Colloidal Assembly of Proteins with Delaminated Lamellas of Layered

Racemic 1-phenylethylamine (98%) and glyceryl triacetate (99%) were from ... using the tapping mode at a resolution of 2 nm in X−Y and 0.1 nm in Z o...
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Colloidal Assembly of Proteins with Delaminated Lamellas of Layered Metal Hydroxide Zhe An, Shan Lu, Jing He,* and Yan Wang State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, China Received April 6, 2009. Revised Manuscript Received July 12, 2009 The colloidal LDH nanosheets have been assembled in aqueous medium with three proteins having different structures and surface charge distributions. In addition to the interfacial adsorption features, the secondary and/or higher level structures of surface-bound proteins are investigated by ATR-FTIR and fluorescence spectroscopic techniques. The structure and conformation of porcine pancreatic lipase (PPL), for which the negative charges are concentrated on the side surface opposite to active sites, are well retained, but the orientations of PPL molecules on twodimensional LDH nanosheets could be lying flat or standing up depending on the PPL/LDH ratio. The bioactivity of PPL lying flat is enhanced in both the hydrolysis and kinetic resolution in comparison with its soluble counterpart. In the case of hemoglobin (Hb), a tetrameric hemeprotein with relatively uniform distribution of surface negative charges, the interfacial assembly might result in the unfolding of its tertiary or quaternary structure, but its secondary structure and redox-active heme groups are not denatured. Although the secondary structure of bovine serum albumin (BSA), for which the negative charges are distributed along the surfaces of linearly arranged domains I and II, is unfolded, the loss of the ordered structure is less than previously found owing to the less curvature of the two-dimensional LDH nanosheet surface. This is the first report related to the investigations of protein structures, conformations, and orientations in the biohybrids consisting of LDH nanosheets.

Introduction The bioinorganic interface chemistry has been intensively studied because the interfacial assembly of proteins with inorganic particles was found to be a fascinating route to create novel bioinorganic hybrid materials combining the physical and chemical features of inorganic particles with the functionalities of proteins.1-4 The bioinorganic interfacial assembly is a complex process involving van der Waals forces, hydrophobic affinity, electrostatic attraction, and hydrogen bonding. Proteins that are biologically active in solution may be deactivated or denatured in the adsorbed state, but the enhancement of bioactivity by interfacial adsorption also occurred in some cases.5 In any research pointed to practical application, whether protein adsorption affects biological functioning is no doubt the main concern. Thus, it is essential to study the physical, chemical, and biological features of surface-bound proteins. The protein interfacial adsorption has been found to differ in quantities, densities, conformations, and orientations, depending on the chemical and physical characteristics of the surfaces.6-10 *To whom correspondence should be addressed. Tel: 86-10-64425280. Fax: 86-10-64425385. E-mail: [email protected].

(1) Katz, E.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 6042–6108. (2) Niemeyer, C. M. Angew. Chem., Int. Ed. 2003, 42, 5796–5800. (3) Srivastava, S.; Verma, A.; Frankamp, B. L.; Rotello, V. M. Adv. Mater. 2005, 17, 617–621. (4) Sui, Z.; King, W. J.; Murphy, W. L. Adv. Mater. 2007, 19, 3377–3380. (5) Reetz, M. T.; Tielmann, P.; Wiesenh€ofer, W.; K€onen, W.; Zonta, A. Adv. Synth. Catal. 2003, 345, 717–728. (6) Sigal, G. B.; Mrksich, M.; Whitesides, G. M. J. Am. Chem. Soc. 1998, 120, 3464–3473. (7) Roach, P.; Farrar, D.; Perry, C. C. J. Am. Chem. Soc. 2005, 127, 8168–8173. (8) Koutsopoulos, S.; van der Oost, J.; Norde, W. Langmuir 2004, 20, 6401– 6406. (9) Wang, X.; Zhou, D.; Sinniah, K.; Clarke, C.; Birch, L.; Li, H.; Rayment, T.; Abell, C. Langmuir 2006, 22, 887–892. (10) Karajanagi, S. S.; Vertegel, A. A.; Kane, R. S.; Dordick, J. S. Langmuir 2004, 20, 11594–11599.

