Site-specific immobilization of cytochrome c on mesoporous silica through metal affinity adsorption to enhance activity and stability

Article abstract of DOI:10.1039/c1nj20255c

We report a hydrothermally stable and highly reactive cytochrome c (cyt c) immobilized in the nanochannels of mesoporous silica (SBA-15) through a metal affinity interaction. Due to the strong affinity of mercury-sulfuric bonds, we modified the SBA-15 surfaces with 4-aminophenylmercuric acetate (APMA) groups. As a result, an enzyme molecule (yeast cyt c) with a cysteine residue (cys-102) demonstrated strong adsorption, which provided high cyt c loading amounts, highly catalytic activity, and high stability against hydrothermal processes and organic solvents. To compare the immobilization of cysteine-containing cyt c through metal affinity interactions and a traditional covalent bond (a disulfide bond), we modified the SBA-15 surfaces with 3-mercaptopropyl-trimethoxysilane (MPTS) for further production of a disulfide bond with the cysteine residue of cyt c. The cysteine residue of the cyt c can covalently link to thiol-modified SBA-15 through the formation of a disulfide bond. In addition, a non-specific coordination from the thiol groups of SBA-15 to the heme Fe(iii) of cyt c may destroy the catalytic center and cause the leaching of Fe(iii) ions. Our previous studies of the molecular model have shown that the immobilization of cyt c through the cysteine residue can provide a correct orientation of the catalytic center, where the active site can easily approach the substrate molecules. Therefore, we have developed rapid and highly efficient approaches to immobilize a cysteine-containing enzyme through APMA ligands, which can both protect the protein folding and control the orientation to optimize the stability and catalytic activity.

Full text of DOI:10.1039/c1nj20255c

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PAPER
Site-specific immobilization of cytochrome c on mesoporous silica
through metal affinity adsorption to enhance activity and stability
a
b
a
a
Shih-Hsun Cheng, Kun-Che Kao, Wei-Neng Liao, Li-Ming Chen,
b
a
Chung-Yuan Mou and Chia-Hung Lee*
Received (in Montpellier, France) 19th March 2011, Accepted 1st June 2011
DOI: 10.1039/c1nj20255c
We report a hydrothermally stable and highly reactive cytochrome c (cyt c) immobilized in the
nanochannels of mesoporous silica (SBA-15) through a metal affinity interaction. Due to the
strong affinity of mercury–sulfuric bonds, we modified the SBA-15 surfaces with
4
-aminophenylmercuric acetate (APMA) groups. As a result, an enzyme molecule (yeast cyt c)
with a cysteine residue (cys-102) demonstrated strong adsorption, which provided high cyt c
loading amounts, highly catalytic activity, and high stability against hydrothermal processes and
organic solvents. To compare the immobilization of cysteine-containing cyt c through metal
affinity interactions and a traditional covalent bond (a disulfide bond), we modified the SBA-15
surfaces with 3-mercaptopropyl-trimethoxysilane (MPTS) for further production of a disulfide
bond with the cysteine residue of cyt c. The cysteine residue of the cyt c can covalently link to
thiol-modified SBA-15 through the formation of a disulfide bond. In addition, a non-specific
coordination from the thiol groups of SBA-15 to the heme Fe(III) of cyt c may destroy the
catalytic center and cause the leaching of Fe(III) ions. Our previous studies of the molecular model
have shown that the immobilization of cyt c through the cysteine residue can provide a correct
orientation of the catalytic center, where the active site can easily approach the substrate
molecules. Therefore, we have developed rapid and highly efficient approaches to immobilize a
cysteine-containing enzyme through APMA ligands, which can both protect the protein folding
and control the orientation to optimize the stability and catalytic activity.
Introduction
stability issues, generating robust biocatalysts. For example,
site-directed mutagenesis of Gly82:Thr102 variant in cyt c showed
Cytochrome c (cyt c), a heme protein in mitochondria, acts
1
as an electron shuttle in living cells. Regarding in vitro
that catalytic activity was improved by ten-fold in comparison
4
with the wild type.
functionality, cyt c shows peroxidase activity, which can
decompose hydrogen peroxide and catalyze the oxidation
Conversely, chemical modification can provide a more
convenient method for improving stability and catalytic activity.
Various chemical methods for modifying the enzyme molecules
were also employed to obtain more stable derivatives such
as amphiphilic polymer conjugation and cross-linked enzyme
of various types of polycyclic aromatic hydrocarbon PAHs
2
a carcinogenic and mutagenic activity). The excellent activity
(
of native enzymes is usually obtained under milder conditions;
however, they lose catalytic activity under extreme conditions.
