Reversible Intercalation of Large-Capacity Hemoglobin into in Situ

Application of Inorganic Layered Materials in Electrochemical Sensors. Sai-Dan XIE , Yang LIU , Zhao-Yang WU , Guo-Li SHEN , Ru-Qin YU. Chinese Journa...
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Langmuir 2004, 20, 10231-10237

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Reversible Intercalation of Large-Capacity Hemoglobin into in Situ Prepared Titanate Interlayers with Enhanced Thermal and Organic Medium Stabilities Qigang Wang, Qiuming Gao,* and Jianlin Shi State Key Laboratory of High Performance Ceramics and Superfine Microstructures, Shanghai Institute of Ceramics, Graduate School, Chinese Academy of Sciences, Shanghai 200050, People’s Republic of China Received June 7, 2004. In Final Form: August 24, 2004 The exfoliated single-layer titanate can rapidly restack and reversibly release heme protein simply by adjustment of the pH value. The composites have regularly layered structure and powdery morphology by their ideal layer-to-layer assembly, which provides the titanate nanosheet an unusual specific intercalation capacity of 5900 mg g-1 for the protein. The bound and released proteins keep active relative to the intact protein. The hemoglobin thermal and organic solvent stabilities are improved by the protective environment of the titanate host.

Introduction The immobilization of active proteins has extensive applications in the field of biocatalyses, medical implants, biosensors, enzyme reactors, etc.1-6 Among the various applications, traditional enzyme immobilization for the purpose of organic biotransformation has been deeply investigated and widely applied.7-11 Immobilized enzymes can fully exploit the technical and economical advantage of biocatalysts, on the basis of the facile separation and reuse of the biocatalysts with decreased operation costs. Easy separation of the enzyme from the products facilitates enzymatic application and supports a reliable and efficient heterogeneous reaction. Reuse of enzymes provides a cost advantage which is often an essential precondition for establishing an enzyme-catalyzed process. There are many available immobilization approaches which span from binding on prefabricated carrier materials to incorporation into in situ prepared carriers. Basically, carrier materials can be divided into inorganic and organic origins. The previous efforts for the binding of proteins have been mostly conducted with natural organic polymers12,13 and synthetic organic polymers.14-16 As carriers, inorganic materials such as glass, alumina, diatomaceous earth, hydroxyapatite, metal and metal * To whom correspondence should be addressed. E-mail: qmgao@ mail.sic.ac.cn. Phone: (86) 21-52412513. Fax: (86) 21-52413122. (1) Hultin, H. O.; Laurence, R. L.; Kittrell, J. R. Enzyme Technol. Dig. 1974, 2, 156. (2) Lvov, Y.; Mohwald, H. Protein Architecture: Interfacing Molecular Assemblies and Immobilization Biotechnology; Marcel Dekker: New York, 2000. (3) Freitag, R. Biosensors in Analytical Biotechnology; Academic Press: San Diego, 1996. (4) Brown, C. L.; Aksay, I. A.; Saville, D. A.; Hecht, M. H. J. Am. Chem. Soc. 2002, 124, 6846. (5) Mrksich, M.; Sigal, G. B.; Whitesides, G. M. Langmuir 1995, 11, 4383. (6) Klibanov, A. M. Science 1983, 219, 722. (7) Schmidtke, J. L.; Wescott, C. R.; Klibanov, A. M. J. Am. Chem. Soc. 1996, 118, 3360 (8) Wong, C. H.; Whitesides, G. M. Enzymes in Synthetic Organic Chemistry; Pergamon: Oxford, 1994. (9) Davies, H. G.; Green, R. H.; Kelly, D. R.; Roberts, S. M. Biotransformations in Preparative Organic Chemistry: The Use of Isolated Enzymes and Whole Cell Systems in Synthesis; Academic Press: London, 1989. (10) Theil, F. Chem. Rev. 1995, 95, 2203. (11) Tischer, W.; Wedekind, F. Top. Curr. Chem. 1999, 200, 95.

