Characterization of Hemoglobin Immobilized in MgAl-Layered Double

Apr 15, 2010 - lamellaires et Nanomat´eriaux hybrides, Facult´e des Sciences de Bizerte, Universit´e 7 Novembre `a Carthage, Tunisia. Received Janu...
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Characterization of Hemoglobin Immobilized in MgAl-Layered Double Hydroxides by the Coprecipitation Method Khaled Charradi,†,§ Claude Forano,† Vanessa Prevot,† Dominique Madern,‡ Abdesslem Ben Haj Amara,§ and Christine Mousty*,† † Clermont Universit e, Universit e Blaise Pascal, Laboratoire des Mat eriaux Inorganiques, CNRS UMR 6002, LMI, F-63177 Aubiere, France, ‡Institut de Biologie Structurale CNRS CEA, UJF, UMR5075 Laboratoire de Biophysique Mol eculaire, F-38027 Grenoble Cedex 1, France, and §Laboratoire de Physique des Mat eriaux lamellaires et Nanomat eriaux hybrides, Facult e des Sciences de Bizerte, Universit e 7 Novembre a Carthage, Tunisia

Received January 11, 2010. Revised Manuscript Received February 25, 2010 Hemoglobin was immobilized in Mg2Al-Layered Double Hydroxides (LDH) by coprecipation method at pH 9.0. Interactions between Hb and LDH particles were investigated by X-ray diffraction patterns, FTIR, UV-vis, circular dichroism, and fluorescence spectroscopies. Morphology and porosity of Mg2Al-Hbcop biohybrid are analyzed from SEM and TEM images and permeability measurement. The direct electron transfer of immobilized Hb was studied by cyclic voltammetry, and the electrocatalytic activity was evaluated at glassy carbon modified with this Mg2Al-Hbcop biohybrid. Even though the percentage of electroactive Hb was less than 2%, this bioelectrode showed a low detection limit (1.5  10-8 M) and a very high sensitivity (37 A/M cm2) for the amperometric detection of H2O2.

1. Introduction Interaction of proteins with solid surfaces is not only important for a fundamental point of view, but it also plays a key role to several biotechnology applications such as biocatalysts or biosensors. The interfacial assembly of proteins with inorganic materials involves van der Waals forces, hydrophobic affinity, as well as hydrogen bonds. Consequently, the interfacial conditions may induce either the denaturation of immobilized proteins or a specific enhancement of the bioactivity. For instance, adsorption of heme proteins or enzyme, such as hemoglobin (Hb), myoglobin (Mb), or horseradish peroxidase (HRP), on cationic clays can provide a suitable microenvironment of the proteins which enhances the direct electron transfer between the electroactive heme sites of the proteins and the underlying electrode.1,2 Layered double hydroxides (LDH) are synthetic solids with positively charged brucite-like layers of mixed metal hydroxides separated by interlayered hydrated anions, defined by the general formula [M1-x2þMx3þ(OH)2]xþ [(A-)x/n, yH2O] (abbreviated as M(1-x/x)2þM3þ - A). LDH present interesting intercalation properties which allow the adsorption of negatively charged biomolecules such as amino acids,3 DNA,4 nucleoside monophosphate,5 and enzymes with isoelectric point varying in a large pH domain.6,7 Various soft chemistry processes such as adsorption, delamination/ restacking, chemical grafting, coprecipitation, and electrodeposition methods have been used to prepare these new biohybrid *Corresponding author. Christine Mousty (Christine.Mousty@ univ-bpclermont.fr) Universite Blaise Pascal (Clermont-Ferrand, France). Fax: 33 473 407 108. Tel: 33 473 407 598. (1) Mousty, C. Anal. Bioanal. Chem. 2010, 396, 315. (2) Charradi, K.; Forano, C.; Prevot, V.; Ben Haj Amara, A.; Mousty, C. Langmuir 2009, 25, 10376. (3) Aisawa, S.; Takahashi, S.; Ogasawara, W.; Umetsu, Y.; Narita, E. J. Solid State Chem. 2001, 162, 52. (4) Choy, J.-H.; Kwak, S.-Y.; Park, J.-S.; Jeong, Y.-J.; Portier, J. J. Am. Chem. Soc. 1999, 121, 1399. (5) Kwak, S.-Y.; Jeong, Y.-J.; Park, J.-S.; Choy, J.-H. Solid State Ionics 2002, 151, 229. (6) Forano, C.; Vial, S.; Mousty, C. Curr. NanoSci. 2006, 2, 283. (7) Li, F.; Duan, X. Struct. Bonding (Berlin) 2006, 119, 193.

