Structure and Catalytic Behavior of Myoglobin Adsorbed onto

Jul 13, 2009 - Tamara Posati , Valentina Benfenati , Anna Sagnella , Assunta Pistone , Morena Nocchetti , Anna Donnadio , Giampiero Ruani , Roberto ...
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Structure and Catalytic Behavior of Myoglobin Adsorbed onto Nanosized Hydrotalcites Francesca Bellezza, Antonio Cipiciani,* Loredana Latterini, Tamara Posati, and Paola Sassi Dipartimento di Chimica, Universit a di Perugia, via Elce di Sotto, 8, 06123 Perugia, Italy, and “Centro di Eccellenza Materiali Innovativi Nanostrutturati” (CEMIN), Universit a di Perugia, via Elce di Sotto 10, 06123 Perugia, Italy Received April 23, 2009. Revised Manuscript Received June 24, 2009 The adsorption of myoglobin (Mb) onto nanosized nickel aluminum hydrotalcite (NiAl-HTlc) surface was studied, and the structural properties of the resulting protein layer were analyzed by using FT-IR, Raman, and fluorescence spectroscopies. Upon adsorption onto the nanoparticle surface, the protein molecules maintained their secondary structure, while the tertiary structure was altered. The fluorescence spectra and anisotropy values of adsorbed Mb revealed that the emitting amino acid residues are affected by different microenvironments when compared to the native protein behavior. Moreover, the decrease of fluorescence decay times of tryptophan indicated the occurrence of interactions among the fluorophores and the constituents of the nanoparticles, such as the metal cations, which can take place when conformational changes of Mb occur. Raman spectra indicated that the interaction of Mb molecules with NiAl-HTlc nanoparticles modified the porphyrin core, changing the spin state of the heme iron from high spin (HS) to low spin (LS). The enzymatic activity of the nanostructured biocomposite was evaluated in the oxidation of 2-methoxyphenol by hydrogen peroxide and discussed on the basis of structural properties of adsorbed myoglobin.

Introduction During the last few decades, the interfacial behavior and the adsorption of biomacromolecule such as proteins on solid inorganic surfaces have attracted much attention.1 The adsorption of a protein onto a nonbiological solid surface is an important phenomenon not only from a fundamental point of view but also because it is the key to several important applications such as artificial implants, protein-purification strategies, biosensors, drug delivery systems, catalysts, and catalyst supports.2 Protein adsorption is a complex process involving many events such as conformational changes, hydrogen bonding, and/or hydrophobic and electrostatic interactions. Although surfaceprotein interactions are not well understood, surface chemistry has been shown to play a fundamental role in protein adsorption.3,4 Proteins adsorb in different quantities, conformations, and orientations, depending on the chemical and physical characteristics of both protein and support surfaces. In the biomaterials field, much research has been devoted to methods that modify the size and textural surface of existing materials in order to achieve more desirable biological integration.5 The exposure of a solid surface to biological fluids normally leads to the adsorption of proteins, and the adsorbed protein layer can further mediate additional responses, such as cell attachment and activation, and can create unpredicted perturbations to *Corresponding author. Tel: 075/5855540. Fax: 075/5855560. E-mail: [email protected]. (1) Gray, J. J. Curr. Opin. Struct. Biol. 2004, 14, 110. (2) (a) He, L.; Dexter, A. F.; Middelberg, A. P. J. Chem. Eng. Sci. 2006, 61, 989. (b) Sels, B. F.; De Vos, D. E.; Jacobs, P. A. Catal. Rev.;Sci. Eng. 2001, 43, 443. (c) Martinez Martinez, V.; De Cremer, G.; Roeffaers, M B. J.; Sliwa, M.; Baruah, M.; De Vos, D. E.; Hofkens, J.; Sels, B. F. J. Am. Chem. Soc. 2008, 130, 13192. (3) (a) Bellezza, F.; Cipiciani, A.; Costantino, U.; Negozio, M. E. Langmuir 2002, 18, 8737. (b) Bellezza, F.; Cipiciani, A.; Costantino, U.; Marmottini, F. Langmuir 2006, 22, 5064. (4) (a) Nath, N.; Hyun, J.; Ma, H.; Chilkoti, A. Surf. Sci. 2004, 570, 98. (b) Roach, P.; Farrar, D.; Perry, C. C. J. Am. Chem. Soc. 2005, 127, 8168. (5) Caruso, F. Adv. Mater. 2001, 13, 11.

