Construction of a Hyperbranched Supramolecular Polymer as a Bifunctional Antioxidative Enzyme Model

Article abstract of DOI:10.1002/mabi.201000478

A HBSP has been designed as a novel bifunctional enzyme model with SOD and GPx activity by host/guest-directed self-assembly of MnTPyP-M-Ad and 6-Te-diCD. The structure of the host/guest complex was elucidated by ¹H NMR spectra, and the HBSP wa

Full text of DOI:10.1002/mabi.201000478

Full Paper
Construction of a Hyperbranched
Supramolecular Polymer as a Bifunctional
Antioxidative Enzyme Model
Shuangjiang Yu, Wei Zhang, Junyan Zhu, Yanzhen Yin, Haiyan Jin,
Lipeng Zhou, Quan Luo, Jiayun Xu, Junqiu Liu*
A HBSP has been designed as a novel bifunctional enzyme model with SOD and GPx activity by
host/guest-directed self-assembly of MnTPyP-M-Ad and 6-Te-diCD. The structure of the host/
guest complex was elucidated by 1H NMR spectra, and the HBSP was characterized by SEM, DLS
and measurement of catalytic properties. In the bifunctional enzyme model, the Mn(III)
porphyrins act as efficient SOD active sites and the tellurol moieties endow GPx activity.
The SOD-like activity (IC50) of this new supramolecular catalyst was found to be 1.05 ꢂ 10ꢀ7 M,
which corresponds to 2.82% of the activity of
the native SOD enzyme. Besides this, the
hyperbranched supramolecular polymer also
shows a higher GPx activity (n0 ¼ 21.7 ꢂ 10ꢀ6
M ꢃ minꢀ1) than other supramolecular enzyme
models.
Introduction
In recent years, there was increasing interest in
mimicking the individual functions of these important
antioxidant enzymes not only for elucidating the catalytic
mechanism but also for potential pharmaceutical applica-
tions, and several attempts have been made to produce
artificial enzymes which mimic the properties of SOD, CAT
and GPx.[5–19]
In biological organisms the antioxidant enzymes super-
oxide dismutase (SOD), catalase (CAT), and glutathione
peroxidase (GPx) dominate the cellular antioxidative
defense system against oxidative stress (Scheme 1).[1–4]
SOD, a metalloenzyme, catalyzes the dismutation of the
superoxide radical anion (Oꢀ2 ꢁ) to H2O2 and dioxygen. H2O2
is then detoxified either to H2O and O2 by CAT or to H2O by
GPx.Studiesrevealedthateachenzymehasaspecificandan
irreplaceable function. More importantly, the enzymes act
in a cooperative or synergistic way, so optimum protection
of cells can be achieved.
In order to further study the cooperation of these
enzymes in antioxidation action, some bifunctional
enzyme models with antioxidant activity have been
constructed; typical examples include synthesized
mimetics of SOD and CAT, the salen/manganese complexes
(EUKs), manganese-4-pyridylporphyrin (Mn-HPyP)-modi-
fied liposomes, and the cross-linking of native SOD and
CAT.[20–23]
Up to now, various antioxidative enzyme models
with controlled catalytic properties were demonstrated.
The combination of supramolecular and nanoscience
approaches seems a potent way to construct new enzyme
models. Among them, hyperbranched supramolecular
S. J. Yu, W. Zhang, J. Y. Zhu, Y. Z. Yin, H. Y. Jin, L. P. Zhou, Q. Luo,
J. Y. Xu, J. Q. Liu
State Key Laboratory of Supramolecular Structure and Materials,
College of Chemistry, Jilin University, Changchun 130012, China
E-mail: junqiuliu@jlu.edu.cn
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120 8C in vacuo. 1-Adamantylbromomethylketone, xanthine oxi-
dase (XOD) and xanthine were acquired from Aldrich Chemical Co.
Cumene hydroperoxide (CumOOH; tech., 80%) and ammonium
hexafluorophosphate (NH4PF6) were purchased from Alfa. Sodium
borohydride and pyrrole were purchased from Fluka (AR grade). 4-
Pyridinecarboxaldehyde was obtained from J&K Chemical, Ltd.,
and used without further purification. Tellurium powder was
purchased from Tianjin Fuchen Reagents Company. Triethylamine
and tetrahydrofuran were rigorously dried with sodium. N,N-
Dimethylformamide(DMF)wasdriedbydistillingfromCaH2 under
reduced pressure. Column chromatography was performed on a
Sephadex G-25 instrument. 1H NMR spectra were recorded on a
Bruker AM-500 spectrometer. The spectrometric measurements
were carried out with a Shimadzu 3100 UV-Vis/near-IR recording
spectrophotometer interfaced with a personal computer. Data
were acquired and analyzed by using UV spectroscopy software.