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enzyme-LDH materials.1,6 In particular, very recently, the immobilization of heme proteins (Hb, Mb, and HRP) in LDH matrices has been reported in the literature. The adsorption of Hb was realized on ZnAl-dodecylsulfonate (DDS) and on exfoliated MgAl-lactate hybrid LDH,8,9 whereas the adsorption of Mb was investigated onto NiAl-Br colloid suspension.10 Finally, the direct electrochemical behavior of HRP was studied at NiAl-NO3 modified electrode.11 All these studies show that specific interactions occur between these proteins and the LDH particles, depending on the particle sizes or on the nature of the interlayer anions. For instance, Li et al. have shown that only LDH functionalized with DDS allows the direct electron transfer of Hb entrapped in this surfactant-LDH.8 No reduction peak was observed at the electrode coated with Hb immobilized in LDH treated with cetyltrimethylammonium (CTA) or poly(ethylene oxide) (PEO). In the present work, the immobilization of Hb in Mg2Al-LDH host matrices was realized by adsorption and the coprecipitation method, respectively, at pH 7.0 and 9.0. The interaction between Hb and Mg2Al-LDH was investigated by XDR, FTIR, UV-vis, fluorescence, and circular dichroism spectroscopies. The morphology the Hb-LDH biohybrids prepared by coprecipitation was characterized by electronic microscopies, and finally, the direct electron transfer of the immobilized Hb was studied by cyclic voltammetry. The electrocatalytic behavior of Hb-LDH biohybrids in the presence of hydrogen peroxide was also investigated, and the H2O2 calibration curves were recorded under amperometric conditions.

2. Experimental Section Products. Hydrogen peroxide (H2O2) solutions (from SigmaAldrich) were freshly prepared before being used. Hemoglobin (8) Li, M.; Chen, S.; Ni, F.; Wang, Y. W. Electrochim. Acta 2008, 53, 7255. (9) An, Z.; Lu, S.; Wang, Y. Langmuir 2009, 25, 10704. (10) Bellezza, F.; Cipiciani, A.; Latterini, L.; Posati, T.; Sassi, P. Langmuir 2009, 25, 10918. (11) Chen, X.; Fu, C.; Wang, Y.; Yang, W.; Evans, D. G. Biosens. Bioelectron. 2008, 24, 356.