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device operations.6 Studies on both natural and synthetic clay minerals including hydrotalcite-like compounds (HTlc’s) have been extensively carried out because of their low toxicity, good biocompatibility, and possible use in pharmaceutical, cosmetic, and biomedical applications.7,8 HTlc’s are layered solids with positively charged layers and interlayered charge balancing anions. Their structure is similar to that of brucite, the naturally occurring Mg(OH)2, in which the Mg atoms are octahedrically coordinated by six OH groups; each OH group is shared by three octahedral cations and points to the interlayer space. The general formula of synthetic HTlc is [M(II)1-x M(III)x (OH)2]x+ [An-x/n]x- 3 mH20, where M(II) is a divalent cation (Mg, Zn, Ni,..), M(III) is a trivalent metal cation (Al, Fe, Cr,...), An- is an anion of charge n, and m is the molar amount of cointercalated water.9 HTlc’s are widely applicable not only to build various supramolecular structures and heterogeneous hybrid systems but also to stabilize and protect biomolecules (DNA, enzymes, oligonucleotides, etc.), and to develop drug delivery systems.10 Although HTlc’s present structures and intercalation properties similar to cationic clays, these materials have been scarcely exploited for the adsorption of biological macromolecules such as proteins and enzymes at the solid-liquid interface. For this reason, a more in-depth knowledge of the properties of protein adsorption of HTlc could be useful to improve their biocompatibility. Many researchers have indicated that an important factor in determining the biological response of solid materials is the (6) Bajpai, A. Polym. Int. 2005, 54, 304. (7) Choy, J. H.; Choi, S. J.; Oh, J. M.; Park, T. Appl. Clay Sci. 2007, 36, 122. (8) Costantino, U.; Ambrogi, V.; Nocchetti, M.; Perioli, L. Microporous Mesoporous Mater. 2008, 107, 149. (9) Rives, V. Layered Double Hydroxides: Present and Future; Nova Science Publishers: New York, 2001. (10) Kwak, S. Y.; Jeong, Y. J.; Park, J. S.; Choy, J. H. Solid State Sci. 2002, 151, 229.

Published on Web 07/13/2009

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particle size. Attention has been focused on nanomaterials, which offer a new pathway for regulating protein behavior through surface interactions because they can provide large surface areas for efficient protein binding, and multivalent functionalities can be grafted on their surfaces to meet the structural complexity of biomolecules.11 Recently, we prepared nanoparticles of nickel aluminum hydrotalcites (NiAl-HTlc) in bromide form with the doublemicroemulsion method.12 In the present study, the adsorption of myoglobin (Mb) onto NiAl-HTlc nanoparticles has been investigated in terms of structural properties and enzymatic activity. Mb was chosen as the heme-protein model because of its well-defined structure and extensively characterized catalytic properties. It has a molecular weight of 17.8 kDa, an isoelectric point of 7.2, and a molecular dimension of 4.5  3.5  2.0 nm3.13 The adsorption of Mb onto NiAl-HTlc nanoparticles was studied as a function of pH, and the catalytic activity was evaluated in terms of peroxidase-like activity, with 2-methoxyphenol (guaicol) and hydrogen peroxide as substrates. The adsorbed protein was characterized by using FT-IR, Raman, and fluorescence spectroscopies to obtain structural information for explaining the observed catalytic activity of the biocomposite. The adsorption and kinetic parameters were also discussed in comparison with those obtained with NiAl-HTlc prepared by the urea hydrolysis method, which allowed one to obtain particles of larger dimension (4-5 μm).14 The HTlc’s in bromide form were obtained by ion exchange; first, titrating the carbonate form with 0.1 M HCl and then equilibrating the chloride form with 2M NaBr.

Experimental Section Materials. Myoglobin (from horse heart, 90% pure, LOT 056K7008), hydrogen peroxide (30% v/v in water), and 2-methoxy-phenol were purchased from Sigma-Aldrich. Synthesis of Colloidal Dispersion of HTlc Nanoparticles. Colloidal aqueous dispersions of NiAl-HTlc were prepared by the double-microemulsion technique as described in ref 12. Briefly, two microemulsions designated A and B were prepared by dispersing 12.5 g (0.034 mol) of CTABr and 15.5 mL (0.169 mol) of n-butanol in 36.2 mL (0.219 mol) of isooctane. The aqueous phase of A was a solution of Ni(NO3)2 3 6H2O (0.4 M) and Al(NO3)3 3 9H2O (0.125 M), while the aqueous phase of B was a NH3 solution (1.25 M). Equal volumes of the two microemulsions, A and B, were mixed to obtain the precipitation of NiAl hydrotalcites in the reverse micelles. The resulting system (μAB) was stirred at room temperature for 15 min, after which it became cloudy and was aged at 75 °C for 15 h. After aging, the particles were recovered by centrifuging (12000 rpm for 10 min), and a semitransparent gel was obtained. The gel, was washed with isooctane (1  30 mL), with water (2  30 mL), and a methanolchloroform mixture (1:1 v/v) (3  30 mL) and then dispersed in water to give a colloidal solution. Adsorption of Mb onto HTlc Nanoparticles. The colloidal dispersions of HTlc were characterized in terms of nanoparticle concentration by drying a fixed volume and weighing the resulting solid. The pH of the dispersion was then measured (5.0), and (11) (a) Katz, E.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 6042. (b) Bellezza, F.; Cipiciani, A.; Quotadamo, M. A. Langmuir 2005, 21, 11099. (c) Bellezza, F.; Cipiciani, A.; Quotadamo, M. A.; Cinelli, S.; Onori, G.; Tacchi, S. Langmuir 2007, 23, 13007. (12) Bellezza, F.; Cipiciani, A.; Costantino, U.; Nocchetti, M; Posati, T. Eur. J. Inorg. Chem. 2009, 2603. (13) Antonini, E.; Brunori, M. In Hemoglobin and Myoglobin in Their Reaction with Ligands; North-Holland Publishing Co.: Amsterdam, 1971. (14) Costantino, U.; Marmottini, F.; Nocchetti, M.; Vivani, R. Eur. J. Inorg. Chem. 1998, 1439.