10704 DOI: 10.1021/la901205c

Layered materials provide large, open, accessible two-dimensional surfaces for protein adsorption because of their facile exfoliation reactions. The large two-dimensional platelike sheet surfaces require fewer modifications of the active conformation of proteins, supposedly avoiding the protein denaturation resulting from inorganic surface curvature.11 Layered zirconium phosphate12-14 and titanate15 have been employed as hosts to encapsulate enzymes through exfoliation and restacking approach. One of the layered metal materials, layered double hydroxides (LDHs), also called anionic clays, is more biocompatible and less toxic than most inorganic nanoparticles.16 The forever positively charged LDH slabs, surrounded with hydrogen-bonding layers, provide electrostatic-driven hydrophilic surfaces, allowing the adsorbed enzyme to mimic the interactions of its natural environment. In fact, the protein adsorption is in nature the complementary surface recognition. The surface recognition between proteins and layered metal hydroxides, driven by the electrostatic attraction of positively charged slabs to negatively charged proteins, is supposed to be affected by protein characteristics. This work thus reports the role of protein surfaces and structures in the interfacial assembly with colloidal LDH slabs. The adsorption density, protein orientation, protein conformations, and secondary or high-level structures are investigated.

Materials and Methods Materials. Porcine pancreatic lipase (PPL, type II) was purchased from Sigma-Aldrich as salt-free dry powders, hemoglobin (11) (12) (13) (14) (15) (16)

Mandal, H. S.; Kraatz, H.-B. J. Am. Chem. Soc. 2007, 129, 6356–6357. Kumar, C. V.; Chaudhari, A. Chem. Commun. 2002, 2382–2383. Kumar, C. V.; McLendon, G. L. Chem. Mater. 1997, 9, 863–870. Kumar, C. V.; Chaudhari, A. J. Am. Chem. Soc. 2000, 122, 830–837. Wang, Q.; Gao, Q.; Shi, J. J. Am. Chem. Soc. 2004, 126, 14346–14347. Choi, S. J.; Oh, J. M.; Choy, J. H. J. Mater. Chem. 2008, 18, 615–620.

Published on Web 08/19/2009

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An et al. (Hb, from bovine blood) from Sigma, and bovine serum albumin (BSA, albumin bovine V, 98%) from Amresco in biotechnology grade. They were all used as received without further purification. Racemic 1-phenylethylamine (98%) and glyceryl triacetate (99%) were from Alfa-Aesar. All other chemicals applied in this work were of analytical purity. Preparation of LDHs Colloids. Mg/Al-LDH intercalated with lactate anion (denoted Mg/Al-lact LDHs or lactate-LDH) was first synthesized by a coprecipitation method and then exfoliated in an aqueous medium. Typically, 0.2 mol of lactic acid (pKa = 3.9) was dissolved by adding 1.25 M NaOH solution to pH = 10. Then a mixture solution of 0.02 mol of magnesium nitrate and 0.01 mol of aluminum nitrate (Mg/Al = 2) in 60 mL of deionized water was added dropwise under vigorous agitation. NaOH solution (1.25 M) was additionally introduced until the pH of the reaction mixture reached 10. The resulting slurry was stirred at refluxing temperature for 10 h. The solid was centrifuged, washed thoroughly with deionized water to pH = 7.5, and then suspended in 600 mL of deionized water. The suspension was refluxed under vigorous agitation until a translucent colloidal solution was produced. The content of LDH nanosheets in the colloidal solution is estimated to be 4 mg/mL. In the whole procedure, an inert atmosphere or sealed container was used to avoid contamination by atmospheric CO2. The deionized water for the preparation of all aqueous solutions was decarbonated by boiling prior to use. Assembly of Proteins with Colloidal LDHs. For the assembly of colloidal LDHs with PPL (pI = 5.0), 7.5 mL of LDH nanosheet colloidal solution was exposed at 303 K to freshly prepared PPL solution in HCl-Tris buffer (pH = 7.5). The PPL concentration was set as 0.375, 0.75, 1.5, 3, 6, 12, 18, 24, 30, 36, 42, 54, 60, 66, 72, 78, 84, and 90 mg/mL, corresponding to an input PPL/LDH mass ratio of 0.125, 0.25, 0.5, 1, 2, 4, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30 (g/g). The mixtures of colloidal LDHs and PPL were oscillated in an incubator at 150 rpm for 10 h until the assembly finished. The precipitate was centrifuged, washed several times with fresh buffer to remove the floating proteins, and then lyophilized. The supernatant was collected for the determination of PPL content by the Bradford method.17 The PPL loading in PPL-LDH hybrids was estimated by subtracting the PPL remaining in the supernatant from the initial PPL content. The assembly of colloidal LDHs with Hb (pI = 6.8) and BSA (pI = 4.8) was carried out at pH 8.5 and 7.4, following procedures similar to that for PPL. It took 3 and 7 h for the assembly of Hb and BSA with colloidal LDHs to achieve the steady or quasisteady state. The protein content in the supernatant was quantified directly by the UV-vis absorbance at 408 nm for Hb and 280 nm for BSA. Characterization. The powder XRD patterns from 2θ = 3-70° were collected on a Shimadzu XRD-6000 instrument using Cu KR radiation with a scan rate of 5°/min. The powder XRD patterns from 2θ = 0.5-5° were obtained on a Rigaku D/MAX2500 X-ray diffractometer with Cu KR radiation and a scan rate of 0.5°/min. The AFM micrograph was taken on a DI nanoscope IV (Digital Instruments Co.) using the tapping mode at a resolution of 2 nm in X-Y and 0.1 nm in Z orientaion. A Shimadzu UV2501PC UV-vis spectrophotometer was used for the protein quantification. FT-IR spectra were recorded in attenuated total reflection (ATR) mode on a Nicolet Nexus 470 spectrometer equipped with Smart OMNI sampler single pass ATR accessory. Proteins or protein-LDH hybrid powders were spread onto a germanium ATR accessory. The data were collected by 256 scans in the absorption mode with a resolution of 4 cm-1. The curvefitting (by Origin 7.0) for multipeaks was done in Gaussian mode for the spectra in the amide I, II region (1500-1700 cm-1). Fluorescence spectra were recorded at room temperature on a Shimadzu RF-5301PC spectrophotometer operating in the spec(17) Verger, R.; Sarda, L.; Desnuelle, P. Biochim. Biophys. Acta 1971, 242, 580– 592.