Published on Web 04/15/2010

DOI: 10.1021/la1001286

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Article (Hb) from bovin blood (Sigma-Aldrich) was used without further purification. Na2HPO4 3 10H2O, NaH2PO4, MgCl2 3 6H2O, and AlCl3 3 6H2O were purchased from Acros. Mg2Al(OH)6Cl 3 nH2O (Mg2Al-Cl) LDH was prepared by the coprecipitation method following the procedure already published.12,13 Typically, a mixed aqueous solution of MgCl2 and AlCl3, with a Mg2þ/Al3þ molar ratio r=2, and a total concentration of metallic cations of 1 M, was introduced with a constant flow into a reactor. The pH was maintained constant at a value of 9.0 during all the coprecipitation by the simultaneous addition of a 2 M NaOH solution. The suspension was aged at room temperature with stirring for 24 h. The final product was centrifuged and washed several times with decarbonated water, and finally dried in air at room temperature for physical characterization or kept as a fresh aqueous gel (20 wt %). All experiments were carried out under a stream of N2, to avoid or at least minimize the contamination by atmospheric CO2. Biohybrid Mg2Al-Hbcop phases with weight/weight Hb/LDH ratio Q = 0.05, 0.25, 0.5, 1, and 2 were prepared by the same coprecipitation method adapted for enzyme solution.14-16 A mixed aqueous solution of MgCl2 and AlCl3, with Mg2þ/Al3þ molar ratio of 2 and a total concentration of metallic cations of 0.1 M, was introduced with a constant flow into a reactor containing a 10-400 mg/Hb in 20 mL H2O. The pH was maintained constant at a value of 9.0 during all the coprecipitation by the simultaneous addition of a 0.2 M NaOH solution. The suspension was aged at room temperature under stirring and N2 atmosphere for 6 h. The final product was centrifuged and washed several times with decarbonated water, and finally kept as a fresh aqueous suspension (20 wt %). UV-vis spectra at 410 nm of supernatant solutions showed that 98% protein was immobilized within the inorganic host material. These samples were systematically stored at -20 °C and diluted in water just before use. Under these storage conditions, the electrochemical activity of Mg2Al-Hbcop sample (Q = 1) was maintained for two months. Instrumentation. Powder X-ray diffraction patterns were recorded on a Siemens D501 diffractometer using Cu KR radiation (λ = 1.5415 A˚). Patterns were recorded over the 2.0-70.0 2θ range in steps of 0.08° with a counting time per step of 4 s and over the 1-20 2θ range in steps of 0.08° with a counting time per step of 12 s. FTIR-ATR spectra were recorded on a Nicolet 5700 (Thermo electron corporation) spectrometer. UV-Vis spectra of thin films coated on quartz plates were recorded using an Evolution 500 UV-visible spectrophotometer (Nicolet). Florescence spectra of free Hb and Mg2Al-Hbcop aqueous suspensions were recorded on a Perkin-Elmer LS55 spectrophotometer with an excitation wavelength of 270 nm. Circular dichroism (CD) was performed on a Jobin Yvon CD6 spectropolarimeter using 0.1 mm quartz curet. The spectra were recorded between 190 and 260 nm at 20 °C. In order to compare the spectrum of each sample, the data were normalized by dividing the crude data in ΔA by the maximal intensity of the negative peaks. Scanning electron micrographs (SEM) of clay samples were recorded on a Zeiss supra 55-VP microscope working at an electron energy of 3 kV. To increase the contrast and avoid charging effect during SEM analysis, the samples were sputtercoated with a thin gold layer. Transmission electron microscopy (TEM) images were taken using a Hitachi 7650 microscope at an acceleration voltage of 80 kV. To prepare the samples, a drop of the solution containing the materials was deposited on a carbon-coated grid and allowed to dry. Hb/LDH film thicknesses (12) Rives, V. Layered double hydroxides: present and future; Nova Sciences Publishers, Inc: New York, 2001. (13) Inacio, J.; Taviot-Gueho, C.; Forano, C.; Besse, J.-P. Appl. Clay Sci. 2001, 18, 255. (14) Vial, S.; Prevot, V.; Leroux, F.; Forano, C. Microporous Mesoporous Mater. 2007, 107, 190. (15) Geraud, E.; Prevot, V.; Forano, C.; Mousty, C. Chem. Commun. 2008, 1554. (16) Mousty, C.; Kaftan, O.; Prevot, V.; Forano, C. Sens. Actuators, B 2008, 133, 442.