oxide, silica gel, and other inorganic matrixes based on sol-gel processes have good mechanical properties, thermal stability, and resistance against microbial attack and organic solvents.14,17-19 However, the immobilizations of other inorganic materials have achieved less success except for high external surface sol-gel materials.19 This is presumably because of the difficulty of fabrication of desirable structures and the absence of reactive functional groups in inorganic materials compared with that of organic polymers. The nonporous inorganic materials, such as metals, metal oxides, etc., only have low binding surfaces. For the organic polymer carriers, in situ polymerization methods can even achieve considerably high enzyme loading (up to 500 mg g-1).20 Inorganic mesoporous silica materials have attracted much attention as promising host materials for the immobilization of globular proteins with similar sizes because of their large surface areas (up to 1000 m2 g-1) and uniform pores or channels (between 2 and 10 nm).21-23 A series of mesoporous silicas were used to physically absorb horseradish peroxidase, which have improved enzymatic activity in organic solvents and enhanced thermostability.22a Another kind of enzyme, R-chymo(12) Kroll, J.; Hassanien, F. R.; Glapinska, E.; Franzke, C. Nahrung 1980, 24, 215. (13) Hertzberg, S.; Kvittingen, L.; Anthosen, T.; Skjak-Brack, G. Enzyme Microb. Technol. 1992, 14, 42. (14) Cao, L.; Bornscheuer, U. T.; Schmid, R. D. J. Mol. Catal. B: Enzym. 1999, 6, 278. (15) Baillargeon, M. W.; Sonnet, P. E. J. Am. Oil Chem. Soc. 1998, 65, 1812. (16) Katchalski-Katzir, E.; Kraemer, D. M. J. Mol. Catal. B: Enzym. 2000, 10, 157. (17) Reetz, M. T.; Zonta, A.; Simpelkamp, J. Biotechnol. Bioeng. 1996, 49, 527. (18) Khare, S. K.; Nakajima, M. Food Chem. 2000, 68, 153. (19) Nguyen, D.; Smit, M.; Dunn, B.; Zink, J. L. Chem. Mater. 2002, 14, 4300. (20) Wang, P.; Sergeeva, M. V.; Lim, L.; Dordick, J. S. Nat. Biotechnol. 1997, 15, 789. (21) (a) Liu, B.; Hu, R.; Deng, J. Anal. Chem. 1997, 69, 2343. (b) He, J.; Li, X.; Evans, D. G.; Duan, X.; Li, C. J. Mol. Catal. B: Enzym. 2000, 11, 45. (c) Deere, J.; Magner, E.; Wall, J. G.; Hodnett, B. K. J. Phys. Chem. B 2002, 106, 7340. (22) (a) Takahashi, H.; Li, B.; Sasaki, T.; Miyazaki, C.; Kajino, T.; Inagaki, S. Chem. Mater. 2000, 12, 3301. (b) Wang, P.; Dai, S.; Waezsada, S. D.; Tsao, A. Y.; Davison, B. H. Biotechnol. Bioeng. 2001, 74, 249. (23) Fan, J.; Lei, J.; Wang, L.; Yu, C.; Tu, B.; Zhao, D. Chem. Commun. 2003, 2140.

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trypsin, was covalently combined on the nanochannels of mesoporous silica via a bifunctional agent, which showed enhanced enzyme stability and activity at elevated temperatures and in organic solvents.22b Rapid and highcapacity (up to 533 mg g-1) immobilization of enzymes within mesoporous silica have also been achieved by finely changing the morphologies of mesoporous silica.23 Inorganic layered materials are attractive carriers because of their flexible interlayer distances, which may adapt the dimensions of the guest molecules. Layered inorganic compounds including graphite, metal oxides, phosphates, and chalcogenides provide a series of supports with varied properties. The direct intercalation of proteins into inorganic layered materials is difficult because of the tremendously large size of the proteins. Recently, the reassembly of the exfoliated inorganic single layers with guest molecules gave an alternative route to bind bulky proteins under mild conditions.24-27 Kumar et al. reported a series of proteins immobilized in layered R-zirconium phosphate (R-ZrP) by reaction of an exfoliated R-ZrP single layer with proteins. The layered R-ZrP host could enhance the activity of bound myoglobin (Mb) with specific substrates and improve the thermal stability of bound proteins. The bound hemoglobin (Hb) could even renature after high-temperature denaturation only by cooling of the sample.24 Gao et al. reported the immobilization of Mb and Hb in the layered manganese oxide colloids with about 10% activity of the free proteins.25 However, most of the above composites were sols or gels, and high-speed centrifugation, solvent vaporization, etc. methods are required to separate them from the solution system. The assembly mechanism of inorganic sheets with proteins was not totally clear. The high cost is also a disadvantage due to the enzyme loss in the homogeneous catalytic process, and a fixed-bed heterogeneous catalytic system is urgently needed. Two-dimensional semiconductor TiO2 is very attractive because it is inexpensive, nontoxic, chemically and thermally stable, and environmentally benign. Recently, the titanate nanosheets have been synthesized by delaminating the layered protonic titanate H0.7Ti1.82500.175O4‚ H2O (0 expresses vacancy) into their elementary layers (0.75 nm thickness, 0.1-1.0 µm in lateral size). Sasaki et al. have summarized the exfoliated titanate nanosheets’ swollen properties and assembly behavior with many guest molecules, which greatly benefits the understanding of the protein-inorganic nanosheet interaction.28 Hb, a standard globular protein for oxygen carriage, has a natural quaternary structure containing a heme group with a molecular weight of 68000 and an isoelectric point (pI) of about 6.8, which can catalyze the oxidation of some toxic compounds such as polycyclic aromatic hydrocarbons, dyes, and phenols.26 Herein, we report the high-capacity immobilization of Hb into layered titanate by host-guest alternate assembly. The protein-titanate composite particle can be rapidly precipitated and reversibly exfoliated by controlling the electrostatic balance via a simple pH (24) (a) Kumar, C. V.; Chaudhari, A. J. Am. Chem. Soc. 2000, 122, 830. (b) Kumar, C. V.; Chaudhari, A. Chem. Mater. 2001, 13, 238. (c) Kumar, C. V.; Chaudhari, A. Microporous Mesoporous Mater. 2003, 57, 181. (d) Bellezza, F.; Cipiciani, A.; Costantino, U.; Negozio, M. E. Langmuir 2002, 18, 8737. (25) Gao, Q.; Suib, S. L.; Rusling, J. F. Chem. Commun. 2002, 2254. (26) Geng, L. N.; Li, N.; Dai, N.; Wen, X. F.; Zhao, F. L.; Li, K. A. Colloids Surf., B 2003, 29, 81. (27) Corma, A.; Fornes, V. Adv. Mater. 2002, 14, 71. (28) (a) Sasaki, T.; Watanable, M.; Hashizume, H.; Yamada, H.; Nakazawa, H. J. Am. Chem. Soc. 1996, 118, 8329. (b) Sasaki, T.; Watanable, M. J. Am. Chem. Soc. 1998, 120, 4682. (c) Sukpriom, N.; Lerner, M. M. Chem. Mater. 2001, 13, 2179. (d) Sasaki, T.; Watanable, M. J. Phys. Chem. B 1997, 101, 10159.

