Structure, Stability, and Activity of Myoglobin Adsorbed onto

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Langmuir 2007, 23, 13007-13012

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Structure, Stability, and Activity of Myoglobin Adsorbed onto Phosphate-Grafted Zirconia Nanoparticles Francesca Bellezza,§ Antonio Cipiciani,*,§ Maria Anna Quotadamo,§ Stefania Cinelli,§,† Giuseppe Onori,§,† and Silvia Tacchi† Centro di Eccellenza Materiali InnoVatiVi Nanostrutturati (CEMIN), Dipartimento di Chimica, UniVersita` di Perugia, Via Elce di Sotto 8, 06123 Perugia, Italy, and Dipartimento di Fisica, UniVersita` di Perugia, Via Pascoli I, +06123 Perugia, Italy ReceiVed May 25, 2007. In Final Form: October 4, 2007 The adsorption of myoglobin (Mb) onto phosphate grafted-zirconia (ZrO2-P) nanoparticles was studied in terms of conformational studies and thermal stability, determined by circular dichroism (CD), differential scanning calorimetry (DSC), and atomic force microscopy (AFM). The changes in protein structure have been correlated with the catalytic activity of free and adsorbed Mb. CD and DSC studies indicate marked rearrangements in Mb structure upon adsorption onto phosphate-grafted zirconia nanoparticles. These structural rearrangements of Mb could be responsible for the loss of catalytic activity observed for the adsorbed Mb. In particular, the conformational changes due to the adsorption process induced a reduction of kcat and KM. AFM measurements indicate that the interaction with the grafted-zirconia nanoparticles also affects the morphology of the bound protein, inducing the nucleation of prefibrillar-like aggregates.

Introduction The adsorption of a protein at a liquid-solid surface usually produces changes in its physicochemical properties, which may affect the biological functioning of the molecules.1 The exposure of proteins to nonbiological solid surfaces may occur in several applications such as artificial implants, proteinpurification strategies, biosensors, and drug delivery systems.2,3 It is well known that these events are influenced by the structural properties of adsorbed proteins. Protein adsorption is a complex process in which the structural stability of a protein, ionic strength, pH, temperature, and the hydrophobicity/hydrophilicity of the surface influence the affinity of the protein for the support.4 The unfolding of protein during the complex formation may lead to a conformational entropy gain. Upon adsorption, a less stable protein (“soft”) will adopt various conformational states ranging from native, through molten globule, to a fully denatured state. Each step gives rise to an increased number of interaction points between the protein and the surface and between the adsorbed proteins themselves.5 Therefore, it is necessary to know the conformational behavior of proteins at solid interfaces in order to understand the mechanism of adsorption and identify the optimal conditions to preserve protein functionality. Knowledge about the physicochemical properties of proteins coupled to different synthetic surfaces is still limited, and a greater understanding of the protein/surface interaction is needed. The focus of this work was to study the adsorption of myoglobin onto phosphate grafted-zirconia (ZrO2-P) nanoparticles in terms of conformation, thermal stability, and retention of biological activity upon adsorption. The protein-nanoparticle biocomposites * To whom correspondence should be addressed. E-mail: [email protected]. Tel.: 075/5855540. Fax: 075/5855560. § CEMIN, Dipartimento di Chimica. † Dipartimento di Fisica. (1) Haynes, C. A.; Norde, W. J. Colloid Interface Sci. 1995, 169, 313. (2) Hlady, V.; Buijs, J. Curr. Opin. Biotechnol. 1996, 7, 72. (3) Nakanishi, K.; Sakiyama, T.; Imamura, K. J. Biosci. Bioeng. 2001, 91, 233. (4) Duinhoven, S.; Poort, R.; Van der Voet, G.; Agterof, W. G. M.; Norde, W.; Lyklema, J. J. Colloid Interface Sci. 1995, 170, 340. (5) Moulin, A. M.; O’Shea, S. J.; Badley, R. A.; Doyle, P.; Welland, M. E. Langmuir 1999, 15, 8776.

