Hybrid Nanoparticles Based on Organized Protein Immobilization on

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Bioconjugate Chem. 2004, 15, 12−15

Hybrid Nanoparticles Based on Organized Protein Immobilization on Fullerenes Pramod Nednoor, Marcello Capaccio, Vasilis G. Gavalas, Mark S. Meier, John E. Anthony, and Leonidas G. Bachas* Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055. Received August 28, 2003

Nanoscale carbon materials (i.e., fullerenes and nanotubes) are an attractive platform for applications in biotransformations and biosensors. The interesting properties displayed by nanoparticles demand new strategies for the manipulation of these materials on the nanoscale. Controlled modification of their surface with biomolecules is required to fully realize their potential in bionanotechnology. In this work, immobilization of a fullerene derivative with a mutant subtilisin is demonstrated, and the effect of the fullerene on the protein activity is determined. The fullerene-conjugated enzyme had improved catalytic properties in comparison to subtilisin immobilized on nonporous silica. Further, the pH profile of free and fullerene-conjugated subtilisin were almost identical.

Among the exciting possibilities of nanotechnology is the use of nanosized materials as building blocks to construct higher order supramolecular architectures. Nanomaterials that have been proposed to be suitable as building blocks include inorganic nanoparticles, quantum dots, fullerenes, and nanotubes/nanofibers from carbon or inorganic materials (1-4). Complementing this is the use of biomolecules as connectors between the various building blocks. Additionally, surface functionalization of nanoparticles for selective molecular attachment has facilitated the development of nanosensors, bioprobes, and other devices (3,5-7). Therefore, development of methodologies to prepare hybrid nanoparticles by controlling the interactions between biomolecules and various nanomaterials should enhance the functionality and range of application of nanoparticles. Although methodologies based on attaching nucleic acids to nanoparticles followed by DNA hybridization have appeared in the literature (8-10), the controlled conjugation of proteins on nanoscale materials has received less attention. We are interested in binding proteins on nanoparticles from a single site on the protein to yield welldefined hybrid nanomaterials with the protein oriented on the surface. For example, we recently demonstrated the preparation of hybrid alumina nanoparticles with phosphorylated pepsin through the interaction of the single phosphoryl group on serine 68 of pepsin with the alumina surface (11). Upon attachment to the alumina nanoparticles the enzyme remained active, while its thermal stability was enhanced. Zhang and Cass proposed a different method to orient proteins: a polyhistidine-tagged alkaline phosphatase was oriented on a titanium dioxide film through the interaction of the polyhistidine tail and surface-immobilized Ni2+ ions (12). Fullerenes should be useful as building blocks of nanostructured materials because of their rich chemistry, electronic properties, and small dimensions (13). Among fullerene derivatives that have found biological applications, a diamido diphenyl fulleroid derivative was used * Corresponding author. E-mail: [email protected]. Phone: (859) 257-6350. Fax: (859) 323-1069.

as inhibitor of HIV protease (14), a fullerene carboxylic acid was used as a light sensitive biochemical probe (15), a C60-PEG conjugate was used for photodynamic tumor therapy (16), a vesicular stomatitis virus was photoinactivated with fullerene-conjugated methoxy poly(ethylene glycol) amine (17), and a malonic acid derivative was shown to have antiapoptopic activity (18). Additionally, it has been recently shown that fullerenes can be used as electron mediators (19), or as coatings on piezoelectric quartz crystals to develop biosensors (20). Herein, a strategy to couple fullerenes with biomolecules has been explored that involves direct attachment of a sitespecifically functionalized protein on the surface of C60. The approach undertaken involves the introduction of a unique functional group (a single cysteine residue) in the protein sequence using protein engineering. The point of mutation was selected to be away from the protein’s active site to avoid deactivation upon coupling to the nanoparticles. Surfaces with maleimides have been previously used for immobilizing site-specifically a variety of proteins and peptides by taking advantage of the presence of a free sulfhydro group in the amino acid cysteine (21-23). A stable thioether bond is formed when the double bond of maleimide undergoes an alkylation reaction with sulfhydryl groups. Because of the inherent reactivity of maleimides for sulfhydro groups, N-(3maleimidopropionyl)-3,4-fulleropyrrolidine (Figure 1, left), a maleimide-containing functionalized fullerene was synthesized. To obtain this compound, N-triphenylmethyl pyrrolidine-C60 was initially prepared according to a previously published procedure (24, 25). To this, trifluoroacetic acid was added to obtain an intermediate fullerene pyrrolidine ammonium salt. An amount of 40 mg of the solid containing the protonated amine (fulleropyrrolidinium salt) was suspended by sonication in 15 mL of dry CH2Cl2. Anhydrous pyridine (0.6 mL) was added to the suspension followed by maleimidopropionic acid (17.6 mg, 2 equiv). Dicyclohexylcarbodiimide (21.4 mg, 2 equiv) was added at 0 °C, and the mixture was stirred in an ice bath for 30 min and then overnight at room temperature. The reaction mixture was diluted to a final volume of 60 mL with CH2Cl2 and filtered