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drops of NaOH solution (0.2 M) were added to get the desired value (6.0 or 9.0). A volume of dispersion, containing about 10 mg of solid, was added to a volume of aqueous solution of protein at different concentrations (1/1 v/v). The mixture was stirred for 1 h at room temperature, and the biocomposite was recovered by centrifuging at 12000 rpm for 10 min. The supernatant clear solution was assayed for protein concentration, and the amount of bound protein was calculated from the difference between the concentration of protein in the initial solution and in the supernatant. The Mb concentration was assayed by UV spectroscopy (λ = 410 nm, ε=160 000 M-1 cm-1). X-ray powder diffraction (XRPD) patterns of the biocomposites are identical to those of the original supports; in particular, no detectable variation in the interlayer distance confirmed that the protein was adsorbed essentially on the external surface of the HTlc nanoparticles. FT-IR Spectroscopy. The biocomposite Mb/HTlc was prepared according the above-described procedure. A 2 mL volume of HTlc colloidal dispersion, containing about 10 mg of solid, was added to a 2 mL volume of aqueous solution of Mb (1.8 mg/mL, 0.1 mM). The mixture was stirred for 1 h at room temperature, and the adsorbed protein was recovered by centrifuging at 12000 rpm for 10 min. The obtained biocomposite (containing about 2.2 mg of Mb) was washed with water (2 mL), centrifuged, and dried. Infrared spectra were measured with an FTIR model IFS113 V Bruker spectrometer, with a resolution of 1 cm-1 in the spectral region 370-5000 cm-1. The measurements were performed using a homemade cell to obtain vacuum spectra (KRS5 circular windows, 2 cm diameter, ∼2 mm thickness). Fluorescence Spectroscopy. The biocomposite Mb/HTlc was prepared according the above-described procedure, and after washing and centrifuging, it was dispersed in water (6 mL). The absorption spectra of this colloidal suspension were recorded by a Varian spectrophotometer equipped with an integration sphere. A fluorimeter (Spex Fluorolog) was used to record corrected fluorescence spectra of the samples using the front face configuration between the excitation and the emission light. The spectra were corrected for the response of instrument components at each wavelength. Polarized fluorescence spectra were recorded, introducing two identical polarizers (Spex FL-1044) in the excitation and the emission paths to produce linearly polarized light. Anisotropy traces were obtained by use of the four possible combinations of the excitation and emission polarization planes. Fluorescence decay profiles were measured by the single photon counting technique (Edinburgh Instrument 199S) using the 270 nm output of a diode laser as light source and collecting the emitted light at 350 nm.15 Resonance Raman Spectroscopy. The biocomposite Mb/ HTlc was prepared according to the described procedure, and after washing and centrifuging, it was cast on quartz plates. After evaporation of the solvent at room temperature, it was placed on the holder of the micro-Raman setup. A 514.5 nm excitation from an ion Argon laser was focused on the sample in a 180° back scattering geometry by means of a 50 objective of an OLYMPUS microscope MOD BX40 connected to an ISA Jobin-Yvon model TRIAX320 single monochromator, resolution ≈2 cm-1. The laser power was adjusted to ca. 2 mW in order to avoid any damage to the protein. The reproducibility of the spectra was always controlled, sampling different points of the film; through the optical microscope analysis, we also verified that no local degradation occurs during laser irradiation. Catalytic Activity for Native and Adsorbed Mb. The peroxidase activity of adsorbed Mb was evaluated by measuring the initial oxidation rates of 2-methoxyphenol by hydrogen peroxide. The biocomposite recovered after centrifuging was (15) Latterini, L.; Amelia, M. Langmuir 2009, 25, 4767.

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Figure 2. Adsorption isotherms of Mb onto NiAl-HTlc nanoparticles at different pH (6.0 and 9.0).

were fitted with the Langmuir model to evaluate the binding affinity and the maximum binding capacity of Mb onto the support. The adsorption isotherms show a sharp initial uptake, indicating a high affinity between the Mb and surface followed by a plateau. In Figure 2, the solid lines represent the best fit of the experimental data using the Langmuir eq 1:17 Qe ¼ Figure 1. Schematic representation of a sequence of layers of NiAl-HTlc. washed with water (3  5 mL) and resuspended in buffer (0.2 M sodium phosphate, pH 6.0), obtaining a dispersion with a known concentration of adsorbed protein. A fixed volume of this dispersion was added to a cuvette containing the substrate to obtain a final volume of 3 mL (0.2 M phosphate buffer, pH 6.0). The kinetics were started by adding hydrogen peroxide. The following concentrations were used: (a) for native protein, [Mb]=1.0 μM, [H2O2]=76 mM, [guaiacol]= 0-30 mM; (b) for adsorbed protein, [Mb]=1.0 μM, [H2O2]=38 -76 mM, [guaiacol]=0-30 mM. The kinetic constants kcat and KM were obtained as described in ref 16.