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adjustment method. The protein structure and activity are undisturbed during the assembly and exfoliation circulation. XRD, SEM, HRTEM, FTIR, and UV-vis are employed to systematically analyze the resulting composite in detail. For valuation of the industrial utilization effect on pollutant decomposition in the possible environments, the protein peroxide catalytic activities are determined in aqueous and water-miscible acetonitrile systems, respectively, which is also helpful to learn the proteins’ general assembly mechanism and their potential application in organic solvents. Experimental Section Cs+-type

titanate Cs0.7Ti1.82500.175O4 was prepared by a conventional solid-state reaction.29 The mixture of Cs2CO3 and TiO2 with a molar ratio of 1.0:5.3 was calcined at 1027 K for 20 h, cooled to room temperature, then grinded, and finally calcined at 1027 K for another 20 h. The resulting powder (10.0 g) was stirred in a hydrochloric acid solution (1.0 L and 1.0 M) at room temperature for 24 h. The H+ derivative was obtained with H+ ions totally superseding Cs+ ions by the acid exchange four times, and the solid materials were washed with copious water to remove the acid residue. Then, the lepidocrocite-type structural protonic titanate (1.0 g) was added to a 2.5 mM TBAOH (Acros Organics) aqueous solution (100 mL).28 The mixture was shaken vigorously at 298 K for 8 d, and the resulting suspensions were centrifuged (8000 rpm) to obtain translucent sols. For immobilizing Hb molecules, the original basic sols (pH about 12) were adjusted to weak acidity (pH about 5.5) by 0.1 M acetic acid solution. The choice of acetic acid as the pH adjustor is based on the fact that the commonly used phosphate buffers may induce the unexpected aggregation of titanate colloid by inorganic cation electrostatic attraction with negatively charged nanosheets. Stock solutions of the Hb (1.0 mg/mL) and the exfoliated titanate sols (1.0 mg/mL) were mixed in different volume ratios of 6:1, 4:1, and 2:1, respectively. The mixture was stirred for 1 h and then left for a further 3 h to precipitate rufous solid. The dry product was collected by filtering the upper liquid, by washing with copious water, and by sequent lyophilizing treatment. At the same time, the residual Hb content in the upper liquid could be determined by its Soret absorption band of UV-vis spectra and its corresponding extinction coefficients at 408 nm. Then the composite particle was suspended in the same volume of water as the reaction systems and titrated dropwise to pH 8.0 by 50 mM TBAOH solution. The composite particles gradually diminished with titration and finally became a colloid mixture. The electrostatic attraction between titanate sheets and Hb molecules is very important in our experiment. The reversible immobilization and release of Hb in the titanate interlayer was carried out by controlling the electrostatic effect. However, high ionic strength salt could suppress the electrostatic interactions in the binding enzyme process.24a So, the solution ionic strength would stay at a low level by avoidance of adding any other substance in our experiments. Powder X-ray diffraction (XRD) patterns were recorded on a Rigaku powder diffractometer equipped with Ni-filtered Cu KR radiation (λ ) 1.5418 Å). For the XRD pattern measurement, the wet Hb-titanate composites were directly mounted on substrate. The morphologies of the composite were observed by scanning electron microscopy (SEM; JSM 6700F NT) and high-resolution transmission electron microscopy (HRTEM; JEOL 200CX, 120 kV accelerating voltage). Prior to the electron microscopical observations, the composite particles in the colloidal mixture were centrifuged and washed with copious water. After the above process was repeated twice, the composite particles were filtrated and washed with a small amount of anhydrous ethanol twice to remove the organic molecules on the particle surface. Prior to the TEM analyses, the samples were treated by suspending the composite particles in water solution, by ultrasonic dispersion, and by dropping them onto a holey carbon film on a Cu grid. The samples prepared for SEM analyses were treated by mounting (29) Grey, I. E.; Madsen, I. C.; Watts, J. A. J. Solid State Chem. 1987, 66, 7.