were characterized by circular dichroism, differential scanning calorimetry, and atomic force microscopy. The far-UV circular dichroism (CD) spectra of protein are sensitive to secondary protein structure. CD can be used to characterize the extent of secondary structure elements and is well suited for the spectral analysis of proteins in both the adsorbed and soluble states.6 A common method for studying the structural stability of proteins is to follow the equilibrium unfolding of the native state by modifying the temperature. Differential scanning calorimetry (DSC) is a powerful tool for performing thermodynamic investigations of protein stability.7,8 DSC measures the temperature-dependent excess heat that is adsorbed upon the unfolding of a protein, and the accumulated excess heat reflects the enthalpy that stabilizes the protein in the native state. Furthermore, the shape of the thermal denaturation curve allows one to draw additional conclusions concerning the structural heterogeneity within a protein population. Finally, AFM is ideally suited for both the visualization of nanostructured materials and measuring the spatial dimensions of features on the surface of nanomaterials. Although adsorption onto solid surfaces has been widely investigated,9-11 information about the correlation of structural data with biological activity measurements, allowing valuable structure-function relationships, is quite rare. Myoglobin (Mb) has been one of the most studied proteins with respect to structure, function, and folding. It is a heme protein characterized by a strong spectroscopic signature that is suitable for monitoring protein binding or redox activity.12 Mb is not an enzyme, but it can oxidize a number of substrates, such as 2-methoxyphenol, in the presence of hydrogen peroxide, although its activity is less than that of native peroxidase.13 (6) Kondo, A.; Mihara, J. J. Colloid Interf. Sci. 1996, 174, 214. (7) Privalov, P. L.; Potekhin, S. A. Method. Enzymol. 1986, 131, 4. (8) Sturtevant, J. M. Annu. ReV. Phys. Chem. 1987, 38, 463. (9) Norde, W.; Giacomelli, C. E. J. Biotechnol. 2000, 79, 259. (10) Vermeer, A. W. P.; Bremer, G, M. G. E.; Norde, W. Biochim. Biophys. Acta 1998, 1425, 1. (11) Giacomelli, C. E.; Norde, W. J. Colloid Interface Sci. 2001, 233, 234. (12) Antonini, E.; Brunori, M. Hemoglobin and Myoglobin in their Reactions with Ligands; North-Holland: Amsterdam, 1971. (13) Redaelli, C.; Monzani, E.; Santagostini, L.; Casella, L.; Sanangelantoni, A. M.; Pierattelli, R.; Banci, L. ChemBioChem 2002, 3, 226.

10.1021/la7015269 CCC: $37.00 © 2007 American Chemical Society Published on Web 11/17/2007

