Platinum nanoparticles encapsulated by aminopeptidase: A multifunctional bioinorganic nanohybrid catalyst

Article abstract of DOI:10.1002/anie.201101833

Two birds with one stone: When platinum nanoparticles are encapsulated by a bacterial aminopeptidase, the novel hybrid combines platinum-catalyzed hydrogenation and peptidase-catalyzed hydrolysis for multistep synthesis (see picture). This concept of bioinorganic integration can be extended to various enzymes and inorganic materials, allowing for the design and fabrication of novel multifunctional materials. Copyright

Full text of DOI:10.1002/anie.201101833

Communications
DOI: 10.1002/anie.201101833
Nanoparticle Hybrids
Platinum Nanoparticles Encapsulated by
Aminopeptidase: A Multifunctional Bioinorganic
Nanohybrid Catalyst**
Boi Hoa San, Sungsu Kim, Sang Hyun Moh, Hyunjoo Lee, Duk-Young Jung, and
Kyeong Kyu Kim*
Angewandte
Chemie
11924
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Angew. Chem. Int. Ed. 2011, 50, 11924 –11929

Biomineralization synthesis of metallic nanoparticles using
biotemplates has been the theme of numerous investiga-
tions,[1] since it allows for the production of high-quality
nanoparticles by altering the physical properties of the
prepared inorganic materials at the ambient temperature.
Most notably, biomineralization using protein shells (PSs) or
cages is being intensely studied for the synthesis of inorganic
nanomaterials that can be applied to electromagnetic devices,
photonics, catalysts, and a variety of other fields.[2] Further-
more, the functional modification of protein surfaces by
genetic or chemical means[3] expands the repertoire of
applying inorganic nanomaterials to medical imaging and
therapy.[3b,4] However, most applications of protein-shelled
nanoparticles (PSNPs) are still limited to their inorganic
properties, and the role of PSs is restricted to that of a
reservoir or, at most, a targeting entity for a specific location,
such as the cell surface.[3b,5] Herein, by employing peptidases
as PSs, we demonstrate that the catalytic activities of both the
proteins and NPs can be combined for multistep synthesis of
the desired compounds. Furthermore, we show that our PSs
are ideal templates for the size-controlled synthesis of
ultrasmall platinum nanoparticles (PtNPs) within the size
range of 0.9–3.2 nm, and that they have higher catalytic
capacities with biocompatibility.
The bacterial aminopeptidase from Streptococcus pneu-
monia (PepA) self-assembles into a well-defined tetrahedral
dodecameric complex with a diameter of about 12 nm at the
exterior and 6 nm at the interior (Figure 1a,b). PepA is a
zinc-dependent metallopeptidase with a substrate preference
for acidic amino acids,[6] and it plays an important role in
catabolizing oligopeptides in bacteria. The active sites located
inside the cavity hydrolyze oligopeptides into free amino
acids. The tetrahedral architecture of PepA reveals a cavity in
the center with four wide channels on the faces of the
tetrahedron and four narrow ones on the edges (Figure 1a).
The wide and narrow channels have diameters of 3 and 1 nm,
respectively, and they are used for substrate entry and product
exit, respectively. Accordingly, these channels are considered
the gates for accepting inorganic molecules during the PtNP
Figure 1. Crystal structure (PDB ID: 3KL9) and TEM images of PepA.
a) The ribbon diagram of a PepA dodecamer. The PepA subunits are
shown in different colors, and the channels with diameters of 1 and
3 nm are indicated by black and red arrows, respectively. b) The charge
distribution on the internal surface of PepA: negative charges are red
and positive charges are blue, and the inside and outside dimensions
are indicated. c,d) PepA–PtNP complexes were analyzed using electron
microscopy with 2% uranyl acetate staining (c) and without staining
(d). The insets are magnified images of the uranyl acetate-stained
PepA–PtNP complexes (c) and PtNPs (d). The lattice fringes show
periodicities of 0.224 (c) and 0.225 nm (d) in the [111] direction (white
arrows). Scale bars are 20 nm unless otherwise noted.
synthesis. The negatively charged interior surface seems to be
crucial for metal deposition in the initial stage of the reaction
(Figure 1b). The geometric and electrostatic complementar-
ity among each subunit results in very tight subunit inter-
action. Owing to these features, PepA is considered an ideal
biotemplate for the synthesis of inorganic nanoparticles
(Figure 1b).
