Immobilization of hexa-arginine tagged esterase onto carboxylated gold nanoparticles

Article abstract of DOI:10.1039/b504184h

Hexa-arginine tagged esterase was efficiently immobilized onto carboxylated gold nanoparticles (AuNP-COOH) and its enzyme activity was investigated by monitoring the absorption spectrum of an enzyme substrate, p-nitrophenol butyrate. The Royal Society of

Full text of DOI:10.1039/b504184h

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Immobilization of hexa-arginine tagged esterase onto carboxylated gold
nanoparticles{
Tai Hwan Ha,a Jin Young Jeongb and Bong Hyun Chung*ab
Received (in Cambridge, UK) 24th March 2005, Accepted 13th June 2005
First published as an Advance Article on the web 6th July 2005
DOI: 10.1039/b504184h
Hexa-arginine tagged esterase was efficiently immobilized onto
carboxylated gold nanoparticles (AuNP-COOH) and its
enzyme activity was investigated by monitoring the absorption
spectrum of an enzyme substrate, p-nitrophenol butyrate.
Biomolecules bound to a metal nanoparticle have demonstrated
quite amazing properties such as highly efficient drug delivery
through the blood–brain barrier,1 facile electron transfer from
glucose oxidase to a metal electrode without a mediator,2
facilitated gene delivery through cell membranes,3 and modulation
of enzyme activities.4 However, the grafting of proteins onto metal
nanoparticles is especially complicated since the conformation of a
protein is liable to change through nonspecific interactions, causing
it to lose its unique enzymatic activities.5 Among protocols that
specifically anchor and separate a concerned protein, recombinant
protein synthesis with an affinity ligand at the C- or N-terminal
might be a breakthrough to avoid nonspecific interactions. A
surface modified by Ni2+–nitrilotriacetic acid (Ni2+–NTA), for
instance, has been widely used for the specific binding of
polyhistidine tagged proteins. Moreover, in recent studies,
oligo(ethylene glycol) has been reported to considerably suppress
the nonspecific adsorption and the subsequent denaturation of
proteins.6 Herein, we report manufacture of a highly water
Fig. 1 (a) A TEM image of citrate reduced gold nanoparticles spread on
a copper grid (inset: size distribution of particles observed in the picture).
dispersive gold nanoparticle (AuNP) that can efficiently immobi-
lize hexa-arginine tagged esterase (Arg6-esterase). While the
carboxylated AuNP (AuNP-COOH) also showed non-specific
interactions, Arg6-esterase bound dominantly onto the surface of
AuNP-COOH in the presence of other proteins and exhibited a
considerable enzymatic activity.
(b) A series of UV spectra as the solution pH varies by dropwisely adding
HCl and NaOH solution sequentially. (c) Schematic illustration of surface
modification of AuNP-COOH with Arg6-esterase and the enzyme reaction
by the immobilized esterase.
conversion to AuNPs, the concentration of AuNP is estimated to
be about 18 nM. The high stability of the AuNP was manifested in
several control experiments. First, AuNP solution in a vial was
dried by continuous blowing of N2 gas and was re-dispersed with
distilled water. Second, the carboxylated AuNPs were washed out
by centrifugation/redispersion cycles twice, and a working solvent
was easily exchanged into methanol, DMF and DMSO. Third, a
local plasmon band red-shifted up to y 630 nm as the pH was
lowered by the addition of HCl. However, it reversibly recovered
to the original band position (y 525 nm) when concentrated
NaOH solution was added dropwise (Fig. 1(b)).8
For the reproducible immobilization of proteins with an
adequate orientation, highly stable gold nanoparticles were
prepared by surface modification of citrate-reduced gold nano-
particles with 16-mercaptohexadecanoic acid (MHA).7 The AuNP
reduced by citrate is known to be sufficiently hydrophilic but has a
tendency to aggregate, depending on the micro-environment. This
vulnerability was substantially improved by protecting AuNP with
a long aliphatic thiol with a carboxyl end group. Fig. 1(a) shows a
TEM image of highly monodisperse AuNPs spread on a copper
grid with an average diameter of 13 nm. Taking the given
concentration of gold salt into consideration and assuming perfect
As shown in Fig. 1(c) schematically, the carboxylated AuNP
functioned as a ‘nano-supporter’ to immobilize recombinant