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Aqueous suspensions of colloidal particles are excellent model systems for studying the adsorption of proteins onto solid surfaces.14 The colloidal size of the nanoparticles gives a large surface area with well-defined properties. Moreover, the surface properties can be designed for special purposes by grafting organic functions onto a monodisperse core particle.15 Previous investigations showed that colloidal particles obtained by grafting phosphate and phosphonates onto zirconia are good supports for the immobilization of myoglobin.16 The nanostructured biocomposites were active in the oxidation of 2-methoxyphenol by hydrogen peroxide, and the catalytic efficiency was similar to that of free Mb. In this study the Mb adsorption characteristics have been considered, especially the changes in protein structure, and related to the catalytic activity of the adsorbed Mb. Protein structure and thermal stability, determined by CD, DSC, and AFM, have been studied, and the results have been correlated with enzymatic activity in solution and in the adsorbed state. Experimental Section Materials. Myoglobin (from horse heart, 90% pure, Lot 044K7006) was obtained from Sigma and purified by gel-filtration chromatography on a Sephadex G-50 column. Synthesis of ZrO2-P Nanoparticles. Grafted zirconia nanoparticles were prepared by a process described in ref 16. In brief, a colloidal suspension of zirconium oxide nanoparticles was prepared by hydrothermal treatment of the zirconium acetate solution. TEM micrographs analysis indicated a system of welldispersed particles with mean diameters of 60 nm. The X-ray diffraction pattern of the solid, collected after drying, was characteristic of monoclinic zirconia and the BET analysis of N2-adsorption isotherms indicated the formation of particles with 113 m2/g surface area. Phosphoric acid can be bound onto zirconia particles by heating a mixture of colloidal ZrO2 and the acid ([ZrO2] ) 0.06 M, [acid] ) 0.03 M, P/Zr ) 0.5) at 110 °C for 4 h. After grafting, the particles remained discrete with a mean diameter similar to that of the starting zirconia. The XRD patterns of the grafted particles (ZrO2-P) were those of the initial zirconia. The absence of a typical lamellar zirconium phosphate pattern indicated that the grafting reaction of zirconia nanoparticles is a surface reaction and that the core of the particles is not attacked. The BET surface area of the dried particles remained large (132 m2/g for ZrO2-P). ZrO2-P does not contain UV-adsorbing groups, hence, because of its negligible adsorption and low scattering in the UV region, this material is particularly suitable to serve as a sorbent for studying the circular dichroism of adsorbed proteins. Adsorption of Mb onto ZrO2-P Nanoparticles. An aqueous solution of Mb was prepared dissolving a fixed amount of Mb in a volume of deionized water without buffer. The resulting pH was measured and a value of 7.0 was found. To examine the Mb adsorption onto ZrO2-P as a function of time, a 2.0 mL volume of the aqueous solution of Mb (C ) 1.4 mg/mL) was added to a colloidal suspension of ZrO2-P nanoparticles (C ) 4 mg/mL, V ) 2.0 mL, pH 7.0 adjusted with LiOH 0.2 M). The mixture was incubated for 30 min, 1 h, 2 h, and 3 h at room temperature, and the adsorbed protein was recovered by centrifuging at 12 000 rpm for 15 min. The pellet was washed, and the supernatants were collected for determination of free protein. The protein concentration was assayed by UV spectra (λ ) 410 nm,  ) 160 000 M-1 cm-1). The amount of adsorbed protein was calculated from the difference between the value of the protein concentration in the (14) Haupt, B.; Neumann, T.; Wittemann. A.; Ballauff, M. Biomacromolecules 2005, 6, 948. (15) Carrie`re, D.; Moreau, M.; Barboux, P.; Boiolot, J. P.; Spalla, O. Langmuir 2004, 20, 3449. (16) Bellezza, F.; Cipiciani, A.; Quotadamo, M. A. Langmuir 2005, 21, 11099.

Bellezza et al. initial solution and that in the supernatants. In all the experiments the same amount of protein was adsorbed (0.28 mg Mb bound/mg support), showing that the adsorption of Mb onto grafted zirconia was very fast. In order to examine the Mb adsorption onto ZrO2-P as a function of protein concentration, a 0.5 mL volume of an aqueous solution of Mb (C ) 40 mg/mL, pH 7.0) was added to a colloidal suspension of ZrO2-P nanoparticles (C ) 30 mg/mL, V ) 0.6-4.8 mL, pH 7.0 adjusted with LiOH 0.2 M). The mixture was incubated for 1 h at room temperature, and the adsorbed protein was recovered by centrifuging at 12 000 rpm for 15 min. The pellet was washed, and the supernatants were collected for determination of free and immobilized protein as described above. The pellet was resuspended in 0.2 M phosphate buffer at pH 6.0; this provided a suspension with a known concentration of immobilized Mb to be used for catalysis. Enzymatic Activity. The enzymatic activity assays for both native and bound Mb were performed by measuring the initial oxidation rates of 2-methoxyphenol (guaiacol) in the presence of hydrogen peroxide according to a described procedure.17 For thermal inactivation studies, the native and bound proteins were first incubated at the fixed temperature for 1 h, and then the kinetic measurement was performed at 25 °C. All the reactions were done at saturation conditions of substrates to measure the maximal reaction rate (Vmax) expressed as micromoles of product formed per micromoles of protein per second. The following concentrations were used: (a) for native protein, [Mb] ) 1.0 µM, [guaiacol] ) 30 mM, [H2O2] ) 76 mM; (b) for bound Mb, [Mb] ) 1.5 µM, [guaiacol] ) 30 mM, [H2O2] ) 570 mM. CD Measurements. The far-UV CD spectra were performed by means of a Jasco model J-810 spectropolarimeter at 25 °C using rectangular quartz cell with a 0.1 cm path length. Water circulating through a jacket around the cell maintained the temperature of the cell. The CD readings were expressed as the mean residue ellipticity [θ] (mdeg cm2 dmol-1). Samples for CD measurements were prepared by adding different volumes of colloidal solution of given concentration to a fixed amount of aqueous solution of Mb in order to obtain mixtures consisting of free and bound Mb. The concentration of bound protein was estimated for each sample by using the method described in previous paragraphs. The investigated ratio of bound/total protein (X) ranged from 0 (protein free) to 0.98. All Mb/ZrO2-P mixtures were then diluted as needed for the CD measurements. Protein desorption upon dilution was checked by UV spectroscopy as previously described. No traces of free protein were found in the reaction medium. The CD spectra of myoglobin adsorbed onto ZrO2-P nanoparticles were recorded after 1 h from their preparation, to be sure that the adsorption process was realized. In all samples the contribution of light scattered by zirconia nanoparticles in the CD signal was small and can be considered negligible. This allowed experiments to be performed in the far-UV. The spectrum of ZrO2-P nanoparticles at an appropriate concentration was subtracted from the CD spectrum of the sample. DSC Measurements. Differential scanning calorimetry was performed using a micro-DSC II Setaram (France), equipped with two cells with a 1 cm3 capacity. The same procedure as that used for CD measurements was used to prepare the protein-nanoparticle biocomposites. DSC thermograms were acquired at a protein concentration of 10 mg/mL. In order to check the independence on concentration, some test measurements were performed in the range between 10 and 30 mg/mL. The mixture was prepared directly in the measurement cell and was stirred for 1 h at room temperature before the measurements were taken. The total mass of the measured sample in the cell was fixed at 0.83 g. The reference cell was filled with an equal mass of colloidal solution of nanoparticles. Sample and reference cells were scanned from 20 to 100 °C at a scan rate of 30 °C h-1. (17) Bellezza, F.; Cipiciani, A.; Costantino, U.; Nicolis, S. Langmuir 2004, 20, 5019.