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Table 1. Detailed Elemental Analyses for the Bound Hb with Maximum Capacity and the Free Hb Na

Fea

N/Cb

Fe/Cb

bound Hb (max) 11.87 38.35 6.25 8.01 0.28 free Hb 14.57 47.43 7.40 0.00 0.34

0.31 0.31

0.0072 0.0072

a

Ca

Ha

Tia

Mass percent content. b Mass ratio.

the composite particles on a carbon film, by volatilization drying, and by coating them with Au for 10 s by E-1030 ion sputtering. Elemental analyses were carried out as follows. Organic components in the lyophilizing samples were determined by a PerkinElmer 2400-type CHN analyzer. The Ti and Fe contents were determined with a Perkin-Elmer model P40 ICP-AES after heat nitric acid decomposition. Electrokinetic potential (ζ-potential) diagrams as a function of the pH of the solution were examined using a ζ-potential analyzer, Zetaplus (Brookhaven Instruments Corp.), by titrating slowly a 0.1 M acetic acid solution to the colloid mixture of titanate sheets and Hb. FTIR spectra were recorded on a Thermo Nicolet FT-IR spectrometer by the standard KBr disk method in the range of 400-4000 cm-1 with a resolution of 2 cm-1. A Shimadzu UV-3101PC spectrophotometer was employed to record UV-vis spectra and estimate the Hb residue concentration by 408 nm Soret absorbance of the upper solution. The catalytic activity changes of Hb were studied using the oxidation reaction of guaiacol to its tetrameric product as a model system.24a The formation of the colored products was monitored as a function of time using the spectrophotometer kinetics mode. The 30 s reaction kinetic curves of guaiacol (2.67 mM) and H2O2 (16.7 mM) catalyzed by various Hb’s (1.25 µM) were monitored at 470 nm with a time interval of 6 s. The initial reaction rate was evaluated by the slope in 30 s. The initial rate of the reaction at varying guaiacol concentration was determined at constant H2O2 concentration. Kinetic constants were determined from the Lineweaver-Burk plots. For evaluation of the effect of solvents on the immobilized Hb, the oxidation reaction of o-phenylenediamine (OPD) to phenazine was carried out in water-miscible acetonitrile solution.30,31 Solutions (50 mL) of 10 mM OPD and 25 mM H2O2 were prepared in acetonitrile solution with different water contents. The reaction was initiated by adding 10.0 mg of freeze-dried powder of free and bound Hb with constant stirring at 298 K. The initial reaction rate was evaluated by detecting the absorbance increase at 450 nm in the first minute.

Results and Discussion Composition and Structure Analyses. A facile coprecipitated procedure for the immobilization of Hb into a titanate interlayer under room temperature is described here. The titanate nanosheets can rapidly assemble with Hb molecules when their colloidal suspension is adjusted to mild acidity (pH 5.5). Equilibrium mixtures of titanate with increasing concentrations of the protein are filtered, and the concentrations of the bound Hb are estimated by monitoring that of the residual Hb in the solution. The results suggest that the titanate nanosheets can completely immobilize Hb with no residual Hb in the solution when the reaction mass ratios of Hb to titanate are less than 6.0. A composite with a maximum bound capacity of Hb 5.9 times greater than that of titanate forms when the reaction mass ratios of Hb to titanate are 6.0 according to the estimated value by subtracting the residual Hb concentration. The detailed compositions of the composite with maximum capacity, as well as free Hb, are listed in Table 1. The composite has the same mass ratios of C to N (0.31) and Fe to C (0.0072) as the free Hb, which indicates that the organic component is mainly Hb molecules. The Hb concentration in the product is calculated to be 81.4 wt %, on the basis of the ratio of C, N, and Fe element contents (30) Bindhu, L. V.; Abraham, T. E. Biochem. Eng. J. 2003, 15, 45. (31) Partridge, J.; Halling, P. J.; Moore, B. D. Chem. Commun. 1998, 841.