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Article trum mode. Unless otherwise noted, the measurements were made on solid powders. For PPL-containing samples, the excitation and emission slit widths were both 3 nm. Trp fluorescence was measured with excitation at 294 nm and emission at 340 nm. For Hb- or BSA-containing samples, Trp fluorescence was measured with excitation at 280 nm using 5 nm/5 nm slit widths. In all cases, background intensities and spectra have been subtracted to obtain the reported values. Activity Assays. The hydrolytic activity of PPL or PPL-LDH nanohybrid (centrifuged, nonlyophilized solid) was determined at 303 K by titrating with NaOH the acetic acid produced in the hydrolysis of triacetin. Control assays were performed to exclude the NaOH consumption by pristine buffer, LDH colloid, and triacetin solution. The kinetic resolution of racemic R-phenylethylamine was performed at 313K using vinyl acetate as acylating reagent. Vinyl acetate (3 mmol), R-phenylethylamine (3 mmol), sodium bisulfite (1.5 mmol), and PPL or PPL-LDH (lyophilized powder, amounting to 20 mg of PPL in each case) were oscillated in 15 mL of diisopropyl ether, sampled at intervals, and centrifuged. The supernatant was analyzed offline on a Shimadzu GCMS-QP2010 instrument to determine the acylate yield. The ee of acylate as product was determined on a Shimadzu LC-10Atvp HPLC using Chiralcel OB-H column (hexane/2-propanol = 85/15, wavelength = 254 nm). The catalytic activity of Hb and Hb-LDH was determined by the oxidation reaction of o-phenylenediamine (OPD) to phenazine with hydrogen peroxide. A 0.3 mmol portion of OPD and 153 μL of 30% aqueous H2O2, Hb, or Hb-LDH (lyophilized powder, amounting to 2.1 mg of Hb in each case) were stirred in 50 mL of Na2HPO4-citric acid buffer (pH = 7.0) or 50 mL of toluene and sampled at intervals. The phenazine concentration in the reaction mixture was monitored on UV-vis spectrometer by measuring the absorbance at 450 nm.

