g at the temperature of 25 °C. The kinetic model applied here considers that at the beginning the substrate is completely uncovered and serves a sink for each arriving macromolecule. By consideration of the boundary conditions for the concentration (c(x ) 0, t) ) 0 and c(x,t ) 0) ) c0) and Fick’s second law, the concentration profile can be described as a function of the diffusion coefficient D. According to Fick’s first law the flux to the surface is determined by the derivative of the concentration profile at x ) 0. The integration of the flux with respect to time leads to eq 3. This model has been described in details by Motschmann and co-workers22 for the adsorption of polystyrene-block-poly(ethylene oxide) onto Si wafers. The apparent diffusion coefficients D has been calculated from the slopes of Γ/time0.5 and eq 322

Γ(t) )

2 cbulkxDt xπ

(3)

The self-diffusion coefficient (Dt) was calculated for the diffusion of CPK dimers, monomers, or unfolded monomers considering the corresponding hydrodynamic radii, Rh, of CPK

Rh ) kT/(6πηDt)

(4)

where k is the Boltzmann’s constant and η is the solvent viscosity, assumed as 1 mPa‚s. AFM. AFM analyses of enzyme covered surfaces were carried out in water, within a liquid cell, with a multimode atomic force microscope connected to a Nanoscope III controller (Digital Instruments, USA) at room temperature. Contact-mode barshaped cantilevers (CSC 12 from NT-MDT, Russia) with an average force constant of 0.6 N m-1 were applied for intermittent contact mode in the liquid cell. This type of cantilevers showed the best performance when adjusted to a drive frequency of about 30 kHz in liquid. The samples were washed with water five times before measurements to avoid adsorption of enzymes on the AFM tip. It is important to avoid that the sample runs dry during the washing procedure. The thickness (d) of the adsorbed layer was also determined by wet AFM using the tipscratch method, where the biofilm was carefully removed using the AFM tip until obtaining a 5 µm × 5 µm area free of enzyme25 (see Supporting Information). The mean roughness (root mean square, rms) values were calculated for 1 µm × 1 µm areas using the Nanoscope software version 5.12r3. ActiVity Measurements. The enzymatic activity of free CPK molecules in solution and of immobilized CPK after 5 h adsorption at pH 4, pH 6.8, and pH 9 was determined by spectrophotometry using a Beckmann Coulter DU 640 UVvis Spectrophotometer at 25 °C. CPK, in the presence of Mg2+ ions, is responsible for catalyzing the transfer of high energy phosphate from creatine phosphate to ATP (eq 5).15 Through a coupled reaction with HK, which catalyses the transfer of the γ-phosphoryl group from ATP to the hydroxyl group on carbon 6 of some hexoses, mainly glucose (eq 6) and glucose-6phosphate dehydrogenase (G-6-PDH) (eq 7), it is possible to indirectly measure the activity of CPK.26 The reduction of NADP+ to NADPH (eq 7), which absorbs at the wavelength of 340 nm, is monitored.26-28 The absorbance at 340 nm was then measured in function of time. By use of the Beer-Lambert equation, the amount of NADPH formed can be obtained, considering NADPH molar absorptivity29 as  ) 6.22 mol-1‚L cm-1 and the optical pathway length as 1 cm. CPK

creatine phosphate + ADP 98 creatine + ATP D-glucose

HK

+ ATP 98 glucose-6-P + ADP

(5) (6)

G6PDH

glucose-6-P + NADP+ 98 gluconate-6-P + NADPH + H+ (7) For the activity measurements, the concentration of each reactant added to the spectrophotometric cell was the following: 0.6 10-3 mol‚L-1 NaCl, 0.03 mol‚L-1 creatine phosphate, 0.0021 mol‚L-1 ADP, 0.046 mol‚L-1 D-glucose, 0.0046 g‚L-1 or 0.011 × 10-6 mol‚L-1 HK, 0.00182 g‚L-1 G-6-PDH, and 0.0038 10-3 mol‚L-1 NADP+. The pH of the reactants mixture was adjusted to 4, 6.8, or 9 by adding HCl, 1 × 10-3 mol‚L-1 NaCl solution or NaOH, respectively. The enzymatic activity experiments have been performed using free CPK concentrations, which correspond to those adsorbed onto Si wafers. The mass of adsorbed CPK onto Si wafers of 1 cm2 in area was calculated by dividing the adsorbed amount (plateau value) by 104. For instance, at pH 6.8 the mass of adsorbed CPK was