[*] B. H. San, S. H. Moh, Prof. Dr. K. K. Kim
Department of Molecular Cell Biology
Samsung Biomedical Research Institute
Sungkyunkwan University School of Medicine
Suwon 440-746 (South Korea)
PtNPs were synthesized within PepA by slowly reducing
K2PtCl4 using NaBH4 as a reducing agent in an aqueous
solution at room temperature. The synthesis was monitored
using transmission electron microscopy (TEM) (Figure 1c,d)
and UV/Vis spectroscopy (Figure 2). The formation of PtNPs
was also visibly monitored by color change from light yellow
to dark brown (Figure S1, Supporting Information). Consis-
tent with other studies,[7] PepA–PtNPs showed broad UV/Vis
absorption spectra in a wide range of wavelengths, but there
E-mail: kkim@med.skku.ac.kr
Homepage: http://smsb1.skku.ac.kr/index.html
B. H. San, S. H. Moh, Prof. Dr. D.-Y. Jung, Prof. Dr. K. K. Kim
Sungkyunkwan Advanced Institute of Nanotechnology
Sungkyunkwan University, Suwon 440-746 (South Korea)
Prof. Dr. D.-Y. Jung
Department of Chemistry-BK21-SAINT, Institute of Basic Sciences
Sungkyunkwan University, Suwon 440-746 (South Korea)
2À
were no peaks corresponding to the absorption of PtCl4 or
the plasmon absorption. UV absorption at 280 nm, which is a
representative fingerprint of proteins, did not shift, but rather,
it gradually increased in intensity and was finally saturated,
implying that the formation of PepA–PtNPs was kinetically
controlled (Figure 2). PepA–PtNPs were purified using high-
speed centrifugation and size-exclusion chromatography
(SEC). The TEM images of PepA–PtNPs stained with 2%
uranyl acetate revealed uniform round PtNPs encapsulated in
S. Kim, Prof. Dr. H. Lee
Department of Chemical and Biomolecular Engineering
Yonsei University, Seoul 120-749 (South Korea)
[**] This work was supported by the Next-Generation BioGreen 21
Program (SSAC PJ008107).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201101833.
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Communications
Figure 2. UV/Vis spectra of PepA–PtNPs in the 250–400 nm range.
PepA and Pt precursors were incubated at a 1:1000 molar ratio. The
spectra obtained from the incubation of 0, 1, 2, 3, 5, 10, 15, 20, 25, 30,
35, 40, 45, 50, 55, 60, and 120 min are differently colored. The
absorption spectra appeared saturated after about 50 min, indicating
the termination of the reaction. The inset panel shows the UV/Vis
spectra of PepA, the Pt precursors, and PepA–PtNP complexes over
the spectral range of 250–800 nm.
tetrahedral PepA shells (Figure 1c). The molar ratio between
the Pt precursors and PepA and the incubation time
determined the size of PtNPs encapsulated by PepA. When
a reaction mixture with a molar ratio of 1000:1 was incubated
until the Pt precursors were completely reduced, PtNPs with
diameters of (2.1 Æ 0.3) nm were synthesized inside of PepA,
and these were used for the physicochemical characterization.
The lattice spacing of the crystalline PtNPs was 0.224 nm,
which can be assigned to the (111) plane of fcc platinum
(Figure 1d). Therefore, the crystalline property of PtNPs
synthesized by PepA is same as that of PtNPs encapsulated by
other protein shells such as ferritin,[7b] DNA-binding protein
of starved cells (Dps),[8] and small heat-shock protein
(sHSP).[2b] The inductively coupled plasma mass spectrometry
(ICP-MS) analysis confirmed that PepA possess smaller
amounts of zinc ions after harboring PtNPs, suggesting that
the zinc ions bound to the active site of PepAwere released or
replaced by platinum ions (Table S1, Supporting Informa-
tion). The SEC profiles of the apo-PepA and PepA–PtNP
complexes monitored at 280 and 350 nm revealed that the
major peaks were eluted at the same volume, thus confirming
that Pt was deposited within the PepA dodecamers and not on
their surfaces (Figure S2, Supporting Information). Fourier-
transform infrared (FTIR) spectroscopy and X-ray photo-
electron spectroscopy (XPS) revealed the binding sites of
PtNPs in PepA and the metallic state of the deposited Pt
(Figures S3 and S4; Table S2, Supporting Information).