Adsorption of Mb onto ZrO2-P Nanoparticles A baseline thermogram, obtained by scanning the colloidal solution in both the sample and reference cells, was subtracted from the sample thermogram in order to minimize systematic differences between the cells. The raw DSC data were converted to excess heat capacity (Cpexc) versus temperature by dividing each datum point by the scan rate and the protein concentration in the sample cell. Atomic Force Microscopy (AFM). Tapping mode AFM imaging was performed in air by using a Solver Pro scanning probe microscope (NT-MDT, Moscow, Russia). Rectangular silicon cantilevers, 100 µm long, with resonant frequencies in the range 190-325 kHz and a nominal force constant between 5.5 and 22.5 N/m (typical 11.5 N/m), were used. Image data, between 5 and 7 µm, were acquired at a scan rate of about 0.5 Hz. The AFM measurements were performed on the native Mb, the pure ZrO2-P nanoparticles, and two Mb/ZrO2-P mixtures, characterized by a bound/total protein ratio of 0.50 and 0.98. The samples were prepared at room temperature and pH 4.7, with the method described in the previous paragraphs. A pH 4.7 value was chosen to allow the binding between the Mb/ZrO-P complex and the mica support. Since the pH value was different from that used for the adsorption experiments (7.0), the amount of Mb bound was checked at pH 4.7, and no significant difference in the bound/total protein ratio was found. Moreover, CD measurement indicated that at pH 4.7 Mb is in the native state. Immediately prior to the AFM measurements, all samples were diluted. The aqueous solution of Mb was diluted to a concentration of 0.01 mg/mL, the colloidal suspension of ZrO2-P nanoparticles to a concentration of 0.06 mg/mL, and finally the two Mb/ZrO2-P mixtures were diluted to a concentration of 0.014 mg/mL of Mb and 0.04 mg/mL of ZrO2-P (X ) 0.50) and of 0.014 mg/mL of Mb and 0.18 mg/mL of ZrO2-P (X ) 0.98), respectively. Protein desorption upon dilution was checked by UV spectroscopy, and no traces of free protein were found in the reaction medium. The samples were then deposited on freshly cleaved ruby mica and blow dried with nitrogen gas. Desorption. The possibility of desorption of Mb from the ZrO2-P nanoparticles was checked in all experimental conditions used in this work by UV spectroscopy as previously described. No desorption was observed in water upon dilution of the samples, in phosphate buffer (0.2 M, pH 6.0), in the temperature range for activity and DSC measurements. Protein desorption was observed in phosphate buffer (0.2 M, pH 8.0), where about 90% of desorbed Mb was found in the supernatant.