Figure 1. Powder XRD patterns of the composite with Hb/ titanate reaction mass ratios of 6.0, 4.0, and 2.0.

in the composite and those in free Hb. The titanate (Ti1.825O40.7-) percent is calculated to be 13.9 wt % by Ti content in the composite. The residue about 4.7 wt % in the composite should be ascribed to the water in the titanate interlayers. A maximum bound capacity of Hb 5.9 times that of titanate in the composite is in accordance with the estimated value by subtracting the residual Hb concentration. On the basis of the composition analyses, the formula is determined to be (Hb)0.013H0.7Ti1.82500.175O4‚ H2O (0 expresses vacancy). XRD pattern analyses of the host and composites with different mass ratios (Figure 1) indicate that the d spacing increases from 0.93 nm for the proton-type titanate to 6.85, 7.12, and 8.35 nm for the composites with Hb/titanate mass ratios of 2.0, 4.0, and 6.0, respectively. The observed d spacing with a mass ratio of Hb to titanate of no more than 4.0 essentially corresponds to the monolayer Hb intercalation with an interlayer distance of 6.05-7.25 nm estimated by adding an Hb size of 5.30-6.50 nm (Hb, 6.50 × 5.30 × 5.40)24b and a titanate sheet thickness of 0.75 nm.28 When the Hb absorption amount is close to the maximum capacity, the observed d spacing is 8.35 nm, evidently larger than the maximum monolayer thickness of 7.25 nm. A more swollen zigzag bilayer structure should form in the titanate interlayers to overcome the steric restriction. The expansion of 7.60 nm for the intercalated Hb composite may be caused by two layers of Hb with a certain degree of intersection. The broad peaks for Hb composites indicate a distribution of orientations. Similar results were also found in protein-intercalated zirconium phosphate systems reported by Kumar et al.24 Such a distribution was important for the substrates to access the active sites of the bound proteins.24a Further morphological and structural characters of the composites can be obtained by SEM (Figure 2a-c) and HRTEM analyses (Figure 2d,e). The SEM image indicates that the composites are comprised of slablike crystals either dispersed or assembled into three-dimensional particles. Parts a-c of Figure 2 are three typical SEM images of the composite particles, which reflect the different morphological characters of their different flanks. The HRTEM image shows a slablike crystal profile and resolved particle border with evident inorganic layers. The interlayer distance is about 7 nm, which is in accordance with the XRD analytic value of 7.12 nm for the composite with an Hb/titanate mass ratio of 4.0. The composites have similar lateral sizes between 0.1 and 1 µm, relative to the single-layer titanate. The expanded height of about 100 nm is due to the aggregation of the composite with 10-20-layer titanate intercalated with Hb. Mechanism of Reversible Immobilization. The electrokinetic potential (ζ-potential) of the mixture of

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Figure 2. SEM images of the composite with a mass ratio of 4.0 (a-c). The SEM images (a-c) of the composite particles reflect the different morphological characters of their different flanks. HRTEM images of the 4.0 composite with a slablike crystal (d) and its particle border (e).

Figure 3. ζ-Potential diagram of the titanate nanosheet colloid with and without Hb as a function of pH in aqueous solution.

titanate and Hb is measured as a function of pH to probe the surface charge variation during the coprecipitated process (Figure 3). The data of a single titanate colloid are also measured as the background. From Figure 3 we can see that the isoelectric point of the mixture is about 5.6, which is in the scope between that of the titanate colloid (1.2) and that of free Hb (6.8). When the pH of the mixture is adjusted to near or less than the mixture isoelectric point, the rapid precipitation can be clearly observed. It is evident that the positively charged Hb surface has electrostatic attraction with the anionic surfaces of titanate nanosheets, which makes it possible for the Hb to alternately immobilize into the titanate nanosheets. To balance the repulsion between negatively charged titanate nanosheets, the mixture has a lower isoelectric point than the free Hb. So the ordered layered composite formation should be due to the alternative assembly of titanate nanosheets with Hb by the electrostatic effect. Additionally, the electrostatic effect is adjustable, and the property may be used to release the bound Hb at a

certain condition. The protein surface charge property can be easily changed by simply adjusting the systemic pH. As shown in Figure 3, both the titanate nanosheet and Hb are negative and electrostatic repulsion between the host and guest will occur when the pH of the mixture is larger than 6.8, which is the Hb isoelectric point. Thus, the composite can be delaminated into individual titanate sheets and negative Hb by base titration. When the pH of the system is larger than 8.0, the negative ζ-potentials of both negatively charged titanate and Hb will increase and the stronger electrostatic repulsion between them will finally lead to the release of Hb from the titanate layers. For an ideal monolayer model, the maximum adsorption amount can be estimated by simple geometric analysis. The minimum cross-sectional area of the intercalated Hb molecules is about 22.5 nm2 (3.14 × (5.35 nm)2/4). The available unit surface area of (Ti1.82500.175O4-0.7) is 0.114 nm2 (ac, a ) 0.38 and c ) 0.3 nm). So the ideal maximum capacity for the monolayer arranged composites is about 4.5 by the formula ((0.114/22.5) × (68000/75.6)). The structural analysis also confirms that the composite with an Hb/titanate mass ratio of 2.0 is in accordance with the ideal monolayer model. A swollen zigzag bilayer structure can be formed to overcome the steric restriction when the Hb/titanate mass ratio is more than 4.5. Additionally, we find that the diffraction peak of the composites with an Hb/titanate mass ratio of 4.0 is broader than those of the composites with mass ratios of 2.0 and 6.0. The diffraction peak onset position of the composite with an Hb/titanate mass ratio of 4.0 is even close to that of the composite with a mass ratio of 6.0. These results indicate that the composite with an Hb/titanate mass ratio of 4.0 has a transitional structure between those of the ideal monolayer and the swollen zigzag bilayer. The Hb/titanate mass ratio