Results and Discussion Adsorption Isotherms of Mb onto HTlc Nanoparticles. NiAl-HTlc of the formula [Ni0.77Al0.23(OH)2]Br0.15 3 0.81H2O were prepared as a colloidal dispersion of nanoparticles by the double-microemulsion technique.12 The brucite-type mixed metal hydroxides obtained are made up of positively charged layers and charge-compensating anions hydrated with water molecules. For the sake of clarity, Figure 1 shows a pictorial representation of the NiAl-HTlc layered structure. Therefore, the adsorption of a protein onto the HTlc surface is expected to occur by an ion-exchange mechanism with the formation of a protein-nanoparticle complex through the surface complementary electrostatic interactions. The adsorption isotherms of Mb onto NiAl-HTlc nanoparticles, at two different pH values (6.0 and 9.0), are shown in Figure 2. The isotherms (16) Bellezza, F.; Cipiciani, A.; Costantino, U.; Nicolis, S. Langmuir 2004, 20, 5019.

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QMAX Ce ð1=aL Þ þ Ce

ð1Þ

where Ce (mg Mb/mL) is the concentration of protein in solution at equilibrium, Qe (mg Mb/g support) is the amount of protein adsorbed onto the support per unit weight, QMAX (mg Mb/g support) is the maximum adsorption capacity of the support per unit weight, and aL (mL/mg Mb) is the Langmuir constant, which is correlated to the affinity between the protein and the support (high values of aL indicate a strong interaction). The sharp rise followed by a plateau is attributed to the formation of a monolayer of protein molecules on the support surface. The fitting of experimental results with the Langmuir isotherm (eq 1) points out that the support surface is energetically homogeneous with identical adsorption sites for the protein, even if recent research has shown that the HTlc exchange sites at the layer edges show a different behavior from that at the sites at the crystal basal planes.18 Figure 2 shows the effect of pH on the maximum adsorption capacity. The binding affinity is not strongly affected by the pH as indicated by the similar aL values determined at the two pH values (Table 1). The highest QMAX value was obtained at pH 9.0, which is about 2 pH units higher than the isoelectric point of protein. This is a general phenomenon in protein adsorption following the normal trend of an electrostatically driven process. The nanoparticle surface is positively charged in the pH range from 6.0 to 9.0, but the protein has a net positive charge at pH 6.0, making less favorable electrostatic interactions; while at pH 9.0, Mb is negatively charged, and electrostatic interactions between (17) Al-Duri, B.; Yong, Y. P. J. Mol. Catal. B: Enzym. 1997, 3, 177. (18) (a) Roeffaers, M. B. J.; Sels, B. F.; Loos, D.; Kohl, C.; Mullen, K.; Jacobs, P. A.; Hofkens, J.; De Vos, D. E. ChemPhysChem 2005, 6, 2295. (b) Roeffaers, M. B. J.; Sels, B. F.; Uji-i, H.; De Schryver, F. C.; Jacobs, P. A.; De Vos, D. E. Nature 2006, 439, 572.

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Table 1. Adsorption Parameters for Mb onto HTlc Nanoparticles with the Langmuir Model pH

aL (mL/mg protein)

QMAX (mg protein/g support)