Results and Discussion In this work, LDH slabs were colloidized by the delamination of lactate-intercalated Mg/Al-LDH in aqueous medium following the procedure reported by Hibino and Kobayashi.18 Consistent with previous reports,18 in the XRD pattern (Figure 1A) for the lactate-intercalated LDH obvious (003), (006), and (009) reflections characteristic of a hexagonal cell of hydrotalcite-like structure are observed. The basal spacing is calculated as 1.34 nm, corresponding to an interdigitated bilayer arrangement of lactate within the interlayer galleries. For the colloidal LDH, the (00l) reflections characteristic of c directional layer-stacking vanish, while the (110) diffraction representative of hydroxide layers in a and b directions is retained. The AFM image (Figure 1B) indicates that the thickness of exfoliated LDH sheets is uniformly around 1.4 nm, approximately corresponding to one single LDH layer with two-side hydrogen-bonding layers (0.48 nm) plus doubleattached lactate anion (2  0.44 nm, calculated by GaussView 3.09). The colloidal LDH nanosheet solution displays apparent Tyndall effect. In the assembly of proteins and colloidal LDH nanosheets, the lactate anions balancing the positive charge of LDH layers are to be replaced partially or completely by the negatively charged proteins through ion-exchange. But diversities might be possible in the interfacial recognition of positively charged LDH layers to negatively charged proteins, in light of the protein structures and surface charge distribution schematically illustrated in Figure 2. For PPL (4.6  2.6  1.1 nm), the negative charges are concentrated on the side surface opposite to active sites. PPL molecules are supposed to be preferably oriented lying flat on the twodimensional LDH surface. But they might be converted to (18) Hibino, T.; Kobayashi, M. J. Mater. Chem. 2005, 15, 653–656.

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Figure 1. (A) XRD patterns of intercalated and delaminated lactate-LDHs; (B) AFM image of colloidal LDH nanosheets.

Figure 2. Top: electrostatic surface potentials for (a) PPL (pI = 5.0) at pH 7.5, (b) Hb (pI = 6.8) at pH 8.5, and (c) BSA (pI = 4.8) at pH 7.4 produced by GRASP 2 (licensed by Dr. Honig’s Lab from the Department of Biochemistry and Molecular Biophysics of Columbia University). Regions of positive charge are shown in blue and those of negative charge are shown in red. Bottom: schematic representations showing the possible orientations of negatively charged PPL (a), Hb (b), and BSA (c) on the positively charged two-dimensional LDH surface.

upstanding at high PPL loading owing to the increase in PPL density. For BSA (14.0  4.0  4.0 nm), the negative charges are distributed along the surfaces of linearly arranged domains I and II. The surface adsorption might occur independently through domain I or II with negligible conformation change or cause the extension of linearly arranged domain I and II chains. For Hb (6.5  5.4  5.3 nm), the negative charges are distributed uniformly on Hb surface, which are supposed to recognize LDH surface readily. The orientation and conformation alterations would be reflected by either the protein amount assembled with LDH nanosheets, the tertiary or even secondary structures of surface-bound proteins, and subsequently the bioactivity, which are to be investigated in this work. Figure 3 displays the profiles of protein adsorption amount (Γ) versus the steady concentration of protein in buffer solution (Ce). The PPL adsorption amount increases with PPL concentration in a shape characteristic of type S. The Γ (defined gram of protein per gram of LDHs) gradually increases first with increasing Ce and then shows a sharp increase since Γ = 12.1 g/g (Ce = 0.20 mg/ mL), and finally reaches a plateau at Γ = 21.6 g/g. Negligible PPL remains free in buffer solution at PPL loadings lower than PPL/ LDH = 10 g/g. The BSA adsorption exhibits typical L-shaped change with BSA concentration, showing a maximum adsorption amount of 0.34 g/g, which is 60 times lower than that of PPL. The 10706 DOI: 10.1021/la901205c

Figure 3. Profiles of adsorption amount of (a) PPL, (b) Hb, and (c) BSA on LDHs versus protein concentration in solution. The adsorption amounts of Hb and BSA were multiplied by 2 and 20.

Hb adsorption gives an L-analogous shape as well with the turning point located around Γ = 3.94 g/g (Ce =0.042 mg/mL). The maximum amount of Hb is markedly lower than PPL but much higher than BSA. As mentioned above, the interfacial assembly requires proteins to gradually replace the lactate anions balancing the charge of LDH nanosheets. For the limitation of LDH layer charge density and protein-protein lateral repulsion, the Langmuir-type adsorptions for Hb and BSA, as well as the Langmuir 2009, 25(18), 10704–10710

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Figure 4. XRD patterns of (a, b) PPL-LDHs with PPL/LDH = 0.5 g/g (a) and 13.8 g/g (b) and (c) Hb-LDHs with Hb/LDH = 1.96 (g/g).