Results and Discussion Adsorption Isotherm. Colloidal ZrO2-P nanoparticles were used for the Mb adsorption studies. The inorganic nanoparticles, with the acidic hydroxyl functions present on the surface, were negatively charged at the pH used for the adsorption experiments (7.0) and had an average diameter of 60 nm, as observed with transmission electron microscopy (TEM).16 The presence of various lysine residues distributed over the surface of horse heart Mb12 (IEP 7.1) could be responsible for the electrostatic interactions with the negatively charged nanoparticles. In the adsorption studies, different protein/particle ratios were used to evaluate the dependence of Mb adsorption on protein concentration under the same conditions used for the CD and DSC experiments. The adsorption isotherm of Mb onto ZrO2-P nanoparticles is shown in Figure 1. As can be observed, the plateau value (0.28 mg Mb bound/mg support) agrees well with the previously reported one and is compatible with a monolayer of adsorbed molecules, according to the Langmuir model.16 Enzymatic Activity as a Function of Temperature. In a previous study,16 the oxidation of 2-methoxyphenol, catalyzed by Mb immobilized onto ZrO2-P nanoparticles, was performed. The rates of substrate oxidation by the bound Mb showed

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Figure 1. Adsorption isotherm of Mb onto ZrO2-P nanoparticles (pH 7.0, T ) 20 °C, t ) 2 h, X ) Mb bound/Mb total, Ce ) residual concentration of Mb in solution after adsorption).

Figure 2. Residual peroxidase activity of native Mb and Mb/ZrO2-P (0.28 mg Mb bound/mg support) as a function of temperature (the activity was reported as a percentage of the initial value obtained at 20 °C).

Michaelis-Menten saturation kinetics demonstrating that the immobilized protein reacts similarly to Mb in solution. The kcat and KM values were affected by the adsorption of Mb onto the nanoparticles, and the values were smaller than those for the free protein (for native Mb, kcat ) 5.5 s-1, KM ) 14.2 mM, and kcat/KM ) 0.39 s-1mM-1; for bound Mb, kcat ) 0.28 s-1, KM ) 1.8 mM, and kcat/KM ) 0.16 s-1mM-1).16 In this work, the effect of temperature on the catalytic activity of myoglobin has been investigated. Both native and bound Mb were heated for 1 h at a fixed temperature and then tested for peroxidase activity in the guaiacol/hydrogen peroxide system at 25 °C. Figure 2 shows the activity changes as a function of temperature expressed as a percentage of the initial value obtained at 20 °C (Vmax ) 1.1 s-1 for free protein and Vmax ) 0.14 s-1 for adsorbed protein). No change in activity was observed for native Mb, below 70 °C, but an abrupt decrease was observed above this temperature. For Mb immobilized onto ZrO2-P nanoparticles, the observed changes in activity as a function of temperature were quite different, with a slight decrease observed at 30 °C. The major decrease was observed at 40 °C, followed by minor decreases up to 100 °C. CD Measurements. In order to investigate if and how the secondary structure of myoglobin is influenced by the adsorption onto the ZrO2-P nanoparticles, the myoglobin spectrum in the far-UV region was recorded with and without particles. CD spectra of free and adsorbed myoglobin for Mb/ZrO2-P mixtures at various bound/total protein ratios X are shown in Figure 3. The far-UV CD spectrum of myoglobin in its native state exhibits a positive ellipticity band below 200 nm, and a double

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Figure 3. CD spectra of myoglobin adsorbed onto ZrO2-P nanoparticles at various ratios X (black, X ) 0; red, X ) 0.27; green, X ) 0.51; blue, X ) 0.71; purple, X ) 0.89; cyan, X ) 0.98). In the inset, CD spectra of heat-unfolded myoglobin and of the mixture characterized by X ) 0.89 are shown.