9998 DOI: 10.1021/la1001286

Charradi et al. were measured with an Alpha-step IQ surface profiler (KLA Tencor). Hydrodynamic LDH particle sizes were measured by photocorellation spectroscopy with a Zetasizer nanoZS Malvern instrument. Cyclic voltammetry and chronoamperometry experiments were carried out with a potentiostat EA161 (EDAQ) connected to a thermostatted cell (20 mL) with a three-electrode system, including an Ag/AgCl reference electrode, a platinum auxiliary electrode, and a glassy carbon disk electrode (GCE) modified with LDH films as working electrode. Rotating disk electrode (RDE from Radiometer) was utilized for chronoamperometry. All electrochemical experiments were performed in 0.1 M PBS (pH 7) degassed by bubbling with argon for at least 30 min before starting the measurements. H2O2 calibration curves were obtained by recording steady-state chronoamperograms (ilim vs time at Eapp = -0.400 V) with a rotating GC disk electrode (A = 0.196 cm2) at 500 rpm. Successive injections of concentrated stock H2O2 solution in the batch cell were performed with a syringe. Procedures. Adsorption Experiments. Adsorption isotherm of Hb on LDH was determined by the batch method at 25 °C in a thermostatted cell. 2.5 mg of Mg2Al-Cl (fresh suspension) was dispersed in 10 mL phosphate buffer solution (0.05 M, pH 7) containing Hb at varying concentrations ranging from 2.5  10-3 to 3.0  10-2 g/L. The mixture was stirred at 100 rpm for 2 h, then centrifuged for 35 min at 10 000 rpm. The initial and equilibrium Hb concentrations were measured in UV-vis spectroscopy at 410 nm (Soret band). The amount of adsorbed Hb on the LDH particles was calculated by the mass balance equation C s ¼ ðCi -mCe ÞV where Ci and Ce are the concentrations of the initial Hb solution and supernatant, respectively (mg/mL); V the volume of the suspensions (mL); and m is the weight of the adsorbent (mg). The adsorption isotherms were obtained by plotting the amount of adsorbed Hb (Cs w/w) versus the Hb equilibrium concentration in the solution (Ce mg/L). The so-called prepared phase was labeled Hb/Mg2Al-Clads. These adsorption conditions are the same as those we used for the adsorption of Hb on cationic clays.2 However, in the case of LDH, a competitive interaction between hydrogen phosphate and protein may occur on the LDH platelets. Preparation of Bioelectrodes. Before use, the glassy carbon electrodes (3 or 5 mm) were polished with alumina particles (0.05 μm), then they were cleaned by ultrasonication in water and ethanol and finally rinsed with water. A first type of bioelectrode was prepared by dropping (100:20 μg) of Hb/LDH aqueous mixture on the electrode surface and drying it in a fridge overnight. Before use, the modified electrodes were exposed 10 min to saturated glutaraldehyde vapor (25%) to cross-link the entrapped biomolecules. Next, they were soaked in 0.1 M PBS (pH 7.0), for at least 25 min to rehydrate the biofilm and remove glutaraldehyde traces. The enzyme leaching into this swelling solution, determined by UV spectroscopy, was calculated at 40% without chemical reticulation and 5% with GA. Another type of bioelectrode was prepared as follows: 200 μg Mg2Al-Hbcop nanohybrid (Q = 1) was deposited on the surface of a glassy carbon electrode and the coating was dried at 4 °C overnight. No protein leaching was observed when this bioelectrode being soaked into the electrolyte solution (PBS pH 7.0); however, a chemical reticulation with GA improved the mechanical stability of the biocoating. Both bioelectrodes were referenced as Hb/Mg 2 Al-Cl ads/ GCE and Mg2Al-Hbcop/GCE.

Results and Discussion Immobilization of Hb on LDH Materials. A reference Mg2Al-Cl LDH was prepared by coprecipiation method at pH 9.0. Its powder XRD pattern (Figure 1a) displays the characteristic diffraction peaks of pure LDH compounds, exhibiting the 00l series, with the hk0 lines characterizing the layer. The 003 reflection corresponds to a basal interlayer spacing d of 0.75 nm, Langmuir 2010, 26(12), 9997–10004

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Figure 1. X-ray diffraction patterns of Mg2 Al-Cl (a) and Mg 2Al-Hb cop with Q = 0.05 (b), 0.25 (c), 0.5 (d), 1.0 (e), 2.0 (f).