saturation eventually reached for PPL adsorption, are not difficult to understand. What is significantly interesting is the S-type adsorption isotherm for PPL and remarkable low adsorption amount for BSA. For PPL adsorption, the steep increase before the final plateau agrees with the orientation alteration of PPL on LDH nanosheets, as predicted in Figure 2a. Due to the electrostatic attraction of LDH nanosheets to PPL as well the hydrophobic affinity between PPL molecules, the gradual replacement of lactate by PPL is supposed to occur to its ultimate extension. With the gradual increase in PPL content, the layer unit area occupied by one PPL molecule has to be reduced. The orientation of PPL is thus forced to alter from flat to vertical. This orientation alteration well explains the shape of PPL adsorption isotherm and also the steep gradient in the adsorption amount. An effort to increase the BSA adsorption amount has been made by tailoring its surface negative charge density. The pH for measuring BSA adsorption isotherm was modulated to 5.3, 5.9, and 6.6, respectively. But only a slight discrepancy in the adsorption amount was found (not shown). Thus, in addition to the protein size, charge distribution, or orientation, the conformational change might be one important reason for the marked difference in the adsorption amounts between the investigated proteins. Based on the electrostatic surface potentials of BSA, it is reasonable to propose the BSA on the nanosheets might unfold its linear structure (Figure 2c), which will be confirmed by the ATR-FTIR and Trp fluorescence spectra hereafter. The XRD patterns, illustrated in Figure 4, reveal the difference between PPL, Hb, and BSA in inducing the layer-restacking of LDH nanosheets in the interfacial adsorption. The layer-stacked structures are observed for all of the PPL-LDH powders by lyophilization, indicating that the layer-restacking took place readily in the presence of PPL. For the PPL-LDH with a PPL loading of 0.5, the basal spacings (d003) are observed at both 2.91 and 1.61 nm (Figure 4a), corresponding to the PPL dimensional sizes of 2.6 and 1.1 nm. For the PPL-LDH with a PPL loading of 13, the basal spacing (d003) is observed at 5.13 nm, related to the PPL dimensional size of 4.6 nm (Figure 4b). The XRD results not only show the ability of PPL to induce the ordered stacking of LDH layers but also provide convincing evidence for the PPL orientations in the LDH interlayer galleries discussed above. For the Hb-LDH nanohybrids, the layerstacked structures are not observed for the powders by lyophilization. When the Hb-LDH hybrid solids are dried by solvent evaporation, the layer-restacking took place (Figure 4c). That means the layer-restacking of LDH nanosheets in the presence of Hb needs the assistance of solvent evaporation. The basal spacing (d003) is observed at 8.50 nm for the Hb-LDH nanohybrid, larger Langmuir 2009, 25(18), 10704–10710