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Figure 1. Adsorption kinetics of CPK on Si surfaces from solutions at pH 6.8 (open squares), pH 4 (open triangles), and pH 9 (open circles). (a) In situ ellipsometric measurements and (b) AFM under water using the tip-scratch method. The lines are just guides for the eyes.

calculated as 2.4 × 10-4g (see Results section). By consideration that the total reactant volume of 658 µL, the concentration of CPK was 3.5 × 10-4 g‚L-1, which was used for the measurement of enzymatic activity for free CPK. Similarly, the amounts of CPK concentration adsorbed at pH 4 and pH 9 were calculated and the corresponding concentrations for a total reactant volume of 658 µL were estimated. The enzymatic activity was measured at pH 4 and pH 9 using free CPK at the concentration of 1.21 × 10-4 g‚L-1 and 6.83 × 10-4 g‚L-1, respectively. Prior to the activity measurements with adsorbed CPK, Si wafers were immersed in CPK solution (c ) 0.005 g‚L-1) at pH 4, pH 6.8, or pH 9 for 5 h. After that period of time, the samples were washed with jets of distilled water and incubated with the reactant mixture as above described. The enzymatic activity was measured at pH 4, 6.8, and 9. The enzymatic activity for free CPK or adsorbed CPK was measured after 2 h of incubation, when one aliquot has been removed and the absorbance at 340 nm was measured.

Figure 2. Adsorption kinetics measured for CPK onto Si surfaces from solutions at (a) pH 4, (b) pH 6.8, and (c) pH 9. Lines show the region used for calculating the diffusion coefficient (D).

TABLE 1: Diffusion Coefficient (D) and Thickness (d) of the CPK Film at the Plateau Determined from the Ellipsometric Dataa PH

d (nm)

D (cm2/s)

4 6.8 9

(0.8 ( 0.1) (2.4 ( 0.1) (4.5 ( 0.1)

1.2 × 10-7 8.0 × 10-7 2.7 × 10-7

a Results obtained for the adsorption of CPK on silicon from CPK solutions 0.005 g/L in NaCl 1mM at pH 4, 6.8, and 9.

TABLE 2: Hydrodynamic Radii (Rh)36 and Diffusion Coefficient (Dt) Estimated for CPK Monomers, Dimers, or Unfolded Monomers, at the Corresponding pH Range

Results The Effect of pH on the Adsorption of CPK. The adsorption behavior of CPK onto silicon wafers at pH 4, pH 6.8, or pH 9 was investigated by means of in situ null ellipsometry and AFM under water. The thickness (d) of CPK films as a function of time was determined by ellipsometry (Figure 1a) and AFM (Figure 1b). At pH 6.8 (squares) and pH 4 (triangles) the thickness values obtained by both methods are in good agreement. At pH 9 (circles), the thickness values obtained by AFM are slightly smaller than those determined by ellipsometry. The present findings corroborate with Gesang and co-workers,25 who observed excellent agreement between ex situ AFM and ellipsometry for continuous films in the thickness range of 1-10 nm. Regardless the pH condition, the thickness of the adsorbed CPK layer increases until the appearance of a plateau, indicating

completely unfolded monomer Monomer Dimmer

pH range

Rh (nm)

Dt (cm2/s)

7

6.1 2.8 3.5

3.3 × 10-7 7.2 × 10-7 5.7 × 10-7

the formation of a monolayer.30 The apparent diffusion coefficients D were calculated from the slopes of Γ/time0.5 (Figure 2). The thickness values found by ellipsometry, for the CPK films formed under different pH conditions at the plateaus, as well as the D values are presented in Table 1. Self-diffusion coefficient (Dt) presented in Table 2 were estimated for the diffusion of CPK dimers, monomers or unfolded monomers considering the corresponding hydrodynamic radii, Rh, of CPK dimers, monomers, or unfolded monomers reported in the literature24 and eq 4. The comparison between D and Dt shows that they are in the same order of magnitude, indicating that