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Figure 5. UV-vis absorption spectra of the free Hb (a), the immobilized Hb with a mass ratio of 4.0 (b), and the colloidal mixture by base exfoliation of the composite with a remaining 0.2 mg/mL titanate content (c). Spectra a and c were recorded in transmission mode, and that of b was recorded in diffuse reflectance mode.

Figure 4. FTIR spectra of the free Hb and the immobilized Hb with maximum capacity (a). Expanded amide region (14001800 cm-1) of the FTIR spectra of the free Hb, the exfoliated Hb, and the immobilized Hb with maximum capacity (b).

of 4.0 of the composite in a practical experiment is comparable to the ideal 4.5 times monolayer maximum capacity, which may be used to explain the transitional structure of the composite. The stronger combination between the titanate individual nanosheet and Hb and the titanate swollen structural character may be the reason that the composite from the preparation system with an Hb/titanate ratio of 6.0 has such a large capacity of 5900 mg g-1. So, quantitative immobilization and release of Hb in the layered titanate can be realized by only adjusting the pH and the Hb/titanate ratio of the reaction system. Because the electrostatic effect is the key factor for the process, this method may be applied to protein immobilization of other kinds of inorganic layered materials. The reversible immobilization of a large amount of proteins can be advantageous in practical industrial biocatalyses. Remaining Protein Structure and Catalytic Activity. The protein native structural changes can be monitored by FTIR and UV-vis absorption measurements. The infrared vibrational bands of the amide group of proteins are sensitive to the protein secondary structure. Accordingly, the amide I (relative to the stretching of the carbonyl coupled to C-N) and amide II (assigned to N-H bending) vibrational bands are chosen to detect the Hb native structural changes. The FTIR spectra of Hb immobilized in the lamellar composite, free Hb, and exfoliated Hb from the lamellar composite are shown in Figure 4). The amide I (1651 cm-1) and II (1523 cm-1) bands of the immobilized protein are almost the same as those of the free protein, which are in accordance with the unchanged secondary structure for the immobilized protein. The absorption peaks at 460 cm-1 for the spectra of the immobilized Hb (in Figure 4a) are assigned to the characteristic Ti-O vibration of the TiO6 octahedra, which further proves the existence of a titanate layer in the composite. The expanded amine region of the FTIR spectrum of the exfoliated Hb is almost the same as that

of the immobilized Hb and the free Hb (in Figure 4b), which indicates that the exfoliated process has no effect on the Hb native structure. The UV-vis absorption spectrum of Hb is sensitive to the microenvironment, substructure, and oxidation state. The absorption spectra of the free Hb, immobilized Hb, and exfoliated Hb from the composite are shown in Figure 5. The position of the Hb Soret band at 408 nm is unaffected by the layered titanate host by comparing these absorption spectra. After two-step immobilization and release processes, the Soret band position of the Hb in the re-exfoliated colloidal mixture also stays constant. About a 10% decrease in the extinction coefficient may be due to Hb loss during the whole experimental operation process. The result indicates that the release of Hb from the bound Hb is effective. The absorption at 266 nm for the spectra of bound Hb and exfoliated Hb is due to the exciton transition of exfoliated titanate nanosheets with about a 1.40 eV energy blue shift compared with that of the layered titanate, 3.24 eV, due to quantum size effects.32-35 Although the singlelayer titanate sheets have been alternatively assembled with Hb in the bound composite particles, they still show evident exciton absorption. For the exfoliated Hb, the titanate exciton absorption of Hb is very intense. Even after dilution by a factor of 100, the exciton absorption internsity of titanate in the exfoliated Hb is still relatively high, which is similar to that of the freshly prepared titanate nanosheets.28 The result also confirms that the Hb/titanate composite has been completely exfoliated into a colloidal mixture of the single-layer titanate sheets and Hb molecules by adjusting the pH to 8.0. Additionally, the spectra of the bound composite particles were recorded in a diffuse reflectance mode. So the intensity of the titanate exciton absorption of the bound Hb cannot be compared with that of the exfoliated Hb, which were recorded in transmission mode. At the same time, the absorption at 266 nm for the free Hb is not evident. To further assay their activity, the Hb peroxidase activities are employed. Oxidation of o-methoxyphenol by Hb in the presence of hydrogen peroxide produces a chromogenic tetramer with an absorption peak at 470 nm, which provides a convenient spectrophotometer (32) Sandroff, C. J.; Kelty, S. P.; Hwang, D. J. Chem. Phys. 1986, 85, 5337. (33) Sandroff, C. J., Hwang, D. M.; Chung, W. Phys. Rev. B 1986, 33, 5953. (34) Smotkin, E. S.; Lee, C.; Bard, A. J.; Campion, A.; Fox, M. A.; Mallouk, T. E.; Webber S. E.; White, J. M. Chem. Phys. Lett. 1988, 152, 265. (35) Liu, C.; Bard, A. J. J. Phys. Chem. 1989, 93, 3232.