In many cases of industrial processes, extreme pH, organic
solvent, and high temperature are required for accelerating
to the products. Increasing the thermal stability of an enzyme
has many advantages such as raising reaction rates, increasing
substrate solubility, and high temperature sterilization to
5
assemblies. Vazquez-Duhalt et al. modified cyt c with poly-
(ethylene glycol) (PEG) to show that the modified cyt c exhibits
cage-like polymer protection; and thus a high activity under high
6
temperature. Enhancement of the stability and activity of an
enzyme molecule could be obtained by immobilization of fragile
enzymes on a solid support. One can also modify the solid surfaces
with applicably functional groups, which can increase enzyme
stability through the protection of solid supports and optimization
3
prevent microbial contamination. Various methods for
modifying the biocatalyst by biochemical and molecular
biology approaches have been developed to overcome the
7
of solvation in their microenvironment.
In recent years, mesoporous silica (MPS) has been widely
8
applied in biotechnology. The uniqueness of large surface areas
a
Department of Life Science and Institute of Biotechnology,
National Dong Hwa University, Hualien 974, Taiwan.
E-mail: chlee016@mail.ndhu.edu.tw; Fax: 886-3-863-3630
Department of Chemistry, National Taiwan University, Taipei,
2
near 1000 m g ), highly ordered pore structure, and adjustable
ꢀ1
(
b
pore size (2–30 nm) extends MPS for applications in immobilizing
9 10
enzymes or enzyme-mimicking compounds. Previously, we
Taiwan 106
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reported catalytic applications with an immobilized model com-
pound (hydroxo-bridged di-nuclear phenanthroline cupric com-
Experimental
11
Chemicals
plex) in Al-MPS, which can mimic catechol oxidases. Many
research groups have also reported that the immobilization of
enzymes on MPS exhibited improved enzyme stability, catalytic
activity, products specificity, and resistance to extreme environ-
Cytochrome c from Saccharomyces cerevisia, 3-aminopropyl-
trimethoxysilane (APTMS), glutaric anhydride (GAC),
(
3-Iodopropyl)trimethoxysilane, 4-aminophenylmercuric acetate
APMA), pyrene, Pluronic P123 (EO20PO70EO20) were all
obtained from Sigma Chemical Co. Hydrogen peroxide
35%), cetyltrimethylammonium bromide (CTAB), KCl, tetra-
1
2
mental conditions. In our previous studies, we have immobilized
cyt c in the nanochannels of MPS through electrostatic attractions
and covalent bonding. The immobilized cyt c can increase both
the catalytic activity and stability; therefore, an increase in the
(
(
ethyl orthosilicate (TEOS), NaH
were all obtained from Acros.
2
PO
4
2 2 4
ꢁH O, and Na HPO
1
3
transformation of PAHs was observed. Thus, the nanochannels
of MPS provide the confined spaces that could prevent cyt c
from unfolding. Recently, we studied the orientation effect of
cyt c within MPS through various covalent conjugations on
Methods of preparation
(a) Synthesis of mesoporous silica. Highly ordered mesoporous
the side groups of the cyt c molecule (–NH , –COOH, and
2
structure of MCM-41 belongs to a family of mesoporous silicates
17a
–
SH site). The use of molecular modeling provided further
(M41S) that was invented by Mobil researchers. The MCM-41
insight into the relationship between the most efficient peroxidase
activities and the orientation of the active site in a favorable
location. The results indicated that the immobilization of cyt c
in their cysteine residue has the highest activity because the
active site of cyt c is pointed away from the silica wall, which
would leave room for the substrate to diffuse into the nano-
channels and react with the active site of cyt c in the nanospace
material with pore diameters of 2.7 nm was prepared according to
17b
a method given previously. Briefly, 27.4 g of (CTAB) was fully
dissolved in 240 g of water. A portion of 35.6 g of sodium silicate
solution was added to the above mixture under stirring. A sample
of 42 g of 1.2 M H
by pipette under stirring for 1 h at 32 1C and then hydrothermal at
00 1C for 2 days in a PTFE-lined autoclave. The final product
2 4
SO was added slowly to the resulting mixture
1
1
4
of channels.
was recovered by filtration, followed by washing twice with
deionized water, dried at 100 1C, and calcined at 540 1C for 8 h.