The temperature for UV time course studies was controlled to
within ꢄ0.5 8C by use of a Lauda compact low-temperature
thermostat RC6 CP. The buffer pH values were determined with
Scheme 1. Catalytic reactions of SOD, CAT and GPx with coopera-
tive actions.
polymers (HBSP) appear to be a new scaffold for the design
of artificial enzymes. In recent decades, a large number of
noncovalent-interaction-based HBSPs have been reported;
they showed potential applications in many fields such as
biochemistry, medicine, and materials science.[24,25] Com-
pared to the classic synthetic hyperbranched polymers,
HBSPs have obvious advantages. For example, their
syntheses are convenient and it is easier to introduce
functional groups, and furthermore their dendritic globular
architecture, lowerviscosity, andbettersolubilityshouldbe
advantageous for constructing artificial enzymes.[26,27]
However, up to now only few studies have been made to
exploit the advantages of HBSPs to construct multi-
functional enzymes with synergistic antioxidative activity.
Herein, we present a novel bifunctional enzyme model
with both SOD and GPx activity. The novel enzyme model
has been constructed by self-assembly of the tellurium-
containing cyclodextrin dimer 6,60-tellurobis(6-deoxy-b-
cyclodextrin) (6-Te-diCD) with adamantane-modified
Mn(III) porphyrin (MnTPyP-M-Ad) through host/guest
interaction (Scheme 2). In this novel artificial enzyme the
Mn(III) porphyrin complex acts as an efficient SOD active
site, and the tellurol moiety endows GPx activity. The
catalytic properties and the kinetics of the bifunctional
enzyme model were investigated in detail.
a
Mettler Toledo 320 pH meter. Molecular weights of the
compounds were determined using matrix-assisted laser deso-
rption/ionization time-of-flight mass spectrometry (MALDI-ToF
MS, LDI-1700, Linear Scientific Inc., USA). The size of the comples
was determined using a Malvern Zetasizer NanoSeries instrument.
Scanning electron microscopy (SEM) observations were carried out
on a JEOL FESEM 6700F scanning electron microscope with a
primary electron energy of 3 kV.
Synthesis of meso-Tetrakis[1-(1-adamantyl methyl
ketone)-4-pyridyl]porphyrin (TPyP-M-Ad)[28]
Synthesis of TPyP-M-Ad followed standard procedures as described
in the literature with some alteration.[29,30] The pyridyl groups in
TPyP (203 mg, 0.328 mmol) were quarternized with 1-adamantyl-
bromomethylketone (408 mg, 1.586 mmol) in DMF (40 mL) for 20 h
at 100 8C to synthesize TPyP-M-Ad under a nitrogen atmosphere.
After being cooled to room temperature, the brown precipitate was
collected by filtration and washed with diethyl ether to yield a
crude product. The crude product was purified by recrystallization
from methanol and chloroform (1:2) to yield 300 mg of the desired
product as a brown solid.
1H NMR(500 MHz, DMSO-d6, 25 8C): d ¼ ꢀ3.03 (s, 2H, ꢀNH), 1.80–
1.86 (s, 24H, adamantane), 2.12 (s, 24H, adamantane), 2.18 (s, 12H,
adamantane), 6.40(s, 8H, ꢀCOCH2ꢀ), 9.142–9.155 (d, J ¼ 5.0 Hz, 8H),
9.182 (d, J ¼ 5.0 Hz, 8H), 9.42(s, 8H). MALDI-ToF MS: Calcd. 1 327.7,
found 1 327.3.
Experimental Part
Materials and Methods
b-Cyclodextrin (b-CD) was obtained from Tianjin Chemical Plant,
recrystallized three times from distilled water, and dried for 12 h at
Scheme 2. Schematic representation of the preparation of the enzyme model.