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Figure 1. (Left) Structure of N-(3-maleimidopropionyl)-3,4-fulleropyrrolidine. (Right) Schematic illustration of the protein conjugated to N-(3-maleimidopropionyl)-3,4-fulleropyrrolidine (shown to scale).

through a Bu¨chner funnel. The filtered solution was then washed with water (2 × 60 mL), a saturated solution of CuSO4, and with water again. The organic phase was dried over MgSO4 and evaporated under reduced pressure. The crude product was then purified by column chromatography (eluant CH2Cl2) yielding 30.8 mg (65%) of pure N-(3-maleimidopropionyl)-3,4-fulleropyrrolidine as a brown solid: 1H NMR (CDCl3/CS2 2:1, 200 MHz) 6.78 (s, 2H), 5.46 (s, 2H), 5.38 (s, 2H), 4.14 (triplet, J ) 7.2 Hz, 2 H), 3.16 (triplet, J ) 7.5 Hz, 2 H); m/z (MS-FAB-) 914 (M-, 45%), 720 (C60, 100%). Subtilisin is a serine protease secreted by members of the genus Bacillus that has high specificity for proteinaceous substrates. Subtilisin BPN′ from Bacillus amyloliquifaciens has 275 amino acids without any cysteine residues. For this study, a mutant subtilisin was produced in which a serine to cysteine (S145C) mutation was carried out by oligonucleotide-directed mutagenesis generating a single sulfhydro group per enzyme molecule. This mutant also has a nonspecific G98C mutation as a result of the polymerase chain reaction. Because there is only one attachment site on the maleimidofullerene and only one reactive group on subtilisin, a 1:1 coupling reaction is expected (Figure 1, right). The mutated subtilisin gene was inserted into a plasmid, and the protein was expressed in Bacillus subtilis. The plasmid was designed to produce a protein that is secreted into the culture medium (26). This helps in the purification of the enzyme, as the culture medium is relatively devoid of other proteins in comparison to whole cell lysate. Subtilisin BPN′ was purified using a BioCAD Sprint perfusion chromatography system (PE Biosystems, Framingham, MA) equipped with a POROS HS cation-exchange column (26). A volume of 1 mL of subtilisin solution was loaded to the column, and 1-mL fractions were collected and tested for protein concentration. The protein content was determined with the Micro BCA method (Pierce, Rockford, IL) using bovine serum albumin solutions as standards. The purified protein was dialyzed against phosphate buffer, pH 6.2, and stored after lyophilization at -20 °C. A Reduce-Imm column (Pierce) was used to reduce any formed intermolecular disulfide bonds and generate the free sulfhydryl group immediately prior to reaction with the maleimidofullerene. The conjugation of maleimidofullerene and subtilisin was performed in phosphate buffer, pH 7.0, a pH that favors the reaction of maleimides with thiols over amines. An amount of 1.6 mg of N-(3-maleimidopropionyl)-3,4-fulleropyrrolidine was added to 5 mL of phosphate buffer (pH 7.0) containing 0.06 µmol of mutant subtilisin, and the mixture was vortexed to