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Figure 6. Reaction course curves (30 s) of o-methoxyphenol (2.67 mM) and H2O2 (16.7 mM) catalyzed by 1.25 µM free Hb, bound Hb (4.0), and bound Hb (maximum capacity) monitored at 470 nm with a time interval of 6 s (a). Lineweaver-Burke plot for the peroxidase activity of the free Hb, the bound Hb (4.0), and the bound Hb (maximum capacity) (b). Table 2. Kinetic Constants of the Various Forms of Hb

free Hb bound Hb (4.0) bound Hb (max)

Km (mM)

Vmax (µM s-1)

Kcat (s-1)

Kcat/Km (s-1 mM-1)

0.109 0.087 0.072

0.228 0.124 0.100

0.091 0.050 0.040

0.835 0.575 0.555

method to assay the peroxidase activity of Hb. Figure 6a shows the peroxidase catalytic kinetic curves of free Hb, bound Hb with a mass ratio Hb to titanate of 4.0, and bound Hb with maximum capacity. The initial reaction rate in the first 30 s was the maximum, and a good linear relationship (all r > 0.995, between absorbance and time) was obtained. Thus, the initial rates were measured using the first slope over 30 s. The bound Hb with a mass ratio of Hb to titanate of 4.0 has a 71% initial rate (0.123 µM s-1) relative to that (0.174 µM s-1) of the free Hb, and the bound Hb with a mass ratio of Hb to titanate of 6.0 has a 63% initial rate (0.099 µM s-1). The mostly retained activities of the bound Hb demonstrate the facile diffusion of the reactants and products through the layered support and the facile access to the active sites of the bound Hb. Encouraged by these activity results, we also compared the other kinetic constants of the immobilized proteins with those of the free Hb. The Lineweaver-Burk plots (in Figure 6b) constructed by the reaction initial rates at different o-methoxyphenol concentrations are used to estimate the kinetic constant values of free and bound Hb. As listed in Table 2, the Vmax value of the lamellar composite with an Hb/titanate mass ratio of 4.0 is 0.124 µM s-1, while the constant value of the free Hb is 0.228 µM s-1, indicating that about 54% of the enzyme activity was kept when Hb was immobilized. In the case of the composite at the maximum absorption capacity, the Vmax value decreases to 0.100 µM s-1, which is about 44% of the enzymatic activity of the free Hb. Immobilizations of Hb

in titanate galleries result in a decrease of its Vmax value, and there is a decrease of this constant along with an increase of the Hb immobilization amount in the titanate. In general, the product diffusion for the enzyme catalytic reaction in the rigid titanate interlayer is more difficult than that for the free enzyme. So, the decrease of the Vmax value should be ascribed to the loss of enzymatic activity due to conformational restriction of the immobilized enzyme in the rigid titanate interlayer. The other three types of kinetic constants for the free and the bound Hb including Km (Michaelis-Menten constant), Kcat (catalytic turnover), and Kcat/Km (catalytic specificity constant)24b,30 are also listed in Table 2. The Michaelis-Menten constants Km are 0.109, 0.087, and 0.072 mM for free Hb, bound Hb with an Hb/titanate ratio of 4.0, and bound Hb with maximum immobilization capacity, respectively. We can see that the bound Hb also has a decrease of its Km value, which continues to decrease, along with an increase of the Hb immobilization amount in the titanate. The MichaelisMenten constant Km reflects the enzyme-substrate affinity and can be indicative of changes in the microenvironment of the immobilized Hb. In general, Km is a measure of the relative affinity of the substrate for the enzyme active site compared with solvent molecules. Low Km reflects the increase in affinity of the substrate for the Hb in titanate interlayers. For the immobilized Hb, the steric restriction of the titanate host can reduce the decomposition rate of the enzyme-substrate composite, and hence enhances the enzyme-substrate interaction stability. Additionally, the negatively charged titanate layers are highly hydrophilic and may combine a large amount of interlayer water molecules. The hydrophilic hosts may improve the hydrophobicity of the Hb active site, and therefore increase the affinity between the bound Hb and the hydrophobic o-methoxyphenol. The catalytic turnover (Kcat ) Vmax/[Hb]) values exhibit the same trend as Vmax values for the free and bound Hb composites with equal Hb concentration used in all kinetic measurements. The catalytic specificity constants (Kcat/Km) are found to have a maximum value of 0.835 s-1 mM-1 for the free Hb. Along with the increase of the Hb immobilization amount, there is a decrease of the catalytic specificity constant value of 0.575 and 0.555 s-1 mM-1 for the bound Hb with an Hb/titanate ratio of 4.0 and bound Hb with maximum immobilization capacity, respectively. Enhanced Thermal and Organic Solvent Stability. To investigate the titanate layer protective effect, the bound protein stabilities to extremely thermal and organic solvent environments have been studied. The various Hb’s were treated at 363 K in buffer solution for different times, and then their peroxidase activities were measured. The results shown in Figure 7 indicate that the bound Hb has improved thermal stability relative to the free Hb. The bound Hb with an Hb/titanate ratio of 4.0 can remain about 25% active after 363 K incubation for 20 min, while the remaining activity of the unsupported Hb is less than 5% after 5 min of treatment, on the basis of the initial reaction rate of the guaiacol oxidation by detecting the absorbance at 470 nm in the first 30 s. Enhanced thermal stabilities of the protein are welcome changes for biocatalytic application in extreme conditions. The use of organic solvents can dramatically increased the diversity of enzyme-catalyzed reactions, because of an increased solubility of nonpolar substrates in organic media. A pervasive problem is the much lower activity of free dry enzymes in organic solvents than in water, which is ascribed to the distorted effect of organic solvents. Herein, the layered titanate host is examined for its stabilizing effect on the Hb catalytic activity in water-