The size of PtNPs was precisely controlled in the range of
0.9 to 2.1 nm by changing either the reaction time (kinetically
controlled synthesis; Figure 3a–e) or the concentration ratio
between Pt precursors and proteins (ratio-controlled syn-
thesis; Figure 3 f–j). The size of PtNPs increased up to 3.2 nm
when PepAwas saturated with excess Pt precursor (Figure S5,
Supporting Information). Smaller nanoparticles are known to
Figure 3. The size-controlled evolution of the PepA-encapsulated
PtNPs and their catalytic activities. a–e) Kinetically controlled synthesis
of PepA–PtNPs. Pt precursors were incubated with PepA at a
1000:1 molar ratio. Varying incubation times of 1 (a), 5 (b), 15 (c), 30
(d), and 5 h (e) yielded average complex sizes of 0.9, 1.4, 1.7, 1.9, and
2.1 nm, respectively (size distribution histogram, Figure S8, Supporting
Information). f–j) Ratio-controlled synthesis of PepA–PtNPs. Pt precur-
sors were incubated with PepA at varying molar ratios of 50:1 (f),
100:1 (g), 250:1 (h), 500:1 (i), and 1000:1 (j) for 60 min, yielding
average complex sizes of 0.9, 1.1, 1.5, 1.7, and 2.0 nm, respectively
(size distribution histogram, Figure S9, Supporting Information). Each
scale bar represents 20 nm. The proteolysis and hydrogenation activ-
ities of the PepA–PtNP complexes synthesized in a–e and f–j are
plotted versus the size of PtNPs ((k) and (l), respectively; Figure S10
and Figure S11, Supporting information). The proteolytic activity of
PepA–PtNPs was monitored by the hydrolysis of substrates over
15 min at 378C, expressed as the relative activity to free PepA. The
hydrogenation activity is denoted by the rate constant.
possess higher catalytic activity,[9] but the synthesis of ultra-
small PtNPs at a subnanometer scale is a challenging issue. To
date, only a few investigations have addressed the kinetic
effects in the size control of PtNPs[10] or the synthesis of
ultrasmall PtNPs,[11] but those efforts were rather limited and
impractical. In this regard, the current study provides a simple
and reliable technique for the synthesis of ultrasmall PtNPs
that are smaller than any other known PtNPs produced in
ambient conditions.[10] Collectively, it is believed that the
tetrahedral protein shell of PepA is an attractive biotemplate
for the syntheses of ultrasmall inorganic nanoparticles.
The peptidase activity of the PepA–PtNPs was monitored
in two ways: the amount of released of p-nitroanilide from
glutamic acid-p-nitroanilide for 15 min was measured (Fig-
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ure 3k,l), and the ration of the catalysis rate constant to the
Michaelis constant (kcat/Km) of PepAwas calculated (Table 1).
As confirmed in the ICP-MS experiments, PepA–PtNPs
Table 1: Peptidase and hydrogenation activities of PepA–PtNPs.
Peptidase activity
cat/Km [mmÀ1 sÀ1
Hydrogenation activity
k
]
rate constant [10À3 sÀ1
]
PepA
14.88Æ0.45
6.29Æ0.49
4.30Æ0.02
3.50Æ0.06
1.66Æ0.01
–
PepA–PtNP ca. 0.9 nm
PepA–PtNP ca. 1.0 nm
PepA–PtNP ca. 1.5 nm
PepA–PtNP ca. 2.0 nm
10.400
4.552
4.200
2.730
contained a smaller amount of zinc ions than the Pt-free
PepA, but they still have the high peptidase activity of PepA–
PtNPs, possibly owing to the bound zinc and platinum ions.