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than either dimensional size of Hb molecule. No layered architecture has been detected for all BSA-LDH nanohybrids. The absence of layer-restacking in the presence of BSA is not difficult to understand if the BSA unfolding occurs. The positive charges on the opposite side against domain I and domain II prevent the further stacking of LDH nanosheets. To investigate the structural and conformational changes of proteins in the assembly with LDH nanosheets, ATR-FTIR and fluorescence spectra were recorded on the hybrid solids for comparison to pure protein powders. The emission wavelength of tryptophan (Trp) residue is sensitive to its environmental polarity. The position of the Trp fluorescence spectrum thus allows study of the protein unfolding or other conformational alterations resulting in the considerable changes in the compactness of protein molecules. In the ATR-FTIR spectra, the amide I band at 1700-1600 cm-1 is largely due to CdO stretching vibrations accompanied by the in-plane NH bending and the CN stretching modes, enveloping R-helix, β-sheet, β-turn, and unordered structure components. This band is sensitive to the changes in secondary structure and has therefore been widely used for protein conformational studies. The widely accepted assignment for the characteristic absorptions of amide I region is that the bands at 1621-1631 cm-1 arise from β-sheets, 1644-1648 cm-1 from random coils, 1650-1658 cm-1 from R-helices, and 1660-1685 cm-1 from β-turns.19-23 The amide II band near 1550 cm-1, sensitive to the hydrogen-bonded environments, is due to the bending and the stretching mode of N-H and C-N vibrations.20 The ATR-FTIR and fluorescence spectra of PPL-LDH and PPL are shown in Figure 5. For pristine PPL, the R-helix appears at 1650 cm-1, β-sheet at 1632 cm-1, random coil at 1642 cm-1, and β-turns at 1659, 1667, 1673, and 1679 cm-1. The assembly with LDH nanosheets results in no visible shift of the amide I band position and less than 2% changes in the relative content of various components, meaning the interfacial assembly with LDH nanosheets causes no visible unfolding of the PPL secondary structure. But the amide II absorption responds to the assembly with LDH nanosheets. For the PPL-LDH with a PPL loading of 0.5, the amide II band shifts to high frequency and broadens in comparison to pristine PPL. The shift means PPL is more hydrogen-bonded, which is supposed to be associated with intercalation and also the flat orientation of PPL within the LDH interlayer galleries. The hydrated LDH layers, interacting with interlayer PPL from both the upper and lower sides, provide an enhanced hydrogen-bonding environment. In the fluorescence spectra, the Trp emission maximum of PPL shows no visible shift after adsorption on LDH nanosheets. For the PPL-LDH with a PPL loading of 0.5, the unvaried Trp fluorescence seems conflicted with the shifted FT-IR absorption of amide II, but it can be rationally understood in light of the PPL structure. PPL has several tryptophan residues as marked in Figure 6, but all are located away from the negative-charge carrying sites. Thus, the interfacial assembly with LDH nanosheets, in which no visible unfolding occurs, causes no resolved changes in the Trp environmental polarity. The ATR-FTIR and fluorescence spectra of Hb-LDH and Hb are shown in Figure 7. For pristine Hb, the R-helix appears at (19) He, P.; Hu, N.; Zhou, G. Biomacromolecules 2002, 3, 139–146. (20) Secundo, F.; Barletta, G. L.; Dumitriu, E.; Carrea, G. Biotechnol. Bioeng. 2007, 97, 12–18. (21) Shang, L.; Wang, Y.; Jiang, J.; Dong, S. Langmuir 2007, 23, 2714–2721. (22) Roach, P.; Farrar, D.; Perry, C. C. J. Am. Chem. Soc. 2006, 128, 3939–3945. (23) Liu, C.; Bo, A.; Cheng, D.; Lin, X.; Dong, S. Biochim. Biophys. Acta 1998, 1385, 53–60.

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Figure 5. (A) Overall ATR-FTIR spectra, (B) fitted component peaks of amide I, and (C) Trp fluorescence spectra of (a) pure PPL powder, (b) PPL/LDH = 13.8 g/g (higher than the turning point at Γ = 12.1 g/g), and (c) PPL/LDH = 0.5 g/g.

Figure 7. (A) Overall ATR-FTIR spectra, (B) fitted component peaks of amide I, and (C) Trp fluorescence spectra of (a) pure Hb powder, (b) Hb/LDH = 3.94 g/g, and (c) Hb/LDH = 1.96 g/g.

Figure 6. Trp residues in PPL marked on the basis of PDB file.

Figure 8. (A) Overall ATR-FTIR spectra, (B) fitted component peaks of amide I, and (C) Trp fluorescence spectra of (a) pure BSA powder, (b) BSA/LDH = 0.34 g/g, and (c) BSA/LDH = 0.24 g/g.

1652 cm-1, β-sheet at 1624 cm-1, random coil at 1636 cm-1, and β-turns at 1663 cm-1. The assembly with LDH nanosheets results in less than 2% change in the relative content of various amide I components, which is similar to the case of PPL. Different from that observed for PPL, the assembly with LDH nanosheets causes no visible influence on the amide II absorption. It is reasonable according to the difference in the XRD results between PPL-LDH and Hb-LDH nanohybrids. The interlayer spacing for Hb-LDH is higher than the monolayer arrangement of Hb, meaning that the interlayer Hb is not interacting with both of the upper and lower LDH layers. In the Trp emission spectra, the emission maximum for Hb shifts to longer wavelength after assembly with LDH nanosheet. Higher is the LDH proportion; more is the red shift. Hemoglobin is a tetrameric hemeprotein consisting of four heme moieties and two pairs of polypeptide chains known as R and β. Each polypetide chain binds with a heme group inserted into a hydrophobic cleft in the protein.24 The (24) Perutz, M. F. Nature 1970, 228, 726–734.