quite similar to that reported previously for chloride-intercalated MgAl LDH.13 Bovine hemoglobin (Hb) is a tetrameric hemoprotein which has a molecular weight of 68 kDa, an isoelectric point (IP) of 7.1, and ellipsoidal diameters of 70  55  55 A˚.17 To get better insight into the affinity between Hb and the Mg2Al-Cl host structure, the adsorption isotherm was performed using a fresh suspension of LDH (0.5 mg/mL) (Figure 2). The isotherm was fitted with the Langmuir model and the thermodynamic parameters were determined from the linear relation of the Langmuir equation Ce Ce 1 Cs ¼ Cm þ KL Cm where KL is the Langmuir adsorption constant (mL/mg Hb) and Cm the maximum adsorption capacity for a monolayer coverage (mg Hb/g LDH). These values are, respectively, 1843 mL/mg and 69 mg/g. It should be noted that the maximum adsorption capacity depends strongly on the LDH sample storing (dry and fresh suspension). Indeed, the Cm value decreased to 9 mg/g when Hb was adsorbed on a dried Mg2Al-Cl sample. Similarly, Zhe An et al. reported a higher Cm value for the adsorption Hb on delaminated Mg2Al-lactate colloids.9 The interest in using colloidal-sized particles was also highlighted for the adsorption of myoglobin on nanosized NiAl-LDH.10 It should be noted that the maximum adsorption capacity on LDH is significantly lower than that obtained for the adsorption of Hb on smectite clay under the same experimental conditions (Cm = 900 mg/g of Nontronite).2 Since the amount of Hb adsorbed on the LDH surface seems to be very limited, the coprecipitation route, performed at higher pH, was used as an alternative immobilization process to adsorption for the preparation of biohybrid Hb-MgAl samples. Indeed, this “soft chemistry” is a very tunable process for the immobilization of enzymes such as urease and alkaline phospatase (AlP).14-16 It allows one to choose variable conditions (pH, temperature, buffer, solvent, reagents concentration) avoiding structural change of the enzyme and denaturation of its catalytic activity. Consequently, direct coprecipitation appears as an alternative method to immobilize biomolecules within LDH matrices. We have prepared a biohybrid Mg2Al-Hbcop LDH by the coprecipitation method at pH = 9.0 with different Hb/LDH ratio (0.05 e Q e 2 w/w). (17) Palecek, S. P.; Zydney, A. L. J. Membr. Sci. 1994, 95, 71.

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Figure 2. (A) Adsorption isotherm of Hb onto a fresh suspension of Mg2Al-Cl (0.25 mg/mL) in 0.05 M PBS (pH 7) for 2 h at 25 °C. (B) X-ray diffraction pattern of Hb/Mg2Al-Clads (Ce = 4 mg/L). (C) ATR-FTIR spectra of Mg2Al-Cl (a) and Hb/Mg2Al-Clads (b).

Considering the IP of Hb, this high pH value will be favorable to interactions between the protein and the positively charged layers of LDH. The amount of protein immobilized for unit weight of LDH in the Mg2Al-Hbcop is nearly the expected value; for instance, for a ratio Q = 1, 980 mg/g of Hb was immobilized in the LDH. This immobilization capacity is DOI: 10.1021/la1001286

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Figure 5. Fluorescence spectra of Hb (a) and Mg2Al-Hbcop (b) in Figure 3. UV-vis spectra of Hb (a) and Mg2Al-Hbcop (b).