In order to immobilize large APMA-cyt c complexes in the
nanochannels of mesoporous silica, we choose large pore
size of mesoporous silica (SBA-15) as the solid support. For
the synthesis of SBA-15, poly(ethylene oxide)-block-poly-
The purpose of this work is mainly to develop a rapid and
efficient method of cysteine-specific linking for immobilization
of enzyme. We apply this method to linking cyt c into the
nanochannels of MPS. Due to the high affinity of the mercury–
sulfuric bonds, considerable research has reported that thiol
groups can efficiently remove mercury ions for environmental
(propylene oxide)-block-poly(ethylene oxide) triblock copolymer
1
5
purposes. The large surface areas of MPS can be modified
with 3-mercaptopropyltrimethoxysilane (MPTMS); thus a
high loading of mercury(II) ions on the solid surfaces can
be achieved. We then demonstrate that the mercury-modified
SBA-15 can both efficiently immobilize cyt c through cysteine
residue and provide a correct orientation for enhancing
stability its peroxidase activity to catalyze the oxidation of
polyaromatic hydrocarbon (PAH). The introduction of a
metal affinity ligand in the nanochannels of SBA-15 to
immobilize the cysteine-containing enzymes is a new approach,
which can be applied to immobilize other type of enzyme
molecules to optimize the orientation for biocatalytic applications
(
EO20PO70EO20, P123) was used as the structure-directing
agent. By the addition of tetraethylorthosilicate (TEOS) under
the acidic condition, the hydrolysis and co-condensation of
silica source were produced the as-synthesized SBA-15. Briefly,
SBA-15 was synthesized by dissolving 4.0 g of Pluronic P123 in
3
3
0 g of water and 120 g of 2 M HCl solution under stirring at
5 1C. Further, 8.5 g of TEOS was added into the above
solution to further stir for 20 h. Then, the mixture was
hydrothermal at 100 1C for overnight. The final product was
recovered by filtration, followed by washing twice with deionized
17c,d
water, dried at RT, and calcined at 500 1C for 6 h.
16
(Scheme 1).
(
b) Synthesis of mercapto-modified mesoporous silica. We
also synthesized mercapto-modified mesoporous silica, which
can immobilize cyt c through the formation of a disulfide
bond. The synthetic condition was as follows. 0.5 g of calcined
MCM-41/ or SBA-15 was placed in 100 ml of toluene and
stirred for 30 min. A 2.5 g sample of mercaptotriethoxysilane
(
MPTS) was added to the resulting mixture. The reaction was
allowed to proceed overnight at 100 1C. The solids were
washed twice with acetone. The filtered sample was dried
under vacuum.
(
c) Synthesis of amine- and carboxylic acid-modified SBA-15.
0
.5 g of calcined SBA-15 was well-dispersed in 100 mL of
toluene, and 2.5 g of APTMS was added to the mixture. After
stirring at room temperature for 1 h and refluxing at 100 1C for
20 h, the solids were centrifuged, washed by acetone, and dried
in vacuum. The further derivation of carboxylic acid on the
Scheme 1 The representation of the synthesis of APMA-modified
SBA-15 and the further immobilization of cyt c through metal affinity
adsorption.
1
810 New J. Chem., 2011, 35, 1809–1816
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surface of amine-modified SBA-15 was achieved by addition
of amine-modified SBA-15 (1 g) in 30 mL of DMF and then
was added. The mixture was shaken again at 250 rpm and 35 1C
for 5 min. After the reaction was finished, the solids were
separated from the reaction mixture by centrifuge. The reaction
extent was determined by the intensity decrease of the absorption
peaks at 335 nm (pyrene) in the solution phase. Specific activity
was determined by measuring the number of moles of substrate
0
.99 g of glutaric anhydride (GAC) was added. After stirring
at room temperature for 1 h, the solution was heated to 60 1C
with stirring for 9 h. The solids were washed twice with
ethanol. The filtered sample was dried under vacuum.
The anchoring of APMA onto the surface of SBA-15 was
accomplished by the following two approaches.
ꢀ
1
oxidized by one mole of cyt c in 1 min and expressed as min
.
The substrate adsorption on the solid supports was deducted from
the measurement of a control blank experiment.
(
d) Synthesis of SBA-15-APMA-1. The APMA-conjugated
3-iodopropyl)trimethoxysilane was synthesized by dissolving
.5 g of APMA in 3 mL DMSO; 50 mL of THF was then
(
Characterization techniques
0
UV-Visible spectra were taken with a GeneQuant 1300 (GE
Healthcare) spectrophotometer. The spectra were collected in
the range of 200 to 800 nm wavelength against a standard. The
added. Separately, 300 mL of (3-iodopropyl)trimethoxysilane
was added to the above APMA solution and stirred at room
temperature for 12 h; then the above APMA-silane-conjugated
solution was added to 0.3 g SBA-15 while stirring at 90 1C
for another 12 h. Samples were collected by centrifuge at
surface area, pore size, and pore volume were determined by N
2
adsorption–desorption isotherms obtained at 77 K on a Micro-
metric ASAP 2010 apparatus. The sample was outgassed at
6
000 rpm for 10 min, washed twice with DMSO and methanol,
ꢀ
3
10
Torr and 120 1C for approximately 3 h prior to the
and further dried under a vacuum.
adsorption experiment. The pore size distribution curves were
obtained from the analysis of the adsorption portion of the
isotherms using the BJH (Barrett-Joyner-Halenda) method. The
structures of mesoporous silica materials were analyzed by powder
X-ray diffraction (XRD) with a Scintag X1 diffractometer using
copper Ka radiation at l = 0.154 nm. A Bruker EMX EPR
spectrometer (X-band) was employed to measure the electron
paramagnetic resonance (EPR) spectra of the samples. We
monitored the EPR spectra of the solid catalysts. The solid
samples used for the analysis were introduced in a quartz tubing
of 4 mm OD. The sample was degassed and sealed under
a vacuum. The spectrometer was equipped with a variable
temperature controller, allowing us to record spectra at low
temperatures (above the boiling temperature of liquid helium).