obtain a homogeneous suspension. The reaction was carried out for 5 h at 4 °C on a rotary shaker. The mixture was centrifuged and rinsed three times with 100 mM Tris-HCl, pH 8.6, buffer to separate the unreacted enzyme. The enzymatic activity was determined in the supernatant and the fullerene-containing solid. The enzyme releases p-nitroaniline form the substrate succinyl-Ala-Ala-Pro-Phe-p-nitroaniline (SAAPF-pNA) (Sigma, St. Louis, MO). The corresponding increase in absorbance at 412 nm was monitored on a HP 8453 Spectrophotometer. To measure the enzymatic activity, immobilized subtilisin was suspended in the buffer (Tris-HCl, pH 8.6), and aliquots of the suspension were transferred to a cuvette containing the substrate and stirred. Initially, studies were carried out to investigate whether there is physical adsorption of subtilisin on unmodified C60. For this, an excess of C60 (2.5 µmol) was added to the mutant subtilisin (0.05 µmol), and the mixture was shaken in a rotary shaker for 5 h as above. After removing from the rotary shaker, the mixture was centrifuged and washed three times. Activity measurement showed that almost the entire enzyme (92%) was present in the supernatant. No enzyme activity was determined on the fullerene fraction indicating the absence of any nonspecific binding on C60. It should be mentioned that a C60 derivative was previously found to inhibit HIV-1 protease by blocking its active site (14). Therefore, we also evaluated the inhibitory effect of C60 on subtilisin. After adding C60 to the enzyme solution, the mixture was vortexed to obtain a homogeneous suspension, which was incubated in a rotary shaker. In this way, C60 was made available to the enzyme. The activity of the enzyme did not change in the presence of C60 indicating the absence of any kind of inhibition. Kinetic studies were carried out to compare the KM and kcat values of free and fullerene-conjugated subtilisin. The amount of enzyme coupled was determined using the Micro BCA protein assay. To determine the KM and kcat values of the homogeneous and immobilized enzyme, the initial rate of the hydrolysis of the substrate SAAPF-pNA for a range of substrate concentrations was measured (Figure 2). The measurements were carried out in 100 mM Tris-HCl buffer (pH 8.6). The values of KM and kcat were obtained from Lineweaver-Burk reciprocal plots. The immobilizeded enzyme demonstrated a higher KM value than the free enzyme, suggesting that there is an influence of coupling on the affinity of the enzyme for the substrate (Table 1); higher KM indicates lower affinity. Although this could be explained by the substrate having less access to the active site, it is more likely that the active site conformation is changed slightly upon

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Figure 2. Initial rate (in units of mM of product formed per second) as a function of substrate concentration for the fullereneconjugated subtilisin. Data shown are average ( standard deviation, n ) 3. The rates were measured at 25 °C. Table 1. Comparing the Kinetic Parameters of Free and Conjugated Enzyme KM (mM)

kcat (s-1)

kcat/KM (M-1 s-1)

free subtilisin 0.17 ( 0.01 51 ( 2 (3.0 ( 0.2) × 105 coupled subtilisin with C60 0.76 ( 0.2 37 ( 2 (4.9 ( 1.3) × 104 immobilized subtilisin on 0.43 ( 0.02 14 ( 0.4 (4.2 ( 0.4) × 104 derivatized silica (26)