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Metallation of TPyP-M-Ad
The metallation of TPyP-M-Ad proceeded following standard
procedures.[31] Briefly, TPyP-M-Ad (100 mg) and MnCl2 ꢃ
4H2O (200 mg, ca. 20 equiv.) were dissolved in methanol/
H2O (6:1, 35 mL) with 1 M NaOH (200 mL) and mixed under refluxing
conditions to form MnTPyP-M-Ad. The degree of metallation was
assessed by monitoring the soret band shift from 426.5 to 463 nm,
with complete metallation observed after approx. 30 min. The
MnTPyP-M-Ad was then purified as described previously,[31] with
the addition of NH4PF6 to the metalloporphyrin precipitate,
followed by washing with H2O. Counterion exchange to Cl– was
then achieved by dissolving the product in acetone and metallo-
porphyrin precipitation upon the addition of tetrabutylammo-
nium chloride (TBAC). The final Cl– form of MnTPyP-M-Ad was then
dessicated for 12 h. MALDI-MS: Calcd. 1 379.7; found 1 376.8.
Synthesis of 6-Te-diCD[32]
Figure 1. 1H NMR spectra of TPyP-M-Ad (59 ꢂ 10ꢀ6 M) and the
complex in D2O (59 ꢂ 10ꢀ6 M)at 25 8C.
6-Te-diCD was synthesized according to the procedure previously
used in our group. The obtained residue was purified on a column
of Sephadex G-25 with distilled H2O as the eluent. The
resulting solution was freeze-dried, to give a white powder
(yield: 40%).
1H NMR (500 MHz, D2O): d ¼ 3.46–3.59 (m, 2-H, 4-H), 3.74–3.92
(m, 3-H, 5-H, 6-H), 4.96–5.02 (m, 1-H); MALDI-MS: Calcd. 2 363.6;
found 2 363.3.
by following the reduction of nitro blue tetrazolium (NBT) to blue
formazane (MFþ) spectrophotometrically.[16,33,34] The appropriate
amount of xanthine oxidase (final concentration 0.025 U ꢃ mLꢀ1
)
was added to an aqueous solution of 10ꢀ4 M NBT, 3 ꢂ 10ꢀ4
M
xanthine, 10ꢀ4 M ethylenediamine tetraacetate (EDTA), and 0.05 M
phosphate buffer (final volume of 1 mL) to cause an absorbance
variationof0.019(DA560 ꢃ minꢀ1) at25 8C. Thereactionratein blank
samplesandinthepresenceofthemodelcatalystwasmeasuredfor
300 s only in order to avoid problems originating from the natural
inactivation of the enzymatic system.
Preparation of the Hyperbranched Supramolecular
Polymer[28]
An aqueous solution of MnTPyP-M-Ad (2 ꢂ 10ꢀ4 M, 10 mL)
was added dropwise to another aqueous solution of 6-Te-diCD
(4 ꢂ 10ꢀ4 M, 10 mL), followed by mild ultrasonication for 2 h, giving
the inclusion complex (the enzyme model), and the polymerization
process was tracked by dynamic light scattering (DLS). A model
compound was prepared which includes TPyP-M-Ad (2 ꢂ 10ꢀ4 M,
2 mL) and 6-Te-diCD(4 ꢂ 10ꢀ4 M, 2 mL) in D2O, according to a similar
procedure as described above. NMR measurements were carried
out by measuring the difference between the chemical shifts of the
TPyP-M-Ad alone and the same guest with increasing the
concentration of 6-Te-diCD (Figure 1). The average size of the
Evaluation of GPx Activity
The GPx activity was assayed by using 4-nitrobenzenethiol (ArSH)
and cumene hydroperoxide (CumOOH) as substrates (see Scheme 3)
according to a modified method reported by Hilvert et al.[35–37] The
reaction was carried out in 1.0 mL of phosphate buffer (pH ¼ 7.0,
0.050 M) containing the enzyme model catalyst (10ꢀ6
M
catalytic centers), 1.20ꢂ 10ꢀ4 M ArSH and 2.50ꢂ 10ꢀ4 M CumOOH
at 25 8C. The initial rates for the reduction of CumOOH were
determined by monitoring the disappearance of ArSH at 410 nm
(e410 ¼ 14 500L ꢃ molꢀl ꢃ cmꢀ1,pH¼ 7.0)withaShimadzu3100UV-vis/
NIR spectrophotometer. Appropriate control of the non-enzymatic
reaction was performed and subtracted from the catalyzed reaction.
hyperbranched supramolecular polymer was detected by
a
Zetasizer Nano instrument. The morphology of the hyperbranched
polymer was characterized via SEM.