6.0 9.0

78 ( 20 73 ( 19

287 ( 16 448 ( 26

the protein and the nanoparticle make the binding more energetically stabilized. For comparison, the adsorption behavior of Mb onto NiAl-HTlc prepared by the urea hydrolysis method and exchanged in bromide form was carried out at pH 7.0. The material is made up of particles with large dimensions (4-5 μm) and small surface area (20-30 m2/g).14 In this case, a maximum adsorption capacity of 30 mg/g was found. These results confirm the importance of particle dimension for the adsorption of a protein onto a surface. Spectroscopic Characterization of Mb Adsorbed onto HTlc Nanoparticles. FT-IR Spectroscopy. FT-IR experiments were carried out in order to investigate the structural properties of Mb adsorbed onto HTlc nanoparticles. The IR absorption bands of the 1500-1800 cm-1 spectral region mainly arise from vibrational modes of the polypeptide amide group and, thus, can be related to the helical structure of the protein. The Amide I (CdO streching) and Amide II (C-N-H bending and C-N streching) vibrational bands are sensitive to hydrogen bonding interactions with the solvent and with the neighboring functionalities; therefore, changes in the protein secondary structure can be followed by monitoring the infrared absorption.19 The Amide I (1655 cm-1) and Amide II (1541 cm-1) bands corresponding to adsorbed Mb are at the same position as those of native Mb, suggesting an unchanged protein helical structure during the formation of the biocomposite (data not shown). Fluorescence Spectroscopy. The fluorescence behavior of aromatic amino acid residues in proteins is a well established tool for investigating the protein conformational changes.20 Horse heart Mb contains two tryptophan residues localized on helix A: one (Trp 14) is in a buried region, and the other (Trp 7) lies near the protein surface.21 The proximity of both residues to the heme results in a partial quenching of the tryptophan fluorescence. The emission properties of aromatic amino acid residues of the Mb/HTlc biocomposite were investigated in colloidal aqueous dispersion and compared to those obtained from the native Mb in the same experimental conditions, in order to obtain information on the Mb conformation upon adsorption onto HTlc nanoparticles, The emission spectrum of the Mb/HTlc biocomposite presented a maximum at 325 nm similar to the one recorded for the native protein (Figure 3). However, when Mb is adsorbed onto HTlc nanoparticles, the fluorescence spectrum is much broader (6550 and 4480 cm-1 for Mb/HTlc and Mb, respectively), suggesting that the shielding/quenching effects forced by compact protein structure are reduced.22 The broadening of the bandwidths upon adsorption indicates a wider distribution of protein conformations due to a larger variety in the local surrounding of the fluorophores; this behavior is generally observed when fluorophores interact with solid matrixes.23 (19) (a) Rahmelov, K; H€ubner, W.; Ackermann, T. Anal. Biochem. 1998, 257, 1. (b) Smith, J. R.; Cicerone, M. T.; Meuse, C. W. Langmuir 2009, 25, 4571. (20) Matys, L.; Szollosi, J.; Jenei, A. J. Photochem. Photobiol., B 2006, 83, 223. (21) Evans, S. V.; Brayer, G. D. J. Mol. Biol. 1990, 213, 885. (22) Tofani, L.; Feis, A.; Snoke, R. E.; Berti, D; Baglioni, P.; Smulevich, G. Biophys. J. 2004, 87, 1186. (23) (a) Latterini, L.; Nocchetti, M.; Aloisi, G. G.; Costantino, U.; De Schryver, F. C.; Elisei, F. Langmuir 2007, 23, 12337. (b) Latterini, L.; Nocchetti, M.; Aloisi, G. G.; Costantino, U.; Elisei, F. Inorg. Chim. Acta 2007, 360, 728.

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Figure 3. Normalized fluorescence spectra of native Mb (dashed line) and the Mb/NiAl-HTlc biocomposite (solid line) in water (λexc=280 nm).

Figure 4. Fluorescence spectra of the Mb/NiAl-HTlc biocomposite in water at different excitation wavelengths.

More information could be gained by investigating the excitation wavelength effect on the spectral shape. Upon increasing the excitation wavelength, the emission spectrum of the biocomposite gradually shifted to the red (Figure 4). These spectral changes, which were not observed for native Mb in water, can be explained regarding the spectrum of Mb/HTlc as the weighted sum of different contributions. It is well-known that tyrosine fluorescence, which is blue-shifted compared to tryptophan emission (emission maximum at 305 and 350 nm for tyrosine and tryptophan in water, respectively), is completely quenched in folded Mb because of efficient energy transfer processes to tryptophan residues and to heme; tyrosine fluorescence was observed only under denaturing conditions.24 Furthermore, excitation at 290 nm or above allows selective excitation of tryptophan residues. On this basis, the Mb/HTlc spectra, recorded upon excitation below 280 nm, can be assigned to the contributions of tyrosine and tryptophan, while only the latter contributes when longer wavelengths are used to excite the composite material. These arguments lead to the hypothesis that the emitting amino acids of adsorbed Mb are more exposed to an aqueous medium compared to the native protein25 as a consequence of a partial tertiary structure loss. (24) Kirby, E. P.; Steiner, R. F. J. Biol. Chem. 1970, 245, 6300. (25) Du, H.; Fuh, R. A.; Li, J.; Corkan, A. L.; Lindsey, J. S. Photochem. Photobiol. 1998, 68, 141.

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Bellezza et al. Table 2. Fluorescence Decay Times of Native Mb and Mb/NiAl-HTlc Biocomposite (λexc ∼280 nm)

Figure 5. Fluorescence spectrum and anisotropy of Mb in water (λexc=280 nm).

Figure 6. Fluorescence spectrum and anisotropy of the Mb/NiAlHTlc biocomposite in water (λexc=280 nm).