Figure 4. Fractions of bound myoglobin (fb) obtained by CD spectra as a function of X. The solid line is the best linear fit.

minima at 210 and 222 nm. These features are characteristic of proteins that are rich in R-helices.18,19 A gradual decrease in the intensity of all bands of Mb/ZrO2-P mixtures CD spectra with increasing particle concentration is observed. Particularly, a dramatic reduction in the intensity of the CD bands of the spectrum corresponding to completely bound protein is evident. It should be observed that the features of this spectrum are similar to those of heat-unfolded myoglobin (Figure 3). This indicates that myoglobin adsorbed onto ZrO2-P nanoparticles is devoid of an ordered secondary structure. All spectra show an isosbestic point at 202 nm. The presence of an isosbestic point is clear evidence of an equilibrium between two optically absorbing species. According to this, we hypothesized the existence of protein in two conformation states in the solution, in both the native free state and the unfolded bound state. That could account for the CD spectra trend as a function of Mb bound percentage. In fact, for any X value, the CD spectrum can be expressed as a linear combination of the spectra of molecules in the native (X ) 0) and bound (X ) 0.98) states:

[θ] ) ff [θf] + fb[θb] where [θf] and [θb] are the CD spectra and ff and fb are the fractions of free and bound molecules, respectively. This procedure provides fb values with an error of ( 0.05. The values of the bound Mb fractions (fb) as a function of X are plotted in Figure 4. We see that with an increase in the adsorbed amount, the quantity shows a linear fit with a slope equal to 1. (18) Woody, R. W. In Circular dichroism. Principles and applications; VHC Publishers: New York, 1994; p 473. (19) Manavalan, P.; Johnson, W. C., Jr. Nature 1983, 831, 305.

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Figure 5. Melting profiles of myoglobin in the absence and presence of grafted zirconia nanoparticles at three selected values of X.

These results indicate that myoglobin undergoes significant structural changes upon adsorption, losing its ordered secondary structure. DSC Measurements. To study the influence of adsorption on the myoglobin stability, the thermal denaturation of Mb/ZrO2-P complexes was investigated by DSC measurements. It is wellknown that differential scanning calorimetry (DSC) is a useful tool for studying the unfolding of proteins and gaining information about protein folding and stability.7,8 For this reason it is a sensitive method for investigating the conformational changes of protein adsorbed onto particle surfaces. Figure 5 shows the specific heat capacity, at constant pressure, Cpexc, as a function of temperature, for Mb/ZrO2-P mixtures at various bound/total protein ratios X. The thermal profile of native myoglobin in water exhibits a single endothermic peak typical of protein unfolding.20 Denaturation occurs at (75.3 ( 0.2) °C with a transition enthalpy of (390 ( 10) kJ/mol. The introduction of ZrO2-P nanoparticles into the protein solution induces significant changes in the thermal profile. In fact, a progressive decrease of the peak height of transition was observed with increasing particle concentration (Figure 5). When the totality of Mb added to the solution is completely adsorbed onto the particle surface, the relative DSC thermogram loses the main peak. At the same time, the thermogram of adsorbed Mb shows the appearance of a broad band at temperatures much lower than the onset of free protein unfolding. This broad transition, probably due to a thermal effect on protein/particle complex, seems to be correlated with the activity decrease as a function of temperature observed for Mb adsorbed onto ZrO2-P nanoparticles (Figure 2). The enthalpy for the thermal unfolding of myoglobin in aqueous solution (∆H0) as well as in the adsorbed state for various adsorption conditions was estimated by integrating the areas under the principal peak. In Figure 6, the values RCAL)(1 ∆HX/∆H0) are plotted as a function of X. The data are well fitted by a straight line with a slope value close to 1. The linear trend of RCAL as a function of X, together with the reduction of the heat capacity features in DSC thermograms of Mb adsorbed to nanoparticles, indicates that the adsorbed protein lacks the native structure conformation. This is in good agreement with the CD results. The slope values equal to 1 (Figures 4 and 6) exclude the presence of a folded-bound state, obviously within the experimental error (5%). (20) Privalov, P. L. AdV. Protein Chem. 1979, 33, 167.

Adsorption of Mb onto ZrO2-P Nanoparticles

Figure 6. Dependence of the calorimetric enthalpy (normalized) as a function of bound/total Mb ratio X.