Results and Discussion
Determination of the Enzyme Activity
Design and Preparation of the Artificial Enzyme
Evaluation of SOD Activity
As a typical host/guest pair, b-CD and adamantane
Superoxide radical anions generated by the xanthine/xanthine
oxidase system at 25 8C, pH ¼ 7.8 (phosphate buffer) were detected
derivatives have induced a lot of interesting development
Scheme 3. Determination of GPx activity by using ArSH as a substrate.
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in the construction of supramolecular polymers.[38–41]
However, only few study focused on the design of HBSPs
with enzymatic functions. Due to their unique topological
architectures, HBSPs could become a new platform for
constructing enzyme models. For the design of multi-
functional artificial enzymes, the self-assembly of enzy-
matic building blocks represents an easy way to combine
multiple enzyme functions.
Porphyrins are a series of compounds with high stability
and chemical versatility. Manganese porphyrins were
demonstrated to be a promising group of compounds for
developing SOD mimics. In our previous study the
cyclodextrin dimers with GPx catalytic sites have been
designed as artificial GPx models. The CDdimers can act as
supramolecular linker for the construction of HBSP. By
combining the building blocks with SOD and GPx active
sites, a bifunctional enzyme model was designed by using
hyperbranched supramolecular polymers as scaffolds. The
enzyme model of HBSP was prepared by the self-assembly
ofaCDdimer(6-Te-diCD)withMnTPyP-M-Adthroughhost/
guest interactions (Scheme 2).
Figure 2. Average diameters of (a) 6-Te-di-CD, (b) MnTPyP-M-Ad
and (c) HBSP determined using a Malvern ZETAS12-ER Nano Series
instrument, at concentrations of (a) 4 ꢂ 10ꢀ4, (b) 10ꢀ4 and
(c) 5 ꢂ 10ꢀ5
respectively.M (with MnTPyP-M-Ad standard) in water at 25 8C,
In order to confirm the host/guest complexation of 6-Te-
diCDandtheadamantane-containingporphyrin(Mn-TPyP-
M-Ad), 1H NMR measurement was carried out
in D2O. Compared with the signals of methine and
methylene protons (a, b, c) of the adamantane group in
TPyP-M-Ad, as shown in Figure 1, the new peaks (a0, b0, c0)
shifted downfield with an increase in the concentration of
6-Te-diCD.[29] Based on our previous work, this indicates
that the host/guest complex of CD and the adamantane
moieties has been formed.[28] It should be noted that,
compared with the NMR spectra of TPyP-M-Ad, there
are some residual peaks of the adamantane group that
were not shifted downfield in the NMR spectra of the
complex. This suggests that some adamantane groups
did not engage in complex formation with b-CD cavities,
possibly because of steric hindering between the host and
guest molecules.
Figure 3. The average diameter of HBSP as a function of increas-
ing MnTPyP-M-Ad content.
of SEM (Figure 4). SEM clearly revealed the presence of
spherical nanoparticles with
a diameter of 120 nm.
According to SEM, the dimensions in the dry state are
reasonably smaller than the average diameter of the
complex in solution which contains the contribution from
the swollen corona.
To investigate the formation of the complex polymer, the
average size of three samples were detected by a Zetasizer
Nanoinstrument. AsshowinFigure2, theaveragediameter
of single 6-Te-di-CD dimers (at 4 ꢂ 10ꢀ4 M) was determined
to be <10 nm, and that of MnTPyP-M-Ad (at 10ꢀ4 M) to be
<25 nm. In contrast, the hyperbranched supramolecular
polymer (at 5 ꢂ 10ꢀ5 M, with the MnTPyP-M-Ad standards)
shows dramatical increase of the average diameter
(ꢅ150 nm). The presence of large-volume species in the
mixture demonstrates the formation of a HBSP. In order to
evaluate the evolution of the HBSP, the polymerization
process was monitored by DLS. As shown in Figure 3, the
average diameter of the polymer increased continuously
from 2.1 to 157 nm with increasing MnTPyP-M-Ad con-
centration. Furthermore, the actual morphology of the
hyperbranched polymer structure was observed by means
SOD Activity
Manganese porphyrins are promising candidates for
development of SOD mimics, and they have been shown
to possess true SOD function by many researchers.[31] As an
efficient SOD active site and a ‘‘supramolecular-linker’’,
MnTPyP-M-Ad can assemble with 6-Te-diCD through host/
guest interactions.The SOD activity of the enzyme model
was quantified using the standard xanthine/xanthine
oxidase assay system first developed by McCord and
Fridovich.[33] Superoxide anion generation by the
xanthine/xanthine oxidase system was detected by
following spectrophotometrically the reduction of NBT to
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close to 80%. Some MnTPyP-M-Ad moleules in the center of
the catalyst may not contact with the larger volume of XOD
and NBT in the activity test system timely.