The reaction rate constant of PepA–PtNPs-catalyzed hydro-
genation was also estimated using p-nitrophenol as a sub-
strate. Since PepA–PtNPs produced from the Pt/protein ratio-
controlled synthesis were catalytically more active (Figure 3l)
than those from the kinetic controlled synthesis (Figure 3k),
their activity is referred to in the discussion, unless otherwise
specified. PepA–PtNPs showed higher catalytic activity than
the citrate-reduced PtNPs[12] or the Tween80-capped PtNPs[13]
of equivalent sizes (Figure S6 and Figure S7, Supporting
Information). The peptidase and hydrogenation activities
were both inversely proportional to the particle size (Figure 3
and Table 1), and the sub-nanometer-sized PepA–PtNPs were
the most efficient catalysts: the proteolytic activity of PepA–
PtNPs was comparable to that of the free PepA in terms of the
amount of released product during a assigned time, and the
hydrogenation activity was fourfold higher than that of the
2 nm PtNPs (Figure 3l). Because they function as an enzyme
and a chemical catalyst, PepA–PtNPs can be denoted as a
bioinorganic nanohybrid catalyst. At the same time, PepA
exerted strong stabilizing effects on PtNPs, since PepA
extended the lifespan of PtNPs more effectively than two
other representative molecular capping agents (citrate and
Tween80, Figure 4a). Furthermore, PepA–PtNPs also exhib-
ited both sorts of activity in organic solvents (Figure 4b),
implying that the diversity of the catalytic conditions may
contribute to expanding the repertoire of applications for this
multifunctional nanocatalyst.
The PepA complexes harboring the 0.9 nm PtNPs, which
possessed the highest catalytic activity, appear to be suitable
for various application purposes. In similar ways, PepA can
possibly be applied to synthesize various inorganic catalytic
materials that have high catalytic activity, as PSs have been
successfully used for the synthesis of a wide range of inorganic
nanoparticles.[1a,c,d,2b,3c,4,14] Moreover, PepA–PtNPs showed
no detectable toxicity on cultured cells in the MTT assay,
which is unlike PtNPs stabilized by Tween80, suggesting that
PepA can reduce the cytoxicity of PtNPs (Figure 4c).
Inorganic materials may acquire biocompatibility through
PepA encapsulation, which is essential for the biomedical
application of nanomaterials. Consistently, it was also con-
firmed that the catalytic reactions do not affect the conforma-
Figure 4. The lifespan catalytic activity of PepA–PtNPs, their activity in
organic solvents, and their biocompatibility. a) Catalytic activities of
!
&
*
PepA–PtNPs ( ), citrate–PtNPs ( ) and Tween80–PtNPs ( ) with
equivalent diameters (2 nm) in a multiple-cycle reaction. The catalytic
activity of each of the PtNPs, as expressed by the rate constant of the
first reaction, was set to 100%, and the relative activities of the next
reactions were plotted. b) The hydrolysis (black) and hydrogenation
(gray) activities of PepA–PtNPs in 50% and 25% tert-butyl alcohol,
25% DMSO (dimethyl sulfoxide), and 25% DMF (dimethyl forma-
mide) are shown relative to the activity in an aqueous solution. c) Cell
!
*
viabilities in the presence of PepA–PtNPs ( ) or Tween80–PtNPs ( ).
(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assays were performed using Chinese hamster ovary (CHO) cells
grown with varying concentrations (10, 25, 50, 100, and 150 mm) of
PepA–PtNPs or Tween80–PtNPs. Values represent the means Æ
standard deviation from six experiments.
tional integrity of PepA–PtNPs, since the SEC profile of
PepA–PtNPs was not changed after multiple reactions either
in water (Figure S2, Supporting Information) or in organic
solvents (data not shown).
The catalytic activities of PepA and PtNPs were tested
simultaneously using Glu-p-nitroanilide as a substrate. PepA-
mediated hydrolysis yielded p-nitroanilide, with the develop-
ment of yellow color (Figure 5). Subsequent hydrogenation of
p-nitroanilide was monitored by the disappearance of visible
color under reduction conditions. When incubated with the
PepA–PtNP complex and NaBH4, Glu-p-nitroanilide
instantly formed the final product p-phenylenediamine, thus
demonstrating that this two-step reaction was completed by
the bioinorganic nanohybrid catalyst.