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previous study25 has found that β 37 Trp is primarily responsible for the fluorescence signal of Hb. The red shift of the wavelength of emission maximum means a greater hydrophilic environment for β 37 Trp residues. That is, the fluorescing tryptophan residues are no longer buried in the nonpolar core of Hb, consistent with observed previously.23 The ATR-FTIR results discussed above indicate that no change of Hb secondary structure has occurred. The fluorescence emission maximum of Hb shifting to longer wavelength thus might result from the dissociation of tetramers, which was previously found to take place by dilution, high temperature and high salt concentration.26 The ATR-FTIR and fluorescence spectra of BSA-LDH and BSA are shown in Figure 8. For pristine BSA, the R-helix appears at 1655 cm-1, β-sheet at 1628 cm-1, random coil at 1646 cm-1, (25) Hirsch, R. E.; Zukin, R. S.; Nagel, R. L. Biochem. Biophys. Res. Commun. 1980, 93, 432–439. (26) Yang, X.; Chou, J.; Sun, G.; Yang, H.; Lu, T. Microchem. J 1998, 60, 210–216.

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Article Table 1. Biocatalytic Activity of PPL acylation of R-phenylethylamine

hydrolysis of triacetin a

PPL/LDH (g/g)

relative activity (%)

reaction time (min)

soluble PPL 100 30 13.8 62 60 0.5 445 15 a Relative to the activity of soluble PPL. b Comparison of ee in the same or similar yield.

acylate yieldb (%)

eeb (%)

3.31 3.04 3.31

40.9 34.1 54.5

Table 2. Biocatalytic Activity of Hb in aqueous medium

in toluene

25 °C Hb/LDH (g/g)

-1

Kcat (s )

90 °C a

rel activity (%)

Kcat (s )

soluble Hb 0.100 100 0.006 1.96 0.039 39 0.067 a Relative to the activity of soluble Hb at 25 °C in aqueous medium.

and β-turns at 1668, 1673, and 1681 cm-1. The vibration at 1638 cm-1 arising from the extended chains of BSA is also observed. The interfacial assembly with LDH nanosheets causes a gradual increase in β-turn content and decrease in R-helix content. The loss of R-helix component reaches 7% for the hybrid with BSA/LDH = 0.24 g/g. The results indicate that the secondary structure of BSA is denatured when adsorbed on the LDH nanosheets, different from observed for PPL and Hb. Its denaturation is not surprising because BSA, mentioned as “soft” protein previously,27 is supposed to easily undergo conformational changes, for example, unfolding. However, the loss of R-helix component observed in this work is less than that found in the adsorption of BSA on sphere substrates.22 A large change in secondary structure was observed for albumin adsorbed onto spheres with radii between ∼10 and 40 nm, with a rise in random structure of about 15% and a 10% loss of helical component on hydrophilic surfaces. We owe the better preservation of BSA secondary structure to the smaller curvature of the two-dimensional surfaces of LDH nanosheets. In the Trp emission spectra, the emission maximum for BSA shifts to longer wavelength, and the red shift is more obvious than Hb. Moreover, the higher the LDH proportion, the greater the red shift. The red shift of emission maximum means the increase in the polarity of Trp microenvironment. BSA has two tryptophan residues embedded in two different domains: Trp-134, located on the surface of domain I, and Trp-212, located within the hydrophobic pocket of domain II.28 The domains I and II are the sites carrying negative charges and thus interacting with LDH nanosheets. The direct exposure of domain I surface to LDH surface surely accounts for the increase in the Trp environmental polarity. The unfolding of BSA molecules, observed in the ATR-FTIR spectra of amide I, might also expose the Trp residue from the hydrophobic pocket of domain II, contributing to the red shift of the fluorescene emission maximum. The bioactivity of PPL, a lipase widely applied in industry, was evaluated by the hydrolysis of triacetin in aqueous medium and kinetic resolution of racemic R-phenylethylamine in organic medium using vinyl acetate as acylating reagent. The results are given in Table 1. The interfacial assembly enhances the hydrolysis activity to a relative activity of 445% at low PPL loading (PPL/ LDH = 0.5 g/g), while in contrast it is lower at high PPL loading (PPL/LDH = 13.8 g/g). Similar results are observed in the kinetic (27) Servagent-Noinville, S.; Revault, M.; Quiquampoix, H.; Baron, M.-H. J. Colloid Interface Sci. 2000, 221, 273–283. (28) Moriyama, Y.; Ohta, D.; Hachiya, K.; Mitsui, Y.; Takeda, K. J. J. Protein Chem. 1996, 15, 265–271.