These conclusions are further supported by steady-state anisotropy measurements carried out on the native protein and on the Mb/HTlc biocomposite (Figures 5 and 6). An anysotropy value of about 0.14 was measured for native Mb, which is in agreement with literature data.26 This value was constant, within experimental errors, all over the region of the emission spectrum since it is due to the residual tryptophan emission. However, for the Mb/HTlc biocomposite the anisotropy value changed over the spectral region likely due to the contribution of different emitting species, and a higher average value (0.24) was determined. The increase of the fluorescence anisotropy could be due to the reduced efficiency of energy transfer processes as a consequence of conformational changes. Further experiments with selective quenchers are currently under investigation to evaluate the change in protein permeability once adsorbed onto the HTlc nanoparticles. The fluorescence decay time measurements were performed with the single photon counting technique to obtain information on the interactions between the protein and the HTlc nanoparticles by selectively monitoring the emission of tryptophan residues. For both samples, the fluorescence of tryptophan residues revealed complex decays which could be satisfactorily fitted by biexponential functions (Table 2). This type (26) Stortelder, A.; Keizers, P. H. J.; Oostenbrink, C.; De Graaf, C.; De Kruijf, P.; Vermulen, N. P. E.; Gooijer, C.; Commandeur, J. N. M.; Van Der Zwan, G. Biochem. J. 2006, 393, 635.

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sample

τF (ns)

τF (ns)

χ2

native Mb Mb/NiAl-HTlc

1.1 (45%) 0.9 (45%)

3.2 (55%) 2.6 (55%)

1.03 1.00

of nonexponential decay is commonly observed for excited aromatic amino acids because of specific interactions with the other protein units.27 A comparison of the fitting parameters between the native and adsorbed protein revealed that upon adsorption the decay times become shorter, suggesting the occurrence of interactions among the fluorophores and the constituents of the nanoparticles, such as the metal cations. Resonance Raman Spectroscopy. Resonance Raman (RR) spectroscopy is a powerful tool in the characterization of structural properties of hemeproteins and their derivatives. It has been recently used to elucidate the interactions between the heme and its axial ligand in Mb-thin film electrode28 and in Mb-composite materials.29 With excitation above 500 nm, nontotally symmetric modes of heme proteins are strongly enhanced, thus revealing the great sensitivity of Raman scattering to the structural properties of the active site. The dominant RR bands of these samples are all between 1100 and 1650 cm-1, where one expects in-plane porphyrin ring modes, involving the stretching of C-C and C-N partial double bonds and the bending of C-H bonds at the methine bridges.30 The intense Raman band at ca. 1370 cm-1 has been proposed as a marker of either iron atom out of plane displacement or oxidation state: the position of this signal decreases from ca. 1375 cm-1 for Fe(III) hemes to ca. 1360 cm-1 for Fe(II) hemes. The 1580 cm-1 band is sensitive to the spin state of the heme iron, while the band at 1630 cm-1 is sensitive to both spin and oxidation states. Large frequency reductions accompany a change from low to high spin iron. In particular, bands at 1630 and 1580 cm-1 shift to lower frequencies when the state of the iron atom changes from low (LS) to high (HS) spin coordination, and the porphyrin skeleton moves from planar to domed configuration with the iron atom lying out of the mean heme plane.31 Raman spectra of native Mb in aqueous solution and dry film (obtained from an aqueous solution of Mb by gradual evaporation of the solvent on quartz plates) are shown in Figure 7. Our results are in perfect agreement with literature data28 and confirm that in both cases the iron atom is in the ferric state. Moreover, the position of higher frequency bands suggested that both spin states are present in two samples but with different contributions; in particular, the position of 1622 and 1564 cm-1 band maxima of the Mb solution were shifted to the maxima at 1630 and 1583 cm-1, respectively, of Mb dry film. This result indicates that Mb in solution is in the HS state and that the spin state changed to LS upon the formation of dry film. The conformational modifications of Mb after adsorption onto NiAl-HTlc nanoparticles have been analyzed, and the results are shown in Figure 8. The scattering features of the adsorbed Mb sample suggest that the interaction with the inorganic surface does (27) (a) Tcherkasskaya, O.; Bychkova, V. E.; Uversky, V. N.; Gronenborn, A. M. J. Biol. Chem. 2000, 46, 36285–36294. (b) Tcherkasskaya, O.; Bychkova, V. E.; Uversky, V. N.; Gronenborn, A. M.; Janes, S. M.; Holtom, G.; Ascenzi, P.; Brunori, M.; Hochstrasser, R. M. Biophys. J. 1987, 51, 653–660. (28) Feng, M.; Tachikawa, H. J. Am. Chem. Soc. 2001, 123, 3013. (29) Iafisco, M.; Palazzo, B.; Falini, G.; Di Foggia, M.; Bonora, S.; Nicolis, S.; Casella, L.; Roveri, N. Langmuir 2008, 24, 4924. (30) Ogoshi, H.; Saito, Y.; Nakamoto, K. J. Chem. Phys. 1972, 57, 4194. (31) Spiro, T. G.; Loehr, T. M. In Advances in Infrared and Raman Spectroscopy; Heyden & Son Ltd.: London, 1975; Vol. 1, Chapter 3.