Figure 4. Circular dichroism spectra of Hb (a) and Mg2Al-Hbcop (b).

higher than the Cm obtained under the adsorption conditions at pH 7.0. Physical Characterization of Interactions between Hb and LDH. X-ray Diffraction Patterns. The X-ray diffraction pattern of the Hb/MgAl-Clads sample is shown in Figure 2B. This sample was prepared by sedimentation on a glass slide of a Hb/MgAl-Clads suspension corresponding to the maximum adsorption capacity (Ce = 4 mg/L). The observed basal spacing (d003) of 7.41 nm is significantly larger than that obtained with the starting material (0.75 nm). This basal spacing value is very close to that reported for the Hb/Mg2Al sample (8.50 nm), obtained by restacking the delaminated MgAl-lactate layers in the presence of Hb.9 This result is evidence that an anisotropic arrangement of LDH sheet can be formed alternatively with the presence of proteins, with the layered structure being based mainly on electrostatic assembly between both inorganic and biological parts. Due to sample preparation by sedimentation on a glass slide, inducing a preferential orientation of the LDH platelets parallel to the support, only 00l lines are observed, the 012 and 110 lines have vanished. However, FTIR spectrum confirms the presence of Mg2Al-Cl support (Figure 2C) (νMO at 736 and 10000 DOI: 10.1021/la1001286

water (λexc = 270 nm).

636 cm-1, νOMO at 447 cm-1). The vibration bands situated at 1061 and 970 cm-1 confirm the partial exchange with hydrogen phosphate. The amount of immobilized Hb was too low to observe the amide vibration bands. Powder XRD patterns of the Mg2Al-Hbcop phases show progressive modifications of crystallinity (Figure 1). When increasing the Hb over LDH mass ratio from 0.05 to 2, we observe a strong decrease and an enlargement of the (00l) diffraction lines intensities. However, the presence of the (012) and (110) diffraction lines evidence the formation of the layered structure. The enlargement of diffraction lines confirms both a reduction of the particle size according the Laue-Scherrer law and a higher disorder of the structure with a net turbostratic effect caused by the presence of the biomolecules. The same phenomenon was observed with the coprecipitated ZnAl-urease and MgAl-AlP phases.14,15 No intercalation of Hb was evidenced on the XRD patterns even at low angle recording. However, we observe a shift of the (003) line from 0.77 to 0.95 nm when increasing the amount of Hb within the hybrid material. Such a larger basal spacing obtained at high Q value may be explained by an interlamellar swelling of protein containing organic impurities. Spectroscopic Characterization. UV-vis, circular dichroism (CD), fluorescence, and Fourier transform infrared (FTIR) spectroscopies are useful techniques for probing the potential conformational changes of the proteins embedded in inorganic matrices. These experiments were carried out on Mg2Al-Hbcop (Q = 1); indeed, the amount of adsorbed protein in Hb/MgAl-Clads phases was too low to obtain accurate spectra. The position, intensity, and shape of the Soret UV-vis absorption band of the heme group in proteins provide information about the symmetry, complexation feature, and oxidation state of the Fe catalytic site and may address the possible denaturation of the adsorbed Hb. Native Hb showed a Soret band at 410 nm. The Mg2AlHbcop (Q = 1) film cast on a quartz slide showed a peak maximum at 404 nm and a shoulder at 350 nm (Figure 3). This blue shift of the Soret band indicates an alteration in the environment of a part of heme sites. However, it is difficult to extrapolate a change of heme environment to precise conformational changes.18 Various mechanisms can be taken into account for altered heme environment, ranging from a minor change involving relaxation of the folded structure of heme pocket to more drastic changes involving loss of heme and complete disruption of all ternary and secondary structure. (18) Anderson, A. B.; Robertson, C. R. Biophys. J. 1995, 68, 2091.

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Figure 6. (A) ATR-FTIR spectra of Hb (a), Mg2Al-Hbcop Q = 2 (b), Q = 1 (c), Q = 0.5 (d), Q = 0.25 (e), and Q = 0.05 (f). (B) Deconvolution of ATR-FTIR spectra of Hb (a), Mg2Al-Hbcop Q = 2 (b), Q = 0.25 (c).