Typical spectrometer settings were: microwave frequency,
9.526 GHz; microwave power, 2 mW; center field, 2600 G;
sweep width, 5000 G; time constant, 40.96 ms; and modulation
amplitude, 5 G.
(
e) Synthesis of SBA-15-APMA-2. 0.5 g of SBA-15 was
placed in 100 mL toluene and stirred for 30 min. 1.0 mL of
3-iodopropyl)trimethoxysilane was then added to the resulting
(
suspension and allowed to react for 20 h at 90 1C. Samples
were washed twice with acetone. The filtered sample was dried
under a vacuum. The conjugation of 4-aminophenylmercuric
acetate (APMA) groups onto the iodine-modified SBA-15 was
achieved through nucleophilic substitution of the SBA-15-
iodine samples with the APMA. First, we dissolved 0.7 g of
APMA in 10 mL DMSO; 25 mL of THF was then added. The
APMA solution was further added to 0.3 g of iodine-modified
SBA-15 while stirring at room temperature for 12 h. Samples
were collected by centrifuging at 6000 rpm for 10 min, washed
twice with DMSO and methanol, and dried under a vacuum
(
Scheme 1).
(
f) Immobilization of cyt c. The immobilization of cyt c in
different SBA-15 modifications was achieved according to the
following procedure. First, a 20 mM cyt c stock solution was
2 4 2 4
prepared by dissolving cyt c in a 0.05 M NaH PO –Na HPO
Results and discussion
buffer solution (pH 7.4) while stirring. Then, 0.15 g of
modified SBA-15 was added to 30 mL of the cyt c solution
while stirring at 4 1C for 3 h. The SBA-cyt c solids were
collected by filtration and dried under a vacuum. The cyt c
loading was determined by monitoring the change of the
strong absorption band at 407 nm.
Characterisations
(
a) Adsorption studies. The nitrogen adsorption–desorption
isotherms of these samples was shown in Fig. 1. We can
observe that the surface area measured from nitrogen adsorption
2
gradually decreased from 679 m g for SBA-15 to 463 and
ꢀ1
2
50 m g for iodine- and thiol-modified SBA-15, and to 350 and
ꢀ1
ꢀ1
4
(
g) Stability of immobilized cyt c under KCl solution. Stability
2
3
16 m g for further modified APMA samples. At the same
time, the pore size decreased from 7.4 nm for SBA-15 to 6.4 and
.2 nm for SBA-15-I and SBA-15-SH, and to 5.3 and 5.4 nm for
of cyt c in the KCl solution was determined by treating 0.05 g
SBA-15-APMA-cyt c solids in 1 M KCl while stirring for 2 h,
and then the samples were centrifuged. The leaching percentages
of cyt c were determined by the Soret band absorption at 407 nm.
6
SBA-15-APMA-1 and –APMA-2. The decreases in surface areas
and pore size indicate that the grafted groups were indeed attached
on the inner wall surfaces of the nanochannels.
(
h) Enzyme activity. It was reported that the cyt c/H
2 2
O
system could catalyze the oxidation of pyrene to form 1,8-
After the immobilization of cyt c (Fig. 1e and 1f), we noticed
a great decrease for nitrogen adsorption (surface areas of the
1
8
pyrenodione and 9,10-anthraquinone. We performed a PAH
assay to determine the enzymatic activity of various immobi-
lized cyt c. Typically, 5 mg of SBA-15-APMA-cyt c solids were
2
SBA-15-APMA-2-cyt c sample: 171 m g and MCM-41-SH-
ꢀ1
2
ꢀ1
cyt c: 136 m g ) apparently because a good fraction of the
surface areas of the channel was now occupied by the cyt c
molecules. Although the pore size of MCM-41-SH (1.9 nm) is
added to 5 mL of 20 mM PAHs solution (CH CN: 20% (v/v)
3
and 0.05 M of NaH PO –Na HPO buffer: 80%) at pH 6.1.
2
4
2
4
1
3
After shaking the mixture for 1 min, 25 mL of 200 mM H O
less than the molecular size of cyt c (2.5 ꢂ 2.5 ꢂ 3.7 nm), the
2
2
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Table 1 Surface area, pore size, amount of cytochrome c adsorbed,
and specific activity
Surface area Pore size Max. loading of Specific activity
2
(m g
ꢀ1
ꢀ1
ꢀ1
)
Sample
)
(nm)
cyt c (mmol g ) (min
a
SBA-15
SBA-15-I
MCM-41
679
463
7.4
6.4
2.7
1.9
6.2
5.3
N.D.
N.D.
N.D.
3.22
N.D.
N.D.
N.D.