Adsorption and Activity of Creatine Phosphokinase

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Figure 3. AFM (tapping mode) images 5 µm × 5 µm of the adsorbed CPK film measured for CPK (c ) 0.005 g/L) onto silicon wafers at pH 4 (a) after 15 min, (b) after 2 h adsorption, and (c) after 16 h adsorption; at pH 6.8 (d) after 15 min, (e) after 2 h adsorption, and (f) after 16 h adsorption; at pH 9 (g) after 15 min, (h) after 2 h adsorption and (i) after 16 h adsorption. The rms values were calculated for zoom images of 1 µm × 1 µm.

the dimensions of diffusing entities are similar to those expected24 at the corresponding pH. AFM images of the silicon surface after CPK adsorption at pH 4 are shown in parts a-c of Figure 3. During the transport, manipulation, and storage of the Si wafers, the Si surfaces suffered some scratches (0.5 ( 0.2 nm deep), which can be seen until after 2 h of adsorption (parts a and b of Figure 3), indicating that after this period of time the bare silicon wafer was not completely covered by the enzyme. After 16 h (Figure 3c) no scratches can be detected anymore and the surface seems to be completely covered by CPK. The mean roughness (rms) values increased considerably after 16 h of adsorption, although the corresponding mean thickness values did not change. The adsorption process at pH 6.8 is faster than at pH 4 because the scratches on silicon, which can still be seen after 15 min of enzyme adsorption (Figure 3d), after 2 h (Figure 3e) cannot be seen anymore. The CPK film morphological features and rms values did not change significantly up to 16 h of CPK adsorption (Figure 3f). Some aggregates (1.3 ( 0.7 nm high) appeared on the surface, consistent with continuous increase of d observed from the ellipsometric data (Figure 1a). Figure 3g shows the AFM topography image of the surface after 15 min CPK adsorption at pH 9. No scratches can be observed on the surface, indicating that after this short period of time a film is already formed onto silicon. For longer adsorption time, the films are smooth without aggregates (parts h and i of Figure 3). At the early stages, when the surface coverage is incomplete (parts a, d, and g of Figure 3) one must be aware that the

thickness values in Figure 1a might present up to 50% of deviation. The deviation stems from the ellipsometric model, which assumes a homogeneous layer of CPK, but as evidenced by AFM the surface is not completely covered by CPK. To estimate the thickness deviations due to false index of refraction, the thickness values were calculated as a function of index of refraction values starting with 1.34 (close to the medium) and finishing with 1.50 (pure protein). The average thickness value showed deviation of 50% in relation to the limiting values, which is in agreement with literature data.31 On the other hand, it is very difficult to state the exact amount of voids in the adsorbed layer as a function of time. Therefore, all calculations were performed considering the index of refraction for the adsorbed protein layer as n ) 1.50, but one should notice that at the early stages the thickness values might present up to 50% of deviation. Especially in cases of heterogeneous or rough layers, AFM is a perfect complement for ellipsometry. As the surface coverage increases, the adsorbed layer becomes homogeneous with mean thickness deviations of 10% (maximum), caused mainly by systematic errors due to the optics and step motors. The Effect of pH on the Enzymatic Activity of Free CPK. The effect of pH on the enzymatic activity of free CPK, expressed as the formation of NADPH as a function of time, is shown in Figure 4. The largest activity was observed at pH 6.8, followed by that at pH 9 and pH 4. This trend goes along with fluorescence data32 for CPK, which indicated that at pH g 7

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Figure 4. NADPH formation of as a function of time catalyzed by free CPK at pH 4 (open triangle), pH 6.8 (solid square), and pH 9 (open circle).

Figure 5. Amount of NADPH formed after 2 h of reaction under pH 4, 6.8, and 9 catalyzed by free CPK and adsorbed CPK on silicon wafers at pH 4, 6.8, and 9.

the CPK native structure is kept, while unfolding was observed under acidic conditions. The Effect of Initial pH on the Enzymatic Activity of Adsorbed CPK as a Function of pH. The enzymatic activities related to the NADPH formation after 2 h of reaction determined for adsorbed CPK under different pH conditions and compared with those in solution are shown in Figure 5. As expected, upon adsorbing the enzymatic activity was in a general way reduced due to conformational changes, causing entropic gain, which drives the adsorption process.33,34 When CPK was adsorbed at pH 4, and then the enzymatic activities measured at pH 4, 6.8, and 9, the maximum value was observed at pH 6.8 and the minimum at pH 4, following the tendency observed for free CPK. However, different behaviors were observed for CPK immobilized at pH 6.8 and 9. The maximum activity was observed at pH 9, while measurements at pH 6.8 and 4 led to lower enzymatic activity values. Therefore, these findings show that CPK might be adsorbed at pH 6.8 or 9, but the optimal condition to determine the enzymatic activity is at pH 9. Control experiments of enzymatic activity were performed with CPK-coated surfaces (under different pH conditions), which were allowed to dry after adsorption. This type of control experiment is important because drying might cause undesirable structural changes.9-11 The activities with dried CPK-covered surfaces were comparable to those obtained with “wet” CPKcoated surfaces, indicating that in this case drying process did not lead to any substantial structural changes.