coupling to C60. The kcat value of the fullerene-conjugated subtilisin was significantly better when compared to the enzyme immobilized on a nonporous silica support (26) from the same amino acid residue. Compared to the free enzyme, the kcat value of the fullerene-conjugated subtilisin was only reduced by 27%, while in the case of the silica support the corresponding reduction was 73%. Both silica and C60 are nonporous supports. Despite the hydrophilic nature of silica, which is typically advantageous in immobilized enzyme systems, the silica-conjugated subtilisin had >2.5-fold reduction in activity compared to the fullerene-conjugated subtilisin. C60 (0.7 nm) is much smaller in size compared to the silica particles (0.5 µm, average diameter), and it binds to only one enzyme molecule per particle. Thus, crowding effects, which tend to reduce activity at high enzyme loadings (27, 28), are avoided. The effect of pH on the activity of free and conjugated enzyme was also studied. The enzyme-catalyzed hydrolysis of SAAPF-pNA was investigated at 25 °C under various pH conditions. Buffer solutions composed of 0.0500 M acetic acid and 0.0500 M Bis-Tris propane were adjusted to pH 6.0, 7.0, 8.0, 8.5, and 9.0 with HCl or NaOH solutions. To each of these solutions, a fixed volume of substrate stock solution (40 mM SAAPF-pNA in DMF) was added to reach a concentration of 1 mM. At each pH value, a volume of 2.00 mL of the substrate solution of the corresponding pH was mixed with a fixed amount of the free or immobilized enzyme (60 µL) in a cuvette, and the initial reaction rate (60 s) was determined as described above. Attachment on conventional substrates causes changes in the pH profile of hydrolytic enzymatic reactions that is characterized by a displacement or broadening of the profile toward more acidic or basic values. When either proton exchange groups or diffusional limitation are present, both the limbs of the pH profile can be broadened. It was found that the pH profile curve for the coupled enzyme (Figure 3B) was similar to that of the free enzyme (Figure 3A). This can be explained by the lack of charged groups on the surface of C60. Therefore, there should be no effect of the fullerene surface on the protonation state of the substrate. Broadened pH profiles can also be seen if diffusion on the

Figure 3. pH profiles of (A) free subtilisin and (B) subtilisin attached to the maleimidofullerene. All measurements were carried out at 25 °C and with a substrate concentration of 1 mM.

substrate to the enzyme is hindered upon immobilization (29). The small size of the C60 molecule makes the active site of the enzyme accessible to the substrate, and there is no diffusional limitation for the substrate to reach the active site imposed by the C60 surface. Thus, with regard to pH sensitivity, the attached enzyme behaves in a similar manner as free enzyme in solution. In summary, nanotechnology has emerged as a powerful tool in the fabrication of materials having superior and often unique properties. To fully realize the potential of nanotechnology it is essential to control the way by which nanomaterials, such as fullerenes, interact with biomolecules. In this work, a maleimidofullerene was successfully attached to subtilisin through site-specific immobilization. The KM and kcat values of fullereneconjugated subtilisin were found to be different from those of the free enzyme, but the kcat was better in comparison to subtilisin immobilized on silica. Thus, the nature of the immobilization support affects the catalytic properties of the enzyme. It was also found that there is a difference in the pH profile of activity between the two supports, which is consistent with the difference in surface ionizable groups. ACKNOWLEDGMENT

We thank the Office of Naval Research (ONR), National Aeronautics and Space Administration (NASA) and National Science Foundation, Division of Materials Research, under grant no. DMR-9809686 for funding this research. P.N. was partially supported by a training fellowship grant no. P42ES07380 from the National Institute of Environmental Health Sciences, NIH, with funds from HUD. LITERATURE CITED (1) Li, Z., Chung, S., Nam, J., Ginger, D. S., and Mirkin, C. A. (2003) Living templates for the hierarchical assembly of gold nanoparticles. Angew. Chem., Int. Ed. 42, 2306-2309. (2) Loweth, C. J., Caldwell, W. B., Peng, X., Alivisatos, A. P., and Schultz, P. G. (1999) DNA-based assembly of gold nanocrystals. Angew. Chem., Int. Ed. 38, 1808-1812. (3) Parak, W. J., Pellegrino, T., Micheel, C. M., Gerion, D., Williams, S. C., and Alivisatos, A. P. (2003) Conformation of oligonucleotides attached to gold nanocrystals probed by gel electrophoresis. Nano Lett. 3, 33-36. (4) Xu, L., Guo, Y., Xie, R., Zhuang, J., and Yang, W. (2002) Three-dimensional assembly of Au nanoparticles using dipeptides. Nanotechnology 13, 725-728.

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