Conformation changes of adsorbed proteins can be also evaluated by circular dichroism (CD). Figure 4 compares CD spectra of free Hb and Mg2Al-Hbcop (Q = 1) suspensions. The R-helical structure of free Hb is characterized in CD by two strong negative ellipticities at 222 nm and 208-210 nm and a strong maximum at 194 nm.19 The entrapment of Hb within the coprecipated matrix (Mg2Al-Hbcop) caused a change in the relative intensities of the two peaks at 222 and 210 nm, suggesting a slight conformational change of the entrapped protein. Fluorescence spectroscopy is a technique for sensing changes in the local environment of a fluorophore. Fluorescence spectra of proteins are attributed by tyrosine (Tyr) and tryptophan (Trp) residues.20 Hb contains three Trp residues in each Rβ dimer. In particular, β37 Trp residues are located at the dimer-dimer interface, wherein the structure differences between ternary states are largest.21 The intrinsic fluorescence of Hb originates mainly from β37 Trp residues.22 The fluorescence spectrum of Mg2AlHbcop in aqueous suspension was compared to that of the native protein solution upon excitation at 270 nm (Figure 5). The (19) Geng, L. N.; Wang, X.; Li, N.; Xiang, M. H.; Li, K. a. Colloids Surf., B 2004, 34, 231. (20) Yang, X.; Chou, J.; Sun, G.; Yang, H.; Lu, T. Microchem. J. 1998, 60, 210. (21) Wang, Y.-Q.; Zhang, H.-M.; Zhang, G.-C.; Zhou, Q.-H.; Fei, Z.-H.; Liu, Z.-T.; Li, Z.-X. J. Mol. Struct. 2008, 886, 77. (22) Hirch, R. E.; Zukin, R. S.; Nagel, R. L. Biochem. Biophys. Res. Commun. 1980, 93, 432.

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emission spectrum of native Hb presented a broad peak with a maximum centered at 330 nm.23 For Mg2Al-Hbcop suspension, two peaks were observed at 302 and 343 nm, respectively. The same spectra were reported by An for Hb/Mg2Al-lactateads powder.9 These peak positions are very close to the emission maximum of tyrosine (305 nm) and tryptophan (350 nm) in water.10,22 These results suggest that the residues of the amino acids in immobilized Hb are exposed to a more hydrophilic environment than in the native protein as a consequence conformational change on ternary structure, i.e., subunit separation. Hb-MgAl interactions were also investigated by attenuated total reflectance infrared spectroscopy (ATR-FTIR). For all the Mg2Al-Hbcop phases, the infrared spectra show at low frequencies (ν < 1800 cm-1) the vibrational features of both enzyme and LDH network. Under increase of Hb loading, the LDH lattice vibration bands (νM-O = 550 cm-1, δO-M-O = 440 cm-1) undergo a strong broadening then confirming the lost of ordering as shown by X-ray diffraction. In the 1200-1800 cm-1 spectral range, the typical amide I and amide II vibration bands of the Hb appear (Figure 6A). Any modification from the FTIR spectrum of native Hb may be explained by structural changes. Figure 6B displays the deconvolution of Hb and Mg2Al-Hbcop (Q = 2 and 0.25) experimental spectra. Positions and intensities of the amide I (23) Liu, T.-Q.; Guo, R. Chin. J. Chem. 2007, 25, 490.

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Charradi et al. Table 1. Physical Characteristics of Hb-LDH Solutions and Films suspension material

pH

film

zeta potential (mV)

particle size (nm)

þ16.2 þ42.2 þ1.89 þ10.7 þ25.6

156 440 670 a 230 (49%) þ1710(22%)

Hb 7.2 10.1 Mg2Al-Cl 7.7 Hb/Mg2Al-Clads Q = 1 7.1 Hb/Mg2Al-Clads Q = 5 9.8 Mg2Al-Hbcop Q = 1 a Percentage in intensity.

group, characteristic of CdO and CN stretching vibrations and in-plane NH bending mode, appear unaffected under immobilization of Hb in LDH; the second derivative curves for this spectral region are identical for all phases. The secondary structure components (R-helix, β-sheet, β-turn, and unordered structure) of the protein do not undergo strong structural changes.24 However, amide II bands at lower frequencies (