esterases with affinity tags (hexa-arginine and hexa-histidine,
respectively) as well as intact ones. As the supporting material
for enzymes, it is noteworthy that the esterases tested in the present
study tended to adsorb onto AuNP-COOH irrespective of the
presence of affinity tag, although the amounts were strongly
dependent on the presence of adequate affinity tag. Furthermore,
no agglomerate was formed during the adsorption of esterase onto
the carboxylated nanoparticle. The high stability is attributed to
aBioNanotechnology Research Center, Korea Research Institute of
Bioscience and Biotechnology, P. O. Box 115, Yuseong, Daejeon,
305-600, Korea
bUniversity of Science and Technology, P. O. Box 115, Yuseong,
Daejeon, 305-600, Korea. E-mail: chungbh@kribb.re.kr;
Fax: 82 42 8798594; Tel: 82 42 8604442
{ Electronic supplementary information (ESI) available: experimental
details, analyses of FTIR-ATR spectra of AuNP-COOH, and kinetic
analyses of enzymes. See http://dx.doi.org/10.1039/b504184h
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Chem. Commun., 2005, 3959–3961 | 3959

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negatively charged AuNP-COOH9 and relatively low pI value of
the concerned protein (for esterase, 5.9) compared to the working
pH 8.0, which makes proteins bear net negative charges and repel
each other. One important merit of protein immobilization onto
AuNP might be the controlled surface roughness, which assures
relatively homogeneous activities of adsorbed enzymes, compared
to conventional polycrystalline gold surfaces.
proteins (Fig. 2). A number of contaminant protein bands as well
as the Arg6-esterase exist in the stock solution (lane 1), but the
intensities of these contaminant protein bands were substantially
reduced or even disappeared (as indicated by the white arrow in
lane 2) after adsorption to AuNP-COOH. This relative specificity
seems to be caused by faster adsorption of polyarginine tag onto
AuNP-COOH than other non-tagged proteins.
The amount of esterase immobilized on AuNP was semi-
quantitatively analyzed by SDS-PAGE. Unlike the agarose gel
used in the recent studies on protein–nanoparticle complexes,6 the
polyacrylamide gel hardly permitted the present AuNPs to traverse
its networked pores due to the smaller pore size; when a part of the
SDS gel entrapping AuNPs was brought to the UV measurement,
no absorption band of a staining dye was detected. As shown in
Fig. 2, esterase immobilized on the AuNP-COOH definitely
appeared at the same position as the proteins in a solution state
(y 42 kDa, black arrows). It is also noteworthy that only 250 ml of
Arg6-esterase containing AuNP-COOH was concentrated and
analyzed (lanes 2 and 3), whereas 1 ml of His6-esterase containing
AuNP-COOH was used for analysis (lanes 5 and 6). Taking the
aforementioned concentration of AuNP-COOH (18 nM) into
consideration, a single AuNP-COOH was estimated to tether
about 25 Arg6-esterases. Similarly, 6 His6-tagged and 4 non-tagged
esterases were immobilized onto each AuNP-COOH. The
enhanced adsorption characteristics of Arg6-esterase might be
attributed to electrostatic attraction between the positively charged
polyarginine tag and AuNP-COOH with a slightly negative
charge. This electrostatic attraction could be released by treatment
with a concentrated electrolyte.10
The enzymatic activities of the esterases on the surface of AuNP-
COOH were investigated by monitoring the UV/Vis absorption
characteristics of the enzymatic substrate, p-nitrophenol butyrate
(pNPB), which develops a new band at 400 nm as it dissociates. In
Fig. 3(a), a series of absorbance measurements indicate that the
enzymatic reaction in the AuNP-COOH solution (18 nM, 1 mL)
containing Arg6-esterase reached a saturation level within 5 min.
The kinetic curves of the hydrolyzed substrate (pNPB, 0.1 mM) at
various concentrations of His6-esterase (in solution) are depicted as
references in gray lines. Although absorbance measurements of
His6- and non-tagged esterases were made at the same concentra-
tion of AuNP-COOH, substantially lower enzyme activities were
observed as shown in Fig. 3(a). The enzymatic activity of Arg6-
esterase immobilized on AuNP-COOH was more clearly seen when
sixteen times diluted Arg6-esterase on AuNP-COOH (1.1 nM) was
introduced into the given pNPB solution (Fig. 3(a)).