Intercalation of Hb into Titanate Interlayers

Figure 7. Thermal deactivation treatment of free Hb and bound Hb (maximum capacity) at 363 K for different incubation times in 0.1 M phosphate buffer (pH 7.0).

Figure 8. Relative rate as a function of aw for the oxidation of OPD catalyzed by the bound Hb with a 4.0 reaction mass ratio and the freeze-dried free Hb.

miscible acetonitrile (CH3CN). According to previous studies, the amount of water bound to a protein in solvent would be expected to be controlled by the thermodynamic water activity, aw. In polar solvents, to achieve a fixed aw, a known concentration of water can be conveniently added to the system. For CH3CN, aw values of 0.05-0.11, 0.22, 0.44, 0.55, and 0.70 can be achieved by adding 0.3%, 0.5%, 1.0%, 2.7%, 4.5%, and 10% water (v/v) to the solvent.31 We therefore compared the catalytic activities of the free Hb and the bound Hb composite as a function of aw. Figure 8 shows a comparison of the initial reaction rates obtained for an o-phenylenediamine oxidation reaction in CH3CN by the free and the bound Hb composite. The activity of the free Hb continues to increase even up to aw ) 0.70, while a maximal and much higher rate is obtained

Langmuir, Vol. 20, No. 23, 2004 10237

for the bound Hb with an Hb/titanate ratio of 4.0 at aw ) 0.44. The different rate profiles could arise because the two forms of the enzyme differ in either water binding or the water required for catalytic activity. In this case a water-catalyzed reorganization process is probably required. For the Hb-titanate composites the absorbed water in the interlayers led to a large proportion of the Hb molecules in the structure being close to the water sites, which are the active sites in the catalyzed process. In the case of the free Hb, the very low reaction rates suggest that most of the enzyme is initially inactive since the surfaces were directly exposed to the organic solvent, many surface water molecules were lost, and the active sites were lower. The inorganic titanate layers provide effective interlayer water, which plays a key role in promoting Hb catalytic activity. Conclusion The exfoliated titanate is an optimal carrier for controlled immobilization and release of a large amount of Hb via a simple pH adjustment method. During the total process, Hb always remains active, which has been confirmed by spectral analyses and peroxidase activity assays. The titanate host protective environment can even improve Hb thermal and organic medium stabilities in the reaction system. The powder composite morphological structural analyses indicate that it has an evident layered structure including 10-20-layer titanate sheets and Hb molecules. The titanate unusual specific capacity (5900 mg g-1) for Hb immobilization should be ascribed to the layer-to-layer alternate assembly and the titanate swollen structure. The reversible electrostatic binding of proteins in the titanate interlayer can be advantageous in industrial biocatalysis under adverse circumstances, and the reversible release of protein enzymes and the host cuts operating costs. Additionally, the morphologies of the composites are powders instead of colloidal, which are insoluble in water and can be quickly separated from the liquid reaction mixture. This result is very important for not only the reuse of the catalyst, but also changing the catalytic reactions from homogeneous to heterogeneous in water solution, greatly benefiting the fixed-bed reaction systems, such as polluted water treatment, etc. Acknowledgment. This work was financially supported by the Chinese National Science Foundation (Grant No. 20201013) and the “Plan of Outstanding Talents” of the Chinese Academy of Sciences. LA048602+