The Effect of pH on the Adsorption Behavior of CPK. To have a better insight into the adsorption behavior of CPK onto Si wafers as a function of pH, one should first describe the influence of pH on the CPK conformation in bulk solution. Liang et al.32 studied the unfolding of CPK induced by acid and described the acid-induced unfolding of CPK as a “threestate” model. Above pH 7 CPK dimers composed of two identical monomers (with 43 kDa each) can be observed, when the pH decreases the CPK dimers start to separate into monomers. Between pH 6.7 and 5.0 equilibrium mixture of dimers and a partially folded monomer (intermediate which has a significant amount of exposed hydrophobic surface area) is found. Decreasing the pH continuously from 5.0 to 3.0, a mixture of the intermediate and an unfolded monomer is observed and at pH 3.0 the monomers are almost fully unfolded. The consistency observed for Dt values (Table 2) and the experimental D data (Table 1) under different pH conditions (Table 1) strongly support Liang’s model.32 Once the effect of pH on CPK conformation in solution was well described and supported by experimental data, the next question concerns the correlation between the CPK conformation in bulk solution with the thickness of adsorbed CPK layer. At pH 4, a mixture of CPK intermediates and unfolded CPK monomers with Rh of 6.1 nm is expected. However, upon adsorbing the monomers spread over the substrate, so that the monolayer reaches the mean thickness of (0.8 ( 0.1 nm). Entropic gain due to the conformational changes upon adsorbing33 and hydrogen bonding between CPK polar residues and surface Si-OH groups probably drive the irreversible adsorption process (no desorption could be observed). If the enzyme is unfolded multipoint attachment to the substrate is expected. The adsorbed enzyme would require very large activation energy to desorb. At pH 6.8, a mixture of CPK dimers and intermediates with a mean Rh of 2.8 nm might be present in bulk solution. From the adsorption kinetic of CPK onto silicon at pH 6.8 (Figure 1), one might suggest that at the first stages, dimers and intermediates diffuse in the solution toward free sites at the surface. After 5 h adsorption, the plateau value of 2.4 ( 0.1 nm (Table 1) corresponds to a monolayer of intermediates (partially folded monomers) and dimers adsorbed in a conformation similar to that in solution. Electrostatic interaction between SiO- groups on the surface and patches of CPK positively charged residues might take place. For longer adsorption times, the substrate is no longer Si wafer but a CPK layer. The continuous increase of d with time indicates the multilayer formation with CPK molecules adsorbing onto the CPK layer. At pH 9, the enzyme keeps its native structure with Rh of 3.5 nm (Table 2) in bulk solution. The thickness values obtained by AFM and ellipsometry ranged from 3.7 to 4.4 nm (Figure 1), indicating that probably the dimers keep their structure after adsorption, forming a monolayer. Similarly to the condition of pH 6.8, both surface and CPK are negatively charged; however electrostatic interaction between patches of CPK positively charged residues and negatively charged surface might contribute to the adsorption process. Similar effects have been reported in the literature.37,38 The adsorption behavior of CPK onto Si wafers depends strongly on pH (Figures 1 and 2), since the pH medium controls the charge density on substrate and adsorbate. The isoelectric points of CPK35 and SiO236 are 6.5 and 6.8, respectively. In the early adsorption stages, at pH 6.8 and 9 electrostatic repulsion