From the reference curves, the amount of the active Arg6-
esterase was estimated and summarized along with the other two
esterases in Table 1. Since 0.65 mg of Arg6-esterase was estimated
As shown in Fig. 2, Arg6-esterase adsorbed onto AuNP-COOH
(lane 2) was soaked with 1 M NaCl for 10 min, centrifuged, and
the resulting pellet of AuNP was analyzed by SDS-PAGE (lane 3).
The amount of Arg6-esterase remaining on the AuNP-COOH
surface was substantially reduced after elution with 1 M NaCl.
However, the amount of esterase with His6-esterase, of which the
histidine tag has a pKa value of 6.5 and thus exists in a neutral state
at the working pH 8.0, was almost identical to that of non-tagged
esterase and was hardly changed by the treatment with 1 M NaCl
(lanes 5 and 6). All these observations stress the importance of
positive charges of the hexa-arginine tag in the large adsorption
onto AuNP-COOH and the maintenance of enzyme activity
(vide infra).10,11 Furthermore, Arg6-esterase seems to be dominant
absorbant onto the AuNP-COOH in the presence of other
Fig. 3 (a) Enzymatic activity assay of Arg6-tagged (concentrated and
diluted), His6-tagged, and non-tagged esterase tethered on AuNP-COOH
versus incubation time with a hydrolyzing substrate, pNPB (0.1 mM). For
Arg6-tagged esterase, both concentrated and sixteen times diluted solutions
are depicted simultaneously. (b) Enzymatic activity plot of Arg6-tagged
esterase on AuNP-COOH (diluted) before and after elution with 1 M NaCl.
Fig. 2 Amounts of Arg6-esterase (lanes 2 and 3) and His6-esterase (lanes
5 and 6) tethered on AuNP-COOH (18 nM) before and after the elution
with 1 M NaCl solution, in comparison to the bands obtained by loading
the tagged esterase in the solution state (lane 1: Arg6-tagged, lane 4:
His6-tagged).
3960 | Chem. Commun., 2005, 3959–3961
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Table 1 Total amounts of recombinant esterases with distinct affinity tags and amounts of the proteins in active form measured by diverse
experimental methods
Elution of immobilized esterase by 1 M NaCl
Affinity tagged
esterase
Amount of immobilized
esterase(SDS-PAGE)
Amount of active esterase
(UV/Vis)
Surviving
activity
aeluted(Bradford)
remaining(SDS-PAGE)
Arg6
His6
none
a
18 mg/ml
4 mg/ml
3 mg/ml
9.5 mg/ml
0.8 mg/ml
0.5 mg/ml
8 mg/ml
3 mg/ml
2 mg/ml
10.4 mg/ml
0.3 mg/ml
0.2 mg/ml
58%
8%
7%
The Bradford method quantified all the proteins eluted by 1 M NaCl, while SDS-PAGE results estimated a specific band (or protein) after
the gel-electrophoresis.
to maintain its activity in the diluted solution, about 10.4 mg of the
esterase existed in an active form in the normalized AuNP-COOH
solution (18 nM, 1 mL). Taking the total amount of the
immobilized Arg6-esterase based on SDS-PAGE analyses into
account, 58% of the esterase on an average was found to maintain
its activity (vide supra). On the other hand, the His6- and non-
tagged esterases on AuNP-COOH demonstrated extremely small
enzymatic activities of 0.3 mg/ml and 0.2 mg/ml, respectively
(Fig. 3(a)), which corresponded to about 8% and 7% of the
adsorbed esterases, respectively.
through multivalent and electrostatic interactions between Arg6-
tag and carboxyl groups.
The Arg6-esterase was efficiently adsorbed onto AuNP-COOH
through electrostatic attraction with its enzymatic activity main-
tained, while most His6-tagged and non-tagged esterases were
nonspecifically adsorbed with a trace of activity. Nonetheless, it
should be noted that a substantial amount of Arg6-esterase was still
nonspecifically adsorbed, and that only 58% of adsorbed esterases
demonstrated an enzymatic activity. The reduced activity appears
to be caused by hydrophobic interactions between proteins and the
underlying hydrophobic region beneath the carboxyl group.12 It is
expected that the hydrophobic interaction, which results in protein
denaturation, can be substantially alleviated by using oligo(ethylene
glycol) as a spacer between the carboxyl end group and methylene
chains.12 In addition, a proton partition effect caused by negative
charges accumulated at the interface can also result in the
deterioration of acid-labile esterase.13 Furthermore, this partition
effect might explain the higher enzyme activity in Arg6-esterase in
that the local acidity at the interface can be effectively reduced
through partial neutralization by more basic arginine groups.