0.011
0.004
0.773
1015
MCM-41-SH 395
SBA-15-SH 450
2.27
3.15
SBA-15-
APMA-1
SBA-15-
APMA-2
350
316
5.4
3.31
0.841
a
N.D. = not determined.
(
b) Powder X-ray diffraction (XRD). The XRD spectra
of various solid mesoporous materials (Fig. 2), including
SBA-15, SBA-15-I, SBA-15-APMA-1, and -APMA-2 samples
give 2D hexagonal P6mm pore structure showing (100), (110),
1
9
and (200) diffraction peaks. The XRD diffraction intensity
was slightly reduced after the modification of iodine and
APMA on the surfaces of SBA-15. The XRD patterns showed
that the modification of SBA-15 surfaces under organic
solvents does not greatly affect the integrity of the well-defined
mesostructure of solid supports. A slight decrease of the intensity
in the SBA-15-APMA-2 sample may arise from the larger contrast
in the density between the silica walls of the empty pores relative to
that between the silica walls of the pores modified with APMA
ligands.
Fig. 1 Nitrogen adsorption–desorption isotherms of (a) SBA-15,
b) SBA-15-I, (c) SBA-15-APMA-1, (d) SBA-15-APMA-2, (e) SBA-15-
(
APMA-2-cyt c, (f) MCM-41-SH-cyt c, and (g) after the calcination of
the MCM-41-SH-cyt c sample at 560 1C for 8 h.
immobilization of cyt c apparently showed a drastic reduction in
the surface areas. In addition, the MCM-41-SH samples also
ꢀ1
showed high loading amounts of cyt c molecules (3.22 mmol g ).
In the immobilization process of the MCM-41-SH-cyt c sample,
we also noticed that the cyt c changed the color from the bright
red of the native cyt c form to the dark yellow of the immobilized
form. This phenomenon may come from the unfolding 3-D
structures and leaching heme groups of cyt c molecules, and thus
the denatured cyt c molecules could easily enter the nanochannels
of MCM-41-SH. We believe that the denatured form of cyt c
molecules may be located both inside and outside the nano-
channels of the MCM-41-SH. The nitrogen adsorption–desorption
isotherms of the MCM41-SH-cyt c sample (Fig. 1f) showed
the loss of the capillary condensation in the adsorption curve
after the immobilization process. We inferred that large
amounts of denaturated cyt c molecules may occupy the
nanochannels or clog the pores. To demonstrate that the
structures of mesoporous silica were not destroyed during
the immobilization process, we calcined the MCM-41-SH-cyt c
sample in 560 1C for 8 h and further measured the nitrogen
adsorption–desorption isotherms. We can observe that the
elimination of organic molecules from the surfaces of mesoporous
(
c) FT-IR spectra. We employed FT-IR spectroscopy to
characterize the chemical bonds and surface organic groups in
our SBA-15 samples (Fig. 3). The spectra of bare SBA-15
ꢀ1
showed a broad band (2700–3800 cm ) from the O–H stretch
2
ꢀ1
silica restored the surface areas (667 m
g
). Therefore, we
confirm that the loss of the mesoporous adsorption curve may
come from the cyt c molecules occupied the nanochannels or
clogged the window of the pore.
Table 1 also shows the maximum adsorption of cyt c in various
solid supports. The MCM-41-SH and SBA-15-SH samples can
immobilize cyt c through a disulfide bond, which showed 3.22 and
ꢀ
1
2
.27 mmol g of maximum loading of cyt c, respectively. The
SBA-15-APMA-2 sample provided a high affinity adsorption,
ꢀ
1
which showed a little higher loading (3.31 mmol g ) of cyt c than
the disulfide bond of SBA-15-SH-cyt c samples.
Fig. 2 XRD powder spectra of the following samples: (a) SBA-15,
(b) SBA-15-I, (c) SBA-15-APMA-1, and (d) SBA-15-APMA-2.
1
812 New J. Chem., 2011, 35, 1809–1816
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Fig. 3 FT-IR spectra of the following samples: (a) SBA-15,
b) SBA-15-I, (c) SBA-15-APMA-1, and (d) SBA-15-APMA-2.
Fig. 4 (a) UV-Vis spectra of native cyt c in 0.05 M NaH
Na HPO buffer solution (pH 7.4). Diffuse reflectance UV-Vis spectra
for the solid mixtures: (b) SBA-15-APMA-1-cyt c, (c) SBA-15-APMA-
-cyt c, and (d) SBA-15-SH-cyt c.