Adsorption and Activity of Creatine Phosphokinase between CPK negatively charged patches and SiO- on the surface might cause delay in the adsorption of the first CPK molecules. After short adsorption time, the substrate becomes a heterogeneous surface composed of SiO- and CPK. This “new” substrate is more attractive for the arriving CPK molecules than the bare SiO-, which existed at t ) 0. This effect can be correlated with the rise in the adsorption rate (slopes) observed in parts b and c of Figure 2. At pH 4, the substrate is silanol rich and CPK carboxylic and amine groups are protonated. In this case, electrostatic interactions play no role and the observed behavior is that expected for noncharged macromolecules. The initial adsorption rate is attributed to the diffusion of proteins from bulk solution to bare substrate (Figure 2a), when all biomolecules that arrive at the substrate are assumed to be immediately adsorbed. After approximately 10 min the adsorption rate decreased. The substrate is no longer a homogeneous surface, it is composed of free sites and sites occupied by already adsorbed CPK molecules. The arriving biomolecules might stick on the latter, due to cooperative binding. Afterward, they might diffuse to the free sites on the substrate, followed by conformational changes,30,33 turning this adsorption stage slower than the first one. Therefore, at pH 6.8 and 9, where the adsorption of CPK onto Si wafers is driven by electrostatic interactions the surface did not induce CPK conformational changes. However, at pH 4, where the adsorption is controlled mainly by H bonding, the substrate seems to induced CPK conformational changes. One could think about some possible reasons for this behavior: (i) enzyme stabilization due to the substrate electrostatic double layer at pH 6.8 or 9, (ii) the nature of interaction between substrate and adsorbate, or (iii) weaker hydration forces at pH 4. The explanations i and ii have been explored by Quiquampoix39 for the adsorption behavior of β-D-glucosidase onto mineral particles, which was contrary to that observed for CPK onto Si wafers. At pH below the β-d-glucosidase isoelectric point (pI), the adsorption was driven by electrostatic interactions, which led to enzyme conformational changes. Close to the pI the adsorption was driven by H bonding, van der Waals forces, and the hydrophobic effect, which were too weak to modify the enzyme conformation. At pH above pI, no adsorption took place. CPK and β-D-glucosidase presented opposite adsorption behavior onto mineral surfaces. It seems to be difficult to propose a universal model for protein adsorption because of their complex nature with variable contents of hydrophilic and hydrophobic residues on the surface. From the experimental point of view, it is not trivial to separate the buried charges from the charges on surface. Moreover, when two hydrophilic surfaces are brought into contact, repulsive forces of about 1-nm range have been measured in aqueous electrolyte between mineral surfaces. Because of the correlation with the low energy of wetting of these solids with water, the repulsive force has been attributed to the energy required to remove the water of hydration from the surface. These forces, termed as hydration forces,40,41 extend over more than two water layers. In fact, such forces are not only due to structured water layers but also to the osmotic effect, which can be expressed as the changes in the number of water molecules associated with the single macromolecules undergoing conformational changes. For instance, water activity affects both glucose equilibrium and HK turnover in such a way that the affinity for glucose increases with decreasing water activity.42 At pH 6.8 and 9 the contribution of strong hydration forces associated to electrostatic interaction might drive the adsorption of less unfolded CPK. At pH 4 weaker hydration forces might favor the release of

J. Phys. Chem. B, Vol. 110, No. 6, 2006 2679

Figure 6. Scheme of the CPK layer formed onto silicon wafers at (a) pH 4, (b) pH 6.8, and (c) pH 9 with the corresponding layer thickness at the adsorption plateau.