Desorption. The protein desorption from a surface has been demonstrated in many systems. A protein that is released from a surface may either retain the conformation it had in the adsorbed state or partly refold into the original native structure. Desorption of Mb molecules from the ZrO2-P nanoparticles was tested in the presence of phosphate buffer. It was found that about 90% of the adsorbed Mb was desorbed in the presence of phosphate buffer at pH 8.0 (0.2 M) after 30 min.

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Desorbed Mb was studied in terms of structure and enzymatic activity. It was seen that the CD spectra of the desorbed Mb were similar to those of the native Mb (data not shown) while the kinetic parameters kcat and KM, obtained in the oxidation of 2-methoxyphenol by hydrogen peroxide, were similar to those of native Mb. It can be concluded that Mb completely regains its native conformation after being desorbed from ZrO2-P nanoparticles; the adsorption process can therefore be defined as reversible. Atomic Force Microscopy (AFM). The AFM technique was used to study the morphology features induced in the Mb by the interaction with ZrO2-P nanoparticles. First, the native protein and the ZrO2-P nanoparticles have been analyzed separately. As can be seen in Figure 7a, the native Mb self-organizes in small globular structures whose height ranges between 10 and 50 nm. The pure ZrO2-P nanoparticles, instead, assemble into very large (200-350 nm tall), irregularly shaped aggregates (Figure 7b). From a statistical analysis of the AFM data, the mean height of the individual ZrO2-P nanoparticles ranged between 50 and 60 nm. Then, the mixture characterized by a bound/total protein ratio of 0.50 was imaged. The ZrO2-P nanoparticles were found to

Figure 7. AFM images taken in air of (a) globular aggregates of native Mb, (b) large and irregularly shaped aggregates of pure ZrO2-P.

Figure 8. AFM images taken in air of Mb/ZrO2-P mixtures characterized by a bound/total protein ratio of 0.50 (a-c) and 0.98 (d). The Z-range of the images in panels c and d has been extremely amplified to highlight the presence of the prefibrillar-like structures, which are very much lower than the ZrO2-P aggregates. (a) Scan area showing the large, irregularly shaped aggregates of ZrO2-P, (b) scan area showing the Mb globular aggregates, (c) scan area showing large assemblies of ZrO2-P surrounded by branching prefibrillar-like aggregates of Mb, and (d) scan area showing large assemblies of ZrO2-P and prefibrillar-like Mb aggregates (marked by arrows). In the inset (Z-range 10 nm) a magnification of a prefibrillar-like aggregate is shown.

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self-organize into aggregates similar to those imaged for the pure ZrO2-P (Figure 8a). Concerning the Mb morphology, two distinct structures were observed: (i) globular aggregates, similar to those imaged for the native protein (Figure 8b), and (ii) very extensive, branching structures of Mb, with morphological properties similar to prefibrillar aggregates (Figure 8c).21-25 Both the appearance and height (2-4 nm) of these latter structures are consistent with prefibrillar-like assemblies previously imaged for myoglobin.26 Similar prefibrillar-like structures have also been found in a mixture with a bound/total protein ratio of 0.98. In such a case, the aggregates are smaller and less abundant, because of a lower percentage of Mb in the sample (Figure 8d and inset). Finally, AFM measurements have been performed by changing the incubation time of mixtures. We found that the morphology and the dimensions of prefibrillar-like aggregates remain stable, while the formation of long and straight fibrillar aggregates has been never observed. The nucleation of prefibrillar-like aggregates, observed in the AFM images, indicates that one part of the Mb present in the mixture undergoes an alteration of its native conformation, due to the interaction with the ZrO2-P nanoparticles. It is well-known, indeed, that Mb can nucleate fibrillar structures only under destabilizing conditions, where it is in at least partially unfolded conformation.27-30 Moreover, it is interesting to note that in both the two mixtures the prefibrillar-like aggregates have been always observed next to the ZrO2-P nanoparticles. This result suggests that prefibrillar-like structures develop from the bound protein, consistant with the DC and DSC measurements, which indicate that the bound Mb is in an unfolded state. (21) Chamberlain, K.; MacPhee, C. E.; Zurdo, J.; Morozova-Roche, L. A.; Hill, H. A. O.; Dobson, C. M.; Davis, J. J. Biophys. J. 2000, 79, 3282. (22) Kad, N. M.; Myers, S. L.; Smith, D. P.; Smith, D. A.; Radford, S. E.; Thomson, N. H. J. Mol. Biol. 2003, 330, 785. (23) Srinivasan, R.; Jones, E. M.; Liu, K.; Ghiso, J.; Marchant, R. E.; Zagorski, M. G. J. Mol. Biol. 2003, 333, 1003. (24) Arimon, M.; Dı´ez-Pe´rez, I.; Kogan, M. J.; Durany, N.; Giralt, E.; Sanz, F.; Ferna`ndez-Busquets, X. FASEB J. 2005, 19, 1344. (25) Gosal, W. S.; Morten, I. J.; Hewitt, E. W.; Smith, D. A.; Thomson, N. H.; Radford, S. E. J. Mol. Biol. 2005, 351, 850. (26) Malmo, C.; Vilasi, S.; Iannuzzi, C.; Tacchi, S.; Cametti, C.; Irace, G.; Sirangelo, I. FASEB J. 2005, 19, 346. (27) Fa¨ndrich, M.; Fletcher, M. A.; Dobson, C. M. Nature 2001, 410, 165. (28) Uversky, V. N.; Fink, A. L. Biochim. Biophys. Acta 2004, 1698, 131. (29) Rochet, J.; Lansburry, P. T., Jr. Curr. Opin. Struct. Biol. 2000, 10, 60. (30) Fa¨ndrich, M.; Forge, V.; Buder, K.; Kittler, M.; Dobson, C. M.; Diekmann, S. Biochemistry 2003, 23, 15463.