2 4
PO -
(
2
4
2
ꢀ
1
of the absorbed molecular H O. The bands at 1077 cm (with a
2
ꢀ1
ꢀ1
21a
shoulder at 1236 cm ) and 790 cm are assigned to Si–O–Si
of the Soret band. The alteration of low-spin configuration
didn’t produce a significant shift in the Q band at lmax 529 nm;
however. The disappearance of another Q-band absorption at 555
nm and the formation of a new Q band at 502 nm indicated that
the low spin hemes of cyt c were converted to the high spin form
ꢀ1
vibration. The band at 1639 cm is presumably caused by H O
2
deformation (Fig. 3a). The (3-iodopropyl)trimethoxysilane-
ꢀ
1
modified SBA-15 showed C–H stretches at 2940 cm
and
980 cm (Fig. 3b). The further conjugation of APMA molecules
on the iodine-modified SBA-15 surfaces showed a CQO stretch
ꢀ1
2
21b,c
(Fig. 4b and c).
Comparing the UV-Vis spectra of native cyt c
ꢀ1
mode (from the APMA molecule) at approximately 1714 cm
Fig. 3d). Other peaks at 1555 are associated with the ring stretch
and immobilized cyt c, we could verify that the cyt c altered the
spin form after immobilization; however, it still retained the
integrity through the immobilization in SBA-15-APMA supports
(
20
absorption (the CQC stretch) of benzene. We can observe that
the peak intensity (from the contribution of APMA molecules) of
SBA-15-APMA-2 sample (Fig. 3d) was higher than the SBA-15-
APMA-1 sample (Fig. 3c). We can attribute the high intensity of
the APMA signal to the high loading of APMA molecules in the
nanochannels of SBA-15. The lower intensity of the vibrational
signals of AMPA molecules in the SBA-15-APMA-1 sample
(the retention of great Soret band intensity). Fig. 4d shows the
spectrum of cyt c immobilized in SBA-15-SH through a disulfide
bond. Although the SBA-15-APMA-cyt c and SBA-15-SH-cyt c
samples have same cyt c loading (2.0 mmol cyt c per g of supports),
the Soret band intensity of the SBA-15-SH-cyt c sample at 407 nm
is a greater decrease in intensity than that of the SBA-15-APMA-
cyt c samples. The decrease in absorption intensity of the SBA-15-
SH-cyt c samples may be due to an attack of the –SH group on
the heme group, leading to the decomposition of cyt c and leach of
the Fe(III) ions.
2
caused the overlap of the CQO stretch and H O deformation
ꢀ
1
1639 cm ); therefore, an asymmetry peak at 1631 cm may
ꢀ1
(
come from the CQO stretch buried beneath.
(
d) Diffuse reflectance UV-visible spectra. The low-spin
Fe(III) center of the heme group in the native cyt c is coordinated
to six ligands, four of which are nitrogen atoms of the heme
group. The axial ligands are Met-80 and His-18. Fig. 4a show
the UV-Vis spectra of native cyt c.
(e) Low temperature EPR studies. To study the stability of
the heme Fe(III) center in SBA-15 supports, we performed EPR
experiments at 10 K. The EPR spectrum of native low-spin-state
cyt c with rhombic symmetry which was obtained in 2 M,
0
The electronic absorption spectrum of native cyt c exhibits a
pH 8.5 of N-(2-hyroxyethyl)piperazine-N -(3-propanesulfonic
Soret band absorption at lmax 409 and a Q band at lmax
1 2
acid) (EPPS) buffer showed spin at g = 3.1 and g = 2.3. In
5
29 nm. Another Q-band absorption at 555 nm only presents
addition, a lower signal at g = 4.3 due to free Fe(III) ions may
in low spin hemes of cyt c molecules (Fig. 4a). After cyt c
immobilized in SBA-15-APMA-1 (Fig. 4b) and -2 (Fig. 4c)
samples, the Soret band absorption at 409 nm was shifted to
come from the deactivation of Heme-Fe(III) from the synthesis
21a
process (Fig. 5a). Our previous studies have shown that the
immobilization of cyt c in Al-MCM-41 through electrostatic
1
3
4
07 nm. Previous studies indicated that the change of the heme
attractions existed mostly in the high-spin Fe(III) state. High-
iron sphere coordination through the weakening of the
coordination of Met-80 to the heme iron and caused the shift
spin Fe(III) is formed when Met-80 is replaced by H O during the
2
immobilized process. Many studies have also reported that such
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Fig. 6 Wash time 1–4: leaching amounts of cyt c after wash with 1 M
KCl for 1–4 times. Cys: Replacement and release of mercury-adsorbed
cyt c by adding L-cysteine.