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The kinetic data were analyzed with the Michaelis-Menten equation: V ¼ k cat ½E0 ½S=ðK M þ ½SÞ

Figure 7. Raman spectra of native Mb in aqueous solution (black), powder (red), and dry film (blue).

Figure 8. Raman spectra of the Mb/NiAl-HTlc biocomposite.

not affect the oxidation state of iron. Besides, the profile of the C-C stretching mode is indicative of both low and high spin states: the positions are similar to those of the Mb solution, but in this case, the maximum intensity at 1564 cm-1 is accompanied by a shoulder at 1580 cm-1. In conclusion, the adsorption of Mb onto HTlc nanoparticles resulted in an increase of the ratio low to high spin state in comparison with that of the native Mb in solution. A further result to be highlighted is that the behavior of the Mb/NiAl-HTlc biocomposite when dried on quartz plates is different from that of Mb aqueous solution. A comparison between Mb spectra of dry-film and commercial powder is shown in the inset of Figure 7. The position of each Raman signal is perfectly reproduced in two solid samples, thus suggesting that both oxidation state and spin configuration are the same. Since dry film was obtained from a Mb aqueous solution, it can be concluded that the drying process change the spin state of Mb solution and brought it back to the spin state of Mb powder from which it has been prepared. On the contrary, when Mb has been adsorbed onto NiAl-HTlc nanoparticles, the drying process preserves the spin state of the native protein, and this could be reasonably due to its interaction with the support. Catalytic Activity of Mb Adsorbed onto HTlc Nanoparticles. The catalytic activity of Mb after adsorption onto NiAlHTlc nanoparticles was evaluated in terms of oxidation of 2methoxyphenol (guaiacol) by hydrogen peroxide. The substrate oxidation follows the mechanism typical of peroxidase, with the formation of two intermediates named compound I and compound II.32 (32) Dunford, H. B. In Heme Peroxidase; Wiley: New York, 1999; Chapters 1-3.

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where [E0] is the initial protein concentration, kcat is the turnover rate constant, and KM is the Michaelis constant. For peroxidases, kcat measures the electron-transfer rate from the substrate to the compound II active site, and it depends on the redox potential of active species and on the mode of interaction between the substrate and the active site of protein. The constant KM provides a measure of the affinity between the substrate and the protein. The parameter kcat/KM is defined as catalytic efficiency, and it represents a measure of the enzyme catalytic performance at low substrate concentration. The kinetic parameters for the oxidation of 2-methoxyphenol by hydrogen peroxide catalyzed by native Mb and Mb adsorbed onto HTlc nanoparticles are reported in Table 3. The adsorption of Mb onto NiAl-HTlc nanoparticles resulted in a decrease of kcat, KM, and kcat/KM values. A lower kcat value shows that the electron-transfer rate from the substrate to the compound II active site of the adsorbed protein is slower than that of native Mb, while a lower KM value indicates an increased affinity of the adsorbed protein for the substrate. The catalytic turnover (kcat) value of the Mb/NiAl-HTlc biocomposite is ca. 7-fold lower than that of native protein, while the constant (kcat/KM) is slightly decreased. A decrease in the KM value of 4-5-fold is largely responsible for this effect. No significant variation in the kinetic parameters was observed with a biocomposite containing a larger amount of adsorbed Mb (Table 3, lines 2 and 3). This finding led us to exclude the formation of a multilayer of adsorbed biomolecules as previously pointed out from the Langmuir isotherms. This consequently excludes the possibility that the catalytic activity of Mb decreases because of the presence of a protein multilayer on the support. For comparison, the catalytic activity of Mb adsorbed onto NiAl-HTlc in the form of microparticles (4-5 μm) was investigated, and it was found that this biocomposite was inactive in the oxidation of 2-methoxyphenol by hydrogen peroxide. This result, together with the low adsorption capacity of large-size HTlc, evidences once again the importance of the surface dimensions for the modulation of protein activity. The loss of activity upon adsorption of protein has been frequently observed, and many factors are reported as possible causes. An important theory pointed out that the immobilization of a protein on a solid surface can result in the loss of native conformation to a significant extent or denaturation in some cases, with a consequent lower or absent catalytic activity. In this work, the information on the conformational state of adsorbed protein obtained through IR, fluorescence, and Raman spectroscopies has been used to comment on the possible causes for the decrease of Mb catalytic activity upon adsorption onto NiAl-HTlc nanoparticles. The results of the performed spectroscopic studies suggest that the adsorption of protein molecules onto the nanoparticle surface alters the tertiary structure of adsorbed protein without changing the secondary structure. This hypothesis is based on the fluorescence properties of tryptophan residues which change upon adsorption on HTlc nanoparticles, revealing modifications of amino acid environments and the occurrence of interactions with the inorganic support. These interactions had the effect to modify the porphyrin core to a great extent, leading the spin state of the heme iron in some protein molecules to change from HS to LS. DOI: 10.1021/la901448a

10923

Article

Bellezza et al.