water molecules upon the CPK adsorption, bringing about a pronounced enzyme denaturation. The hypotheses proposed above can be related to the enzymatic activity observed for the adsorbed CPK. However, one must keep in mind upon increasing the medium pH, not only CPK and substrate turn more negative but also HK and G-6-PDH, which are both involved in the NADPH formation (eq 5-7), so that each reaction (eq 5-7) might be affected by pH variation. By comparison of the amounts of NADPH formed with the control experiment using free CPK at pH 4 with those obtained with the CPK adsorbed at pH 4, 6.8, and 9, one observes that CPK adsorbed at pH 4 presented the lowest activity, 15% of NADPH formed with the control experiment. On the other hand, CPK adsorbed at pH 6.8 and 9 catalyzed 60 and 53% of NADPH in comparison with the control. Regardless the pH at which CPK was adsorbed, the amount of NADPH produced at pH 6.8 was approximately 10% of that produced with the control at pH 6.8. At pH 9, the amounts of NADPH formed in the presence of CPK adsorbed at pH 4, 6.8, and 9 corresponded to 18, 51, and 44% of that obtained with free CPK at pH 9. By consideration of these comparisons and the absolute amounts of NADPH formed with the corresponding standard deviations (Figure 5), one concludes that for practical purposes, as sensors development, CPK should be adsorbed at pH 6.8 or 9 but that the activity should be measured at pH 9. Standard procedures for the activity determination in solution of CPK recommend performing reactions (eq 5 to 7) at pH 7.4 (probably because of the stability of HK (eq 6) is affected under alkaline conditions); however, the present findings indicate the shift of optimal pH for activity of immobilized CPK. Such shift of optimal pH for activity has been recently reported for acid phosphatase and alkaline phosphatase, which were entrapped in silica gel matrixes.43 The activity behavior observed for immobilized CPK onto Si wafers under different pH conditions can be correlated with the adsorbed CPK layers characteristics. Incubation of CPK in solution at low pH (pH ≈ 4) induced to intermolecular β-sheets structures, as evidenced by IR spectroscopy, to decrease of R-helix structures, as observed by circular dichroism measurements, and CPK monomers formation.17 From ellipsometric data and AFM analysis a very thin film was observed (Figure 6a). In accordance with the D values shown in Table 1, at pH 4 CPK monomers present in solution diffuse to the substrate and adsorb, forming the biolayer. Weak hydration forces facilitate

2680 J. Phys. Chem. B, Vol. 110, No. 6, 2006 H bonding between CPK and Si wafers, causing denaturation upon adsorbing and low enzymatic activity. In the pH range of 5-7, a mixture of dimers (native structure of CPK) and intermediates present in solution might adsorb onto Si wafers, so that the dimers portion is large enough to yield considerable activity (Figure 6b). At pH 9 the CPK films onto Si wafers presented thickness corresponding to a monolayer formed by dimers, which is the CPK native structure, and the highest activity (Figure 6c). Hornemann and co-workers44 proposed that the enzymatic cooperativity between the monomers within the stable dimer improve the catalytic properties. Under alkaline conditions, the contribution of strong hydration forces associated to electrostatic interaction might weaken the CPK unfolding upon adsorption onto Si wafers. Conclusions The combination of AFM in the liquid and in situ ellipsometry yielded the thickness, the adsorbed amount, and the topography of the immobilized CPK layer onto Si wafers under variable pH conditions. At pH 4, monomers present in solution adsorb, forming a very thin (∼0.8 nm) layer. Upon increasing the pH to 6.8, the adsorbed layer is composed of a mixture of CPK dimers (native structure) and intermediates, increasing the film thickness (∼2.4 nm). At pH 9, CPK dimers form monolayer with the highest thickness (∼4.0 nm). The nature of interactions between CPK and Si wafers associated with the hydration force seems to control the degree of CPK unfolding upon adsorbing. The optimal pH value for enzymatic activity of free CPK determined with the help of coupled reactions, where HK and G-6-PDH are also involved, was 6.8. The activity of adsorbed CPK at pH 4 was unsatisfactory, because the low amount of NADPH formed, regardless the medium pH. On the other hand, when CPK was adsorbed at pH 6.8 or 9, the amounts of NADPH indicated the shift of the optimal pH toward alkaline pH. This behavior might be correlated to the effect of pH on the CPK conformation upon adsorbing onto Si wafers, as proposed in the model (Figure 6). However, one must be aware that this model attempts to describe the particular system CPK/Si wafer. It might suit other substrate/protein systems, but as Quiquampoix39 argued, any hypothesis of conformational changes in enzymes or proteins must take into account electrostatic forces, which can vary with the particular amino acid sequence. The present results show a way to obtain CPK covered Si wafers as advantageous devices for creatine or creatinine detection because they can be easily prepared, transported, and applied. Acknowledgment. The authors gratefully acknowledge financial support from FAPESP, CNPq, and DAAD. Supporting Information Available: Section analysis. This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) Alonso, J.; Barredo, J. L.; Armise´n, P.; Dı´ez, B.; Salto, F.; Guisa´n, J. M.; Garcia, J. L.; Corte´s, E. Enz. Microb. Technol. 1999, 25, 88. (2) Slomkowski, S. Prog. Polym. Sci. 1998, 23, 815. (3) Levey, A. S.; Perrone, R. D.; Madias, N. E. Annu. ReV. Med. 1998, 39, 465. (4) Hao, X. Enzymatic substrate chip, its manufacture and application. Patent CN 1459635, 2003.

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