GPx Activity
6-Te-diCD has been reported by our group to be an excellent
GPx enzyme model previously.[32] Now, in order to
construct a HBSP enzyme model, the dimer of 6-Te-diCD
was used as a ‘supramolecular linker’ to form a HBSP
through host/guest interaction. The GPx-like activity of the
catalyst was studied following a modified method reported
by Hilvert et al.[37] using 4-nitrobenzenethiol as a thiol
substrate. The relative activities of the mimics are
summarized in Table 1. For the peroxidase activity, the
enzymatic rates were corrected for the background (non-
enzymatic) reaction between hydroperoxide and thiol. The
initial rate of the background (non-enzymatic) reaction
between CumOOH and ArSH alone proceeds slowly
(Figure6a);thecompoundcatalyst(10ꢀ6 M catalyticcenters)
exhibits a remarkable rate enhancement (n0 ¼ 21.7 ꢂ
10ꢀ6 mol ꢃ Lꢀ1 ꢃ minꢀ1) (Figure 6d). Compared with the
activity of the host molecule 6-Te-diCD, which was found to
be 30.2 ꢂ 10ꢀ6 mol ꢃ Lꢀ1 ꢃ minꢀ1 (Figure 6e), the GPx-like
activity of the HBSP catalyst shows a slightly lower activity.
Consideringthat6-Te-di-CD hasa specificbinding cavityfor
recognizing the substrate ArSH, it is surprising that
the model catalyst in which the CD cavities were
replaced by adamantyl moieties display such high catalytic
activity.[17,32] To investigate the source of the high GPx-like
activity, a control experiment was carried out by using 6-
Te-di-CD solution while adding an excess of adamantane-
carboxylic acid. The GPx-like activity was estimated under
the same condition as mentioned above, and the initial rate
Figure 4. SEM images for the HBSP catalyst (5 ꢂ 10ꢀ5 M, with
MnTPyP-M-Ad standard).
MFþ at 560 nm. In order to determine the concentration of
the catalyst which achieves 50% inhibition (IC50) of the
reaction (a generally used indicator for comparing the
efficiencies of enzymes and enzyme mimics), the percen-
tageofinhibition againstmodelcatalystconcentrationwas
plotted in Figure 5, and the concentration of complex
catalyst (IC50) which inhibits the rate of the reduction of
NBT by 50% (IC50) was found to be 1.05 ꢂ 10ꢀ7 M which
corresponds to 2.82% of the activity of the native SOD
enzyme (Table 1). Compared with the IC50 of the guest
molecule MnTPyP-M-Ad, the SOD activity of HBSP catalyst
was slightly higher. However, as seen from Figure 5, the
percentage of inhibition rate showed premature saturation
Figure 6. Plots of absorbance vs. time during the catalytic
reduction of CumOOH (2.50 ꢂ 10ꢀ4 M) by ArSH (1.20 ꢂ 10ꢀ4 M)
at pH ¼ 7.0 and 25 8C: (a) no catalyst, (b) 10ꢀ6 M MnTPyP-M-Ad, (c)
6-Te-diCD by adding an excess adamantanecarboxylic acid (10ꢀ6
M catalytic centers), (d) HBSP catalyst (10ꢀ6 M catalytic centers)
and (e) 10ꢀ6 M 6-Te-di-CD.
Figure 5. Percentage of inhibition of superoxide radicals formed
by xanthine/xanthine oxidase enzyme assayed by MFþ absorp-
tion at 560 nm in the presence of the supramolecular polymer
catalyst (3 ꢂ 10ꢀ4 M xanthine, 0.025 U xanthine oxidase and
10ꢀ4 M NBT, 50 ꢂ 10ꢀ3 M phosphate buffer, pH ¼ 7.8, 25 8C).
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Table 1. SOD and GPx activities of the catalysts (n.d.: not determined).