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-1

25 °C a

-1

rel activity (%)

Kcat (s )

rel activitya (%)

5.5 67

0.024 0.089

24 89

resolution of R-phenylethylamine in diisopropyl ether. The hybrid with PPL/LDH = 0.5 g/g takes 15 min to attain the same acylate yield as achieved in 30 min by soluble PPL and in 60 min by the hybrid with PPL/LDH = 13.8 g/g. The resolution rate on the hybrid with PPL/LDH = 0.5 g/g is twice that for soluble PPL and 4-fold equal to that on the hybrid with PPL/LDH = 13.8 g/g. We owe the enhancement of biocatalytic activity for the PPL/LDH hybrid with low PPL loading to the PPL orientation. In the case of PPL/LDH = 0.5, PPL molecules are orientated flat with the active sites facing to the hydrated LDH layers and the hydrogenbonded environment enhanced. The hydrogen-bonded environment neighboring the active sites favors the biologic functioning of PPL. For the PPL-LDH with PPL/LDH = 13, however, the upstanding PPL molecules are arranged densely like protein aggregates, with the active site of one PPL facing another neighboring PPL. To our surprise, the enantiomer excess (ee) observed for the PPL/LDH hybrid with PPL/LDH = 0.5 g/g is also higher than observed for both soluble PPL and the PPL/LDH = 13.8 hybrid. Although the exact reasons for the ee observation cannot be explained yet, it signals a possibility that the interfacial adsorption on the LDH nanosheets improves the activity and stereoselectivity of at least some lipases. The heme groups of Hb can be converted to a reactive oxoiron(IV)-protein radical29 in the presence of hydrogen peroxide, being able to oxidize the reductive substrate. The oxidation reaction of o- phenylenediamine (OPD) to phenazine is thus used to study the effects of interfacial adsorption on the bioactivity of Hb in this work. The results are given in Table 2. The interfacial assembly with LDH nanosheets reduces the redox activity of Hb in 60% in aqueous medium at 25 °C. It is not surprising because of the tetramer dissociation, as hinted by the fluorescence spectra, but the bioactivity reduction resulting from the interfacial adsorption is less than the deactivation caused by exposure to high temperature (heated to 90 °C) or to organic medium, for example, toluene. The heating makes soluble Hb lose its activity by more than 90%, and exposure to toluene reduces the activity of soluble Hb by more than 70%. For the Hb/LDH hybrid, however, elevating the reaction temperature or altering the reaction medium to toluene not only does not deactivate the surfacebound Hb but also enhances the activity to approach the intrinsic level of its soluble counterpart (at 25 °C in aqueous medium). The protection of the interlayer water molecules for the essential water layer of the immobilized Hb could be one most possible reason for the retained bioactivity of Hb/LDHs exposed to 90 °C or toluene. (29) Everse, J. Free Radical Biol. Med. 1998, 24, 1338–1346.

DOI: 10.1021/la901205c

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An et al.

This phenomenon was also observed previously.12,15 The high temperature and organic solvent could effectively accelerate the diffusion of the substrates to contact the immobilized Hb, probably accounting for the observed increase in Kcat as well.

Summary In conclusion, in the assembly of negatively charged proteins with colloidal positively charged layered metal hydroxide slabs, the conformations and orientations of proteins on the twodimensional surfaces of LDH nanosheets are diversified depending on the charge distribution and structural features of bound proteins. The conformation and structure of PPL are well retained while the PPL molecules could lie flat or stand up on the LDH nanosheets. The bioactivity of PPL bound in low proportion is

10710 DOI: 10.1021/la901205c

enhanced in both the hydrolysis and kinetic resolution in comparison to its soluble counterpart. Although the tertiary or quaternary structure of Hb might be altered in the interfacial assembly, the secondary structure and redox-active heme groups are not denatured. The secondary structure is unfolded in the case of BSA because the negative charges on the BSA surface are distributed along the linearly arranged domain I and domain II. However, the loss of ordered structure is less than found previously owing to the less curvature of the two-dimensional LDH sheet surface. Acknowledgment. We acknowledge financial support from NSFC, Program for Changjiang Scholars and Innovative Research Team in University (IRT0406), project 111 (B07004), and 973 project (2009CB939802).

Langmuir 2009, 25(18), 10704–10710