SBA-15-APMA-1, and SBA-15-APMA-2). The isoelectric point
(pI) of cyt c is 9.8, and therefore cyt c carries positive charges
under neutral pH. For this reason, we chose the negative surfaces
of bare SBA-15 and SBA-15-COOH to immobilize cyt c
Fig. 5 EPR spectra at 10 K of cyt c (a) in 2 M EPPS buffer, pH 8.5,
b) immobilized on SBA-15-APMA-1-cyt c, (c) SBA-15-APMA-2-cyt
c, and (d) SBA-15-SH-cyt c.
molecules. The pK value of the carboxylic acid and silanol group
a
(
24
in the SBA-15 surface was 4.2 and 3.0, respectively. Therefore,
the bare SBA-15 surfaces showed higher percentages of negative
charge than the SBA-15-COOH sample. We observed a very
strong electrostatic attraction between the SBA-15 surfaces and
the cyt c molecules; therefore, only 1–2% of cyt c leached from the
bare SBA-15 surfaces after washing with 1 M KCl for 1–4 times
(Fig. 6). On the other hand, the lower negative charged surfaces of
carboxylic acid-modified SBA-15 showed higher leaching
amounts of cyt c after washing with 1 M KCl for two cycles
(1st: 43% and 2nd: 36%). By our new approach to immobilize cyt
c through metal affinity interactions, the APMA molecules
showed high efficiency to regulate the binding affinity between
the SBA-15 surfaces and the cyt c molecules. The leaching
amounts of cyt c from SBA-15-APMA-2-cyt c sample in 1 M
KCl (1–4 cycles) were only 1.3–2.8%. However, SBA-15-APMA-
1-cyt c sample showed higher leaching amounts of cyt c (8–11%)
after 1–4 cycles. Due to the one step modification of SBA-15
surfaces with the APMA-propyltrimethoxysilane complexes of the
SBA-15-APMA-1 sample, a high steric hindrance of APMA
complexes leads to less modification in the internal surface of
SBA-15. Thus, more cyt c were immobilized in the external
surface of the solid supports; therefore, relatively high leaching
amounts of cyt c were observed.
2
2
high spin species could increase both the activity and stability.
When Met-80 was detached, the heme groove can fully open in
the high spin configuration. Thus, the active center is easily
accessible to the substrate and, therefore, an increase in the
catalytic activity was observed. For our EPR spectra of SBA-
1
5-APMA-1-cyt c and SBA-15-APMA-2-cyt c samples, we can
observe the appearance of two signals at g = 6.0 and 2.0, which is
assigned to high-spin Fe(III). In addition, a lower intensity of the
signal at g = 4.3 due to free Fe(III) ions which also appeared in the
native cyt c sample (Fig. 5b and 5c). Fig. 5d shows the EPR
spectrum of cyt c immobilized in the thiol-modified SBA-15
sample (SBA-15-SH-cyt c). We observed the following signals:
g = 6.0 and 2.0 (assigned to high spin), g = 2.3 (low spin), and
23
g = 4.3 (non-heme Fe(III)). For the immobilization of cyt c
through the formation of a disulfide bond, the thiol group of the
SBA-15-SH sample may bind covalently to the heme iron, break
down the heme structure, and ultimately free the Fe(III) ion,
which will result in denaturation of the active site. Therefore,
2
the high-spin Fe(III) is formed when Met-80 is replaced by H O;
the low-spin configuration (g = 2.3) originates from the coordi-
nation of the thiol group (SBA-15-SH) to Fe(III).
Our EPR spectra indicated that the use of APMA as
an affinity ligand to immobilize heme protein is a suitable
approach, which can preserve the integrity of the heme
structure of cyt c. However, the thiol-modified SBA-15 may
poison the Fe(III) heme and leach the Fe(III) ions.
Furthermore, we added a 0.5 M L-cysteine solution to replace
and elute the adsorbed cyt c. We observed that SBA-15-APMA-1-
cyt c and SBA-15-APMA-2-cyt c samples could efficiently release
the residual cyt c (24.3% and 58.8%, respectively) by L-cysteine
through the exchange of adsorbed cyt c molecules. However, the
cyt c release from SBA-15-cyt c and SBA-15-COOH-cyt c samples
under L-cysteine solution was much less when compared with that
of SBA-15-APMA-1-cyt c and SBA-15-APMA-2-cyt c samples.
We could verify that the immobilization of cyt c was a metal
affinity interaction through the mercury-adsorbed cyt c. By the
development of this new strategy, we can reuse the solid supports
Binding affinity and catalytic activity of Cyt c
(
a) Binding affinity in high concentration of KCl. We
compared the strength for the binding affinity of cyt c in various
SBA-15-modified surfaces (bare SBA-15, SBA-15-COOH,
1
814 New J. Chem., 2011, 35, 1809–1816
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or enzyme molecules. Furthermore, we can apply to purify
or separate the cysteine-containing enzymes through the size
exclusion and metal affinity interactions.
(
b) Catalytic activity. In the catalytic reaction of pyrene
oxidation, we compared the catalytic activity of cyt c immobilized
on MCM-14-SH and SBA-15-SH samples. Due to the small
pore size of MCM-41-SH (1.9 nm), the native cyt c cannot
easily enter the nanochannels of MCM-41-SH; therefore, the
MCM-14-SH samples showed higher loadings of cyt c molecules
in the external surfaces of mesoporous silica than the SBA-15-SH
samples. Although the different ratio of cyt c existed in the
external and internal surfaces of MCM-41-SH and SBA-15-SH
samples, the specific activity of cyt c in the MCM-14-SH and
SBA-15-SH samples all exhibited extremely low activity (0.011
Fig. 7 Residual catalytic activity for pyrene oxidation of the SBA-15-
APMA-1-cyt c and SBA-15-APMA-2-cyt c samples after hydrothermal
for 12 h and 24 h, and EtOH treatment for 24 h.