In summary, this paper describes the efficient immobilization of
recombinant esterase with a polyarginine affinity tag onto AuNP-
COOH, which will find wide applications in diverse research fields
including biosensors, bioimaging, and proteomics.
Although the reduced activity of the immobilized Arg6-esterase
(i.e. 58%) seems to be caused by a surface-induced conformation
change, diffusional limitation in this bound enzyme system might
also affect the apparent activity without actually fouling the
structure of individual protein; the present esterase–AuNP
complex can be an extreme case of the immobilized enzyme
system compared to conventional polymer beads (with a diameter
of y 10 mm). To address this issue, a kinetic experiment was
performed that measured initial reaction rates for several pNPB
concentrations with fixed amounts of free and immobilized Arg6-
esterases; we assumed Michaelis–Menten type kinetics for the
present system. As can be seen in Fig. S3 in the ESI, both free
and bound Arg6-esterases had the same Km values (x-intercepts)
in the double reciprocal plots for four pNPB concentrations
(. 0.05 mM). Accordingly, the esterases on the surface of AuNP
can be concluded not to be restricted by the substrate diffusion at
the working concentration (y 0.1 mM).
This work was supported by a grant from the KRIBB Initiative
Program.
It is not clear yet whether all the Arg6-esterases on AuNP-
COOH were homogeneously down-regulated through a surface-
induced conformation change, or whether the immobilized
esterases tested had quite diverse activities. In order to resolve
this ambiguity, the Arg6-esterase was eluted from the surface of
AuNP-COOH with 1 M NaCl as shown in Fig. 3(b). When the
Arg6-esterase was eluted from the surface of AuNP-COOH with
1 M NaCl (see Fig. 3(b)), the enzymatic activity of the eluted Arg6-
esterase from AuNP-COOH (62.5 ml, 18 nM) was almost the same
as that of the immobilized one before the elution. On the other
hand, the remaining esterases on AuNP-COOH, which were
resistant to the elution, were totally inactive. The eluted esterase
was further quantified by the Bradford method and summarized
in Table 1. It is noteworthy that the amount of the eluted esterase
(y 9.5 mg/ml) was quite comparable with the value (y 10.4 mg/ml)
estimated by its activity before the elution. Associated with the
kinetic experiments, the result suggests that the immobilized
esterases represent all-or-none type activity profiles. Most of the
active esterase on AuNP-COOH seemed to be rescued intact with
a concentrated electrolyte. This observation, in turn, supported the
idea that the active esterases are bound on the AuNP-COOH
Notes and references
1 J. M. Koziara, P. R. Lockman, D. D. Allen and R. J. Mumper, Pharm.
Res., 2003, 20, 1772.
2 Y. Xiao, F. Patolsky, E. Katz, J. F. Hainfeld and I. Willner, Science,
2003, 299, 1877–1881.
3 M. Thomas and A. M. Klibanov, Proc. Natl. Acad. Sci. USA, 2003,
100, 9138–9143.
4 R. Hong, T. Emrick and V. M. Rotello, J. Am. Chem. Soc., 2004, 126,
13572.
5 N. O. Fischer, C. M. Mclntosh, J. M. Simard and V. M. Rotello, Proc.
Natl. Acad. Sci. USA, 2002, 99, 5018–5023.
6 M. Zheng and X. Huang, J. Am. Chem. Soc., 2004, 126, 12047–12054.
7 K. Aslan and V. H. Perez-Luna, Langmuir, 2002, 18, 6059–6065.
8 G. Li, M. Lauer, A. Schulz, C. Boettcher, F. Li and J.-H. Fuhrhop,
Langmuir, 2003, 19, 6483–6491.
9 The detailed FTIR-ATR analyses of AuNP-COOH are given in the
Electronic Supplementary Information and these data show the surface
of AuNP-COOH is slightly negative-charged.
10 K. Terpe, Appl. Microbiol. Biotechnol., 2003, 60, 523.
11 S. V. P. Barreira and F. Silva, Langmuir, 2004, 19, 10324.
12 R. Hong, N. O. Fischer, A. Verma, C. M. Goodman, T. Emrick and
V. M. Rotello, J. Am. Chem. Soc., 2004, 126, 739.
13 J. Ricard, G. Noat, M. Crasnier and D. Job, Biochem. J., 1981, 195, 357
and ref. 10 in the ESI.
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