Although PSs have previously served as templates and
carriers in the synthesis and transfer of inorganic nano-
particles,[1a,e,3a,15] they have not been considered as active
components. Herein, PepA was catalytically active and func-
tional even with PtNPs deposited inside. PepA could be an
excellent component of robust biomaterial, as it not only
performs the enzymatic function but also stabilizes the
performance of inorganic catalysts and substantially reduces
the cytoxicity of PtNPs. In these regards, PepA–PtNPs are
differentiated from the other known bioinorganic hybrids,
which are mostly simple conjugates of biomolecules and
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[1] a) F. C. Meldrum, V. J. Wade,
D. L. Nimmo, B. R. Heywood,
S. Mann, Nature 1991, 349,
684; b) F. C. Meldrum, B. R.
Heywood, S. Mann, Science
1992, 257, 522; c) S. Mann,
D. D. Archibald, J. M. Didy-
mus, T. Douglas, B. R. Hey-
wood, F. C. Meldrum, N. J.
Reeves, Science 1993, 261,
1286; d) T. Douglas, D. P.
Dickson, S. Betteridge, J.
Charnock, C. D. Garner, S.
Mann, Science 1995, 269, 54;
e) T. Douglas, M. Young,
Nature 1998, 393, 152.
[2] a) B. Dragnea, C. Chen, E. S.
Kwak, B. Stein, C. C. Kao, J.
Am. Chem. Soc. 2003, 125,
6374; b) Z. Varpness, J. W.
Peters, M. Young, T. Douglas,
Nano Lett. 2005, 5, 2306;
c) R. J. Tseng, C. Tsai, L. Ma,
J. Ouyang, C. S. Ozkan, Y.
Figure 5. The multistep reaction catalyzed by the PepA–PtNP complexes. a) Hydrolysis of Glu-p-nitro-
anilide (dash dotted line) to p-nitroanilide (dotted line) was monitored by the change in the absorption
peak from 310 to 380 nm. Subsequent hydrogenation of p-nitroanilide to p-phenylenediamine resulted in
the disappearance of the absorption peak at 380 nm and the emergence of a new peak at 305 nm (dashed
line). A solid line indicates the absorption spectrum of the PepA–PtNP complexes. b) Reaction schemes
for the PepA–PtNP-catalyzed productions of p-phenylenediamine from Glu-p-nitroanilide by proteolysis (1)
and hydrogenation (2). Glutamic acids were not hydrogenated under the experimental conditions.
Yang,
Nat.
Nanotechnol.
2006, 1, 72.
[3] a) M. L. Flenniken, D. A. Wil-
lits, S. Brumfield, M. J. Young,
T. Douglas, Nano Lett. 2003, 3,
1573; b) M. Uchida, M. L.
Flenniken, M. Allen, D. A.
Willits, B. E. Crowley, S. Brumfield, A. F. Willis, L. Jackiw, M.
Jutila, M. J. Young, T. Douglas, J. Am. Chem. Soc. 2006, 128,
16626; c) H. Wu, J. Wang, Z. Wang, D. R. Fisher, Y. Lin, J.
Nanosci. Nanotechnol. 2008, 8, 2316.
inorganic nanomaterials. Moreover, the use of PepA facili-
tated stringent control over the size of nanoparticles, which
enabled us to reproducibly synthesize PtNPs with diameters
ranging from 0.9 to 3.2 nm. Therefore, with these advantages,
PepA surpasses the other known PSs for designing new
functional materials.[1a,e,3a,16] Considering the higher biocom-
patibility of PepA–PtNPs and the functionality of PSs
acquired by genetic or chemical modification of proteins, it
is expected that the current study can be extended to the
construction of nanobiofactories to produce complex mole-
cules for the purpose of biomedical application. In a similar
approach, various inorganic catalysts could be combined with
enzymes with PS-like architectures, such as oligomeric
acyltransferases,[17] aldolases,[18] decarboxylases,[19] and pyro-
phosphatases,[20] to diversify the repertoires of bioinorganic
nanohybrids for the synthesis of chemical compounds. Con-
sidering the increasing demand for enzymes to perform in
organic synthesis,[21] this study could expand the scope of
synthetic organic chemistry in either aqueous or organic
solvents.