ꢀ1
and 0.004 min ). Therefore, we attribute the very low activity
to the poison of catalytic ability of heme Fe(III) through the
coordination of thiol groups (Scheme 2a). On the contrary, the
immobilization of cyt c through metal affinity interactions of
SBA-15-APMA-1 and -2 samples showed high activities (0.773
structure of cyt c in an imaginary cubic box, we can observe
that the active site of the Fe(III)-heme center is pointed away
1
4
from the silica wall and fully accessible to the substrate.
ꢀ
1
and 0.841 min ). Our previous molecular modeling studies
have shown that the different conjugation approaches to
immobilize cyt c can expose the catalytic active sites (Fe(III)-
heme) in different environments. When cyt c is immobilized in
the SBA-15-APMA-2 sample, there is only one mercapto
group from cys-102 (cys-14 and cys-17 formed disulfide bonds
to conjugate with the heme group), which would form Hg–S
bonds with the SBA-15-APMA-2 sample. Since cys-102 is on
the C-terminal helix, it can easily be attached to the pore
surface of the SBA-15-APMA-2 samples. From the crystal
Therefore, the immobilization of cyt c in our new approach
can leave room for the substrate to diffuse into the nanochannel
and react with the active site of cyt c in the void space of
channels (Scheme 2b).
(
c) Hydrothermal stability. It has been hypothesized that the
heme Fe(III) center breaks down irreversibly during hydro-
thermal heating at 100 1C and leaches out Fe(III) ions, which is
evidenced by the irreversible decrease of Soret band absorption
and loss of the catalytic activity. Fig. 7 shows the residual
percentile catalytic activity of SBA-15-APMA-1-cyt c and
SBA-15-APMA-2-cyt c samples after 12 and 24 h of hydro-
thermal treatment at 100 1C and the ethanol treatment at
room temperature. The heat-treated SBA-15-APMA-1-cyt c
sample retained 82% and 58% of residual activities after 12
and 24 h, respectively. The SBA-15-APMA-2-cyt c sample had
high residual activity of 95% and 76% after 12 and 24 h,
respectively. In addition, the two samples can retain most
residual activity (92% and 99%) after the treatment of ethanol
for 24 h. We can refer the high residual catalytic activity of the
SBA-15-APMA-2-cyt c sample to the protection of cyt c from
the strong affinity of APMA molecules and the confined space
of SBA-15. Through this design, we can both increase the
stability of the immobilized cyt c against extreme conditions
and optimize the orientation to increase the reaction rate with
the substrate molecules.
Conclusion
We demonstrate that the immobilization of cyt c in the
nanochannels of SBA-15 through APMA molecules can provide
both confined spaces and metal affinity interactions to stabilize
the enzyme molecules against leaching under the high concen-
tration KCl and hydrothermal processes. We summarize
schematically the structural and bonding differences among
cyt c immobilized in the nanochannels of SBA-15 through a
disulfide bond and a metal affinity interaction from the
APMA-modified SBA-15 in Scheme 2a. Our previous molecular
modeling studies have shown that the peroxidase activity of
Scheme 2 (a) Immobilization of cyt c in the nanochannels of SBA-15
through a disulfide bond or a metal affinity interaction from the
APMA molecule. (b) Orientation and specific binding of cyt c in
SBA-15-APMA: active site (heme group) is pointed away from the
silica wall and fully opened to the substrate (less steric hindrance).
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immobilized cyt c is related to the structural orientation of the
cyt c in the SBA-15 nanochannels. The immobilization of cyt c
through the cysteine residue leads the active site pointing away
from the silica wall, which would leave room for the substrate
to diffuse into the nanochannel and react with the active site of
cyt c in the void space of channels (Scheme 2b). We therefore
examined the two immobilized approaches through the cysteine
residue of cyt c. While the cysteine residue can covalently link
to thiol-modified SBA-15 through a disulfide bond; a non-
specific coordination from the grafted thiol groups to the heme
Fe(III) of cyt c can also destroy the catalytic center and cause
the leaching of Fe(III) ions. The immobilization of cyt c
through SBA-15-APMA showed that the confined space and
metal affinity interactions from the APMA-modified SBA-15
indeed provided the stability toward the leaching of enzyme
molecules under a high concentration of salts. In addition, the
immobilization of cyt c through the APMA-modified SBA-15
can provide a robust catalytic center, where the active site can
easily approach the substrate molecules. Thus, the SBA-15-
APMA provided a correct orientation that could position the
active site in a favorable location in the pores to facilitate its
activity.
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