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Conclusions In this study DSC and CD spectroscopy have been combined with catalytic activity studies to investigate the relationship between the structural modification of Mb upon adsorption on ZrO2-P nanoparticles and its biological functioning. Mb adsorption from aqueous solution onto ZrO2-P nanoparticles is a fast process due to the high affinity of protein for the nanoparticle surface. The adsorption process strongly influences the catalytic activity and thermal stability of Mb. CD and DSC studies indicate drastic rearrangements in the Mb structure upon adsorption onto ZrO2-P nanoparticles (loss of native structure with a CD spectrum feature similar to that of the heat-unfolded protein). These structural rearrangements of Mb could be responsible for the loss of catalytic activity observed for the adsorbed Mb and its temperature dependence. In particular, the conformational changes due to the adsorption process result in an appreciable reduction of kcat and KM. The variation of kcat and KM after immobilization alters the catalytic efficiency, expressed by the kcat/KM ratio, although the obtained value is similar to that of free protein. Last, the AFM measurements reveal that the interaction with the ZrO2-P nanoparticles affects the morphology of the bound protein, inducing the nucleation of prefibrillar-like aggregates. This result is particularly interesting for its biological implications. It is well-known that fibrillar structures are associated with many debilitating human disorders such as Alzheimer’s disease and transmissible spongiform encephalopathies. In most cases, including Mb, prefibrillar aggregates have been shown to display the highest cytotoxicity whereas mature fibrils appear less toxic or even harmless.31-35 Acknowledgment. We are grateful to Dr. Silvia Vilasi for helpful discussions. LA7015269 (31) Walsh, D. M.; Selkoe, D. J. Protein Pept. Lett. 2004, 11, 213. (32) Lambert, M. P.; Barlow, A. K.; Chromy, B. A.; Edwards, C.; Freed, R.; Liosatos, M.; Morgan, T. E. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 6448. (33) Lashuel, H. A.; Hartley, D.; Petre, B. M.; Waltz, T.; Lansbury, Jr. Nature 2002, 418, 291. (34) Nilsberth, C.; Westlin-Danielsson, A.; Eckman, C. B.; Condron, M. M.; Axelman, K.; Forsell, C.; Stenh, C.; Luthman, J.; Teplow, D. B.; Younkin, S. G.; et al. Nat. Neurosci. 2001, 4, 887. (35) Walsh, D. M.; Klyubin, I.; Fadeeva, J. V.; Cullen, W. K.; Anwyl, R.; Wolfe, M. S.; Rowan, M. J.; Selkoe, D. J. Nature 2002, 416, 535.