[4] J. F. Hainfeld, Proc. Natl. Acad. Sci. USA 1992, 89, 11064.
[5] M. L. Flenniken, D. A. Willits, A. L. Harmsen, L. O. Liepold,
A. G. Harmsen, M. J. Young, T. Douglas, Chem. Biol. 2006, 13,
161.
[6] D. Kim, B. H. San, S. H. Moh, H. Park, D. Y. Kim, S. Lee, K. K.
Kim, Biochem. Biophys. Res. Commun. 2010, 391, 431.
[7] a) J. A. Creighton, D. G. Eadon, J. Chem. Soc. Faraday Trans.
1991, 87, 3881; b) J. Fan, J. J. Yin, B. Ning, X. Wu, Y. Hu, M.
Ferrari, G. J. Anderson, J. Wei, Y. Zhao, G. Nie, Biomaterials
2011, 32, 1611.
[8] S. Kang, J. Lucon, Z. B. Varpness, L. Liepold, M. Uchida, D.
Willits, M. Young, T. Douglas, Angew. Chem. 2008, 120, 7963;
Angew. Chem. Int. Ed. 2008, 47, 7845.
[9] C. Wang, H. Daimon, T. Onodera, T. Koda, S. H. Sun, Angew.
Chem. 2008, 120, 3644; Angew. Chem. Int. Ed. 2008, 47, 3588.
[10] a) Y. Li, G. P. Whyburn, Y. Huang, J. Am. Chem. Soc. 2009, 131,
15998; b) Y. J. Li, Y. Huang, Adv. Mater. 2010, 22, 1921.
[11] K. Yamamoto, T. Imaoka, W. J. Chun, O. Enoki, H. Katoh, M.
Takenaga, A. Sonoi, Nat. Chem. 2009, 1, 397.
[12] J. Turkevich, R. S. M. Jr., L. Babenkova, J. Phys. Chem. 1986, 90,
4765.
Received: March 15, 2011
Revised: June 16, 2011
Published online: September 1, 2011
[13] H. Tsuji, Method for Producing Platinum Colloid, and Platinum
Colloid Produced By The Same, U.S. Patent 6455594 B1, 2002.
[14] a) M. Allen, D. Willits, J. Mosolf, M. Young, T. Douglas, Adv.
Mater. 2002, 14, 1562; b) M. T. Klem, D. Willits, D. J. Solis, A. M.
Belcher, M. Young, T. Douglas, Adv. Funct. Mater. 2005, 15,
1489.
[15] T. Douglas, E. Strable, D. Willits, A. Aitouchen, M. Libera, M.
Young, Adv. Mater. 2002, 14, 415.
[16] M. L. Flenniken, M. Uchida, L. O. Liepold, S. Kang, M. J.
Young, T. Douglas, Viruses Nanotechnol. 2009, 327, 71.
Keywords: aminopeptidase · bioinorganic catalysts ·
nanoparticles · platinum · protein shells
.
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[17] I. Mathews, M. Soltis, M. Saldajeno, G. Ganshaw, R. Sala, W.
Weyler, M. A. Cervin, G. Whited, R. Bott, Biochemistry 2007,
46, 8969.
[20] C. A. Wu, N. K. Lokanath, D. Y. Kim, H. J. Park, H. Y. Hwang,
S. T. Kim, S. W. Suh, K. K. Kim, Acta Crystallogr. Sect. D 2005,
61, 1459.
[18] S. Bauer, A. K. Schott, V. Illarionova, A. Bacher, R. Huber, M.
Fischer, J. Mol. Biol. 2004, 339, 967.
[21] F. Raimondi, G. G. Scherer, R. Kotz, A. Wokaun, Angew. Chem.
2005, 117, 2228; Angew. Chem. Int. Ed. 2005, 44, 2190.
[19] H. J. Chen, T. P. Ko, C. Y. Lee, N. C. Wang, A. H. J. Wang,
Structure 2009, 17, 517.
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