Catalyst
GPx activitya)
SOD activityb)
v0(CumOOH) [10ꢀ6 mol ꢃ Lꢀ1 ꢃ minꢀ1
]
IC50 [10ꢀ6 M]
Relative activity [%]
Mn(III)-TPyP-M-Ad
6-Te-di-CD
n.d.
30.2
21.7
–
0.132
n.d.
2.25
–
HBSP
Cu,Zn-SODc)
0.105
1.3 ꢂ 10ꢀ3
2.82
100
a)GPx activity was assessed in phosphate buffer (0.05 M, pH ¼ 7.0) at 25 8C with ArSH (1.20 ꢂ 10ꢀ4 M) and CumOOH (2.50 ꢂ 10ꢀ4 M), and was
corrected for the spontaneous oxidation in the absence of catalyst; b)SOD activity was determined in solution (1 mL) containing 0.050 M
phosphate buffer (pH ¼ 7.8), 0.025 U xanthine oxidase, 10ꢀ4 M NBT, 3 ꢂ 10ꢀ4 M xanthine, 10ꢀ4 M EDTA, and an appropriate amount of
[33,34]
catalyst at 25 8C; c)Ref.
ðappÞ
n(max) ¼ 50.5 ꢂ 10ꢀ6 mol ꢃ Lꢀ1 ꢃ minꢀ1
,
k
¼ 50.5 minꢀ1
,
cat
ðappÞ
Km,CumOOH ¼ 0.224 ꢂ 10ꢀ3 M,
k
/Km,CumOOH ¼ 2.25 ꢂ
cat
105 L ꢃ molꢀ1 ꢃ minꢀ1 (the initial concentration of ArSH
was fixed to 1.20 ꢂ 10ꢀ4 M).
Conclusion
A novel bifunctional antioxidant enzyme model was
designed and prepared that utilized the advantages of
HBSP. This new supramolecular enzyme model shows
bifunctional antioxidative enzyme activities in which the
Mn(III)porphyrinactsasanefficientSODactivesite,andthe
tellurol moiety endows GPx activity. Compared with other
methods, this artificial enzyme shows obvious advantages:
a simple preparation process and easier combination of
multiple functions. We anticipate that this supramolecular
strategy will provide a new way for the design of
multifunctional enzymes.
Figure 7. Plots of initial rates (n ) at different concentrations of
0
CumOOH in the presence of HBSP catalyst (10ꢀ6 M catalytic
centers) at pH ¼ 7.0 and 25 8C. The initial concentration of ArSH
was fixed to 1.20 ꢂ 10ꢀ4 M. The concentration of CumOOH was
0.01, 0.1, 0.25, 0.5, 1.0 and 2.0 ꢂ 10ꢀ3 M, respectively.
was found to be 15.9 ꢂ 10ꢀ6 mol ꢃ Lꢀ1 ꢃ minꢀ1 (Figure 6c).
Compared with the HBSP catalyst, it exhibits
a
5.8 ꢂ 10ꢀ6 mol ꢃ L ꢃ minꢀ1 decrease in activity. This result
suggests that the 3D cavities of the supramolecular
hyperbranched polymer play a positive role during the
catalytic process, and compared with some similar enzyme
models which we have reported previously, the HBSP
catalyst exhibits a visible rate enhancement.[28,42,43]
ꢀ1
Acknowledgements: Financial support from the Natural Science
Foundation of China (No: 91027023, 20874036, 20921003,
21004028), the NSFC for Outstanding Younger Scientists (No.
20725415), the National Basic Research Program (2007CB808006)
and the Key Laboratory of RareEarth Chemistry and Physics,
Changchun Institute of Applied Chemistry (RERU2011008) is
gratefuly acknowledged.
Furthermore, to probe the mechanism of the model
catalyst promoting the peroxidase reaction, the saturation
kinetics of the catalyst for the peroxidase-like reaction were
studied at the individual concentrations of ArSH and
CumOOH, which indicated that this model shows typical
saturation kinetics and acts as a real catalyst for a
peroxidase-like reaction (Figure 7). In the presence of the
HBSP catalyst (10ꢀ6 M catalytic centers) at pH ¼ 7.0 [0.05 M
phoshate-buffered saline (PBS)] and 25 8C, the apparent
kinetic parameters of the catalyst were obtained as
Received: December 4, 2010; Revised: January 25, 2011; Published
online: March 11, 2011; DOI: 10.1002/mabi.201000478
Keywords: enzymes; host-guest interactions; hyperbranched;
supramolecular structures
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