Table 3. Kinetic Constants of Native Mb and Mb Adsorbed onto HTlc Nanoparticles in the Oxidation of 2-Methoxyphenol by Hydrogen Peroxide (0.2 M Phosphate Buffer, pH 6.0, 25 °C) biocatalyst

Ca (mg Mb/g supp)

[H2O2] (mM)

67 native Mbb Mb/ Ni-Al-HTlc 142 76 Mb/ Ni-Al-HTlc 267 38 a Amount of immobilized protein (pH 6.0) per unit mass of support. b Ref 16.

kcat (s-1)

KM (mM)

kcat/KM (mM-1 s-1)

5.5 ( 0.2 0.8 ( 0.1 0.9 ( 0.1

14.2 ( 1.6 2.9 ( 0.9 3.2 ( 0.6

0.39 ( 0.05 0.27 ( 0.09 0.28 ( 0.06

The spin state of myoglobin is relevant for the catalytic activity of the protein because only the five-coordinate high-spin (5c-HS) form of the heme iron can activate exogenous species such as hydrogen peroxide by coordination to the free sixth octahedral position. The increase in the ratio LS to HS due to the increase of the LS state for the adsorbed protein could be responsible of the low catalytic activity (kcat) of the Mb/NiAl-HTlc biocomposite. The partial loss of tertiary structure of adsorbed Mb evidenced from fluorescence studies leads to a modified conformational state that facilitates the formation of the enzyme-substrate complex with consequent KM lowering. It is interesting to note that any factor that affects the protein structure, such as interactions between external amino acids and the surface, could have an impact on the heme pocket internal structure as a consequence of a kind of domino effect. A plausible hypothesis is that the electrostatic interaction, which is the initial driving force of the protein adsorption onto the NiAl-HTlc nanoparticles, gives rise to further chemical interactions such as charge transfers between the metal ions of the hydrotalcite surface and aromatic groups of protein. All of these interactions could affect the Mb structure until drastic modifications in the heme pocket are observed. Desorption of Mb. A protein adsorbed onto a surface has been demonstrated to desorb in many systems, and the released protein may either retain the conformational state it had in the adsorbed state or refold into the original native structure. Desorption of Mb molecules from NiAl-HTlc nanoparticles was tested, and it was found that all of the adsorbed biomolecules were desorbed in the presence of phosphate buffer (pH 8.0, 0.2 M, 1 h). This result indicates that the electrostatic interactions play a key role in forming the Mb/NiAl-HTlc biocomposite. Desorbed Mb molecules were analyzed in terms of structure and enzymatic activity. It was seen that the IR, UV-vis, and Raman spectra of the desorbed Mb were the same as those of the native Mb, and the kinetic parameters kcat and KM were similar to those of native myoglobin (data not shown). It can been concluded that the protein molecules come back to their native conformational state after desorption from the NiAlHTlc nanoparticles.

interaction between Mb and NiAl-HTlc has been elucidated, and the adsorption isotherm can been described by the Langmuir model. The nanostructured biocomposite is active in the oxidation of 2-methoxyphenol by hydrogen peroxide, and the observed enzyme kinetics follows the Michaelis-Menten mechanism. The catalytic turnover (kcat) and the Michaelis constant (KM) values of adsorbed Mb are lower than those of the native protein, while the catalytic efficiency (kcat/KM) of the adsorbed protein is slightly decreased. The absence of catalytic activity for Mb adsorbed onto NiAlHTlc prepared with the urea hydrolysis method, together with the low adsorption capacity of these large-size HTlc particles, evidences the importance of the surface dimensions for the modulation of protein activity. The IR spectra for the Mb/NiAl-HTlc biocomposite indicated an unaltered protein secondary structure, while the fluorescence spectra evidenced a partial loss of tertiary structure. The broad spectrum assigned to different emitting species and the increase of the anisotropy value for adsorbed Mb were related to protein conformational changes induced by the adsorption process. Moreover, the occurrence of interactions among the tryptophan residues and the constituents of the nanoparticles, such as the metal cations, was proven by the decrease of the fluorescence decay time values after adsorption. Data from Raman spectroscopy investigations indicated an increase in the ratio LS to HS of the heme iron atom due to the increase of the LS state for the adsorbed protein. The partial loss of tertiary structure on the Mb/HTlc biocomposite leads to a modified conformational state that could be responsible for the low affinity enzyme-substrate (KM), while the increase in the ratio LS to HS of the heme iron atom for the adsorbed protein is responsible for the low catalytic activity (kcat) of Mb adsorbed on NiAl-HTlc nanoparticles. The conformational modifications observed for adsorbed Mb are reversible since the protein restored its native conformational state after desorption from the NiAl-HTlc nanoparticles.

Conclusions

Acknowledgment. We express our gratitude to Professor U. Costantino and to Dr. M. Nocchetti for helpful discussions and suggestions.

The Mb molecules are able to interact with NiAl-HTlc nanoparticles forming a stable biocomposite. The mechanism of

10924 DOI: 10.1021/la901448a

Langmuir 2009, 25(18), 10918–10924