Investigation of solvent-dependent catalytic behaviour of hydrophobic guest artificial glutathione peroxidase

Article abstract of DOI:10.14233/ajchem.2014.15802

To reveal the relation between the catalytic rate of artificial glutathione peroxidase (GPx) and the property of solvent used in the determination of catalytic behaviour of glutathione peroxidase (ADA-Te-ADA) was investigated. Ethanol, DMSO, DMF and CH

Full text of DOI:10.14233/ajchem.2014.15802

Asian Journal of Chemistry; Vol. 26, No. 8 (2014), 2311-2314
ASIAN JOURNAL OF CHEMISTRY
http://dx.doi.org/10.14233/ajchem.2014.15802
Investigation of Solvent-Dependent Catalytic Behaviour of
Hydrophobic Guest Artificial Glutathione Peroxidase
1,2
1
1
1
1
1
Y.Z. YIN , Y. WEI , Z.F. SHI , H.X. SHI , X.X. HU , S.F. JIAO1,* and D.H. CHEN
1School of Chemistry and Chemical Engineering, Qinzhou University, No. 89, Xihuan Nanlu, Qinzhou 535000, P.R. China
2Guangxi Experiment Centre of Science and Technology, Guangxi University, Nanning 530004, P.R. China
*Corresponding author: Tel: +86 777 2807716; E-mail: jiaoshufei2013@163.com
Received: 20 May 2013;
Accepted: 24 September 2013;
Published online: 15 April 2014;
AJC-15015
To reveal the relation between the catalytic rate of artificial glutathione peroxidase (GPx) and the property of solvent used in the determination
of catalytic behaviour of glutathione peroxidase (ADA-Te-ADA) was investigated. Ethanol, DMSO, DMF and CH3CN were selected as
the co-solvent in the determination of catalytic rates. It was proved that ADA-Te-ADA exhibited the typical solvent-dependent catalytic
behaviour. Moreover, compared with other co-solvents, the higher catalytic rate was observed when polar protic solvent (ethanol) was
used. It suggested that the polar protic solvent was the appropriate co-solvent for the catalytic activity assay of hydrophobic artificial
glutathione peroxidase. The strong polarity of polar aprotic solvent played an important role in the enhancement of glutathione peroxidase
catalytic activity. This study explains the understanding of the catalytic behavior of hydrophobic guest artificial glutathione peroxidase
when co-solvent was used.
Keywords: Artificial enzymes, Biomimetics, Enzyme activity, Glutathione peroxidase, Catalytic behaviour.
solvent mixture is less reported, which has largely limited the
further development of novel supramolecular self-assembled
artificial GPx. Therefore, the elucidation of relation between
the catalytic rate of artificial GPx and the property of solvent
mixture is still a significant goal.
INTRODUCTION
Glutathione peroxidase (GPx, Ec.1.11.1.9) is an important
selenium-containing enzyme among the antioxidative enzyme
system. Glutathione peroxidase functions to protect various
living organism from aerobic oxidative stresses by catalyzing
the reduction of reactive oxygen species (ROS) using gluta-
thione (GSH) as reducing substrate1. Usually, overproduced
reactive oxygen species lead to many human oxidative stress-
related diseases2,3. As the member of antioxidative defense
system, GPx checks the overproduced ROS. Owing to its biolo-
gically crucial role, some artificial GPxs have been designed
based on macromolecular scaffolds4,5.
Therefore, to meet such significant challenge, a hydro-
phobic guest artificial GPx (ADA-Te-ADA) was employed
and the catalytic behaviour of it was investigated. This method
highlights the further development of novel supramolecular
self-assembled artificial GPx using hydrophobic GPx as
building block.
EXPERIMENTAL
Among artificial GPxs with antioxidative catalytic ability
constructed previously, artificial GPxs based on small mole-
cules scaffolds have attracted more attentions6-8. The accurately
catalytic elements of GPx with designable structure can be
anchored to small molecules artificial GPx7. Recently, using
the small molecules artificial GPx as building block, self-
assembled supramolecular artificial GPxs are prepared9.
Generally, the determination of the catalytic activity of small
molecules artificial GPx and the construction of the supra-
molecular self-assembled artificial GPx are achieved in solvent
mixture. However, up to now, the investigation of relation
between the catalytic rate of artificial GPx and the property of
Cumene hydroperoxide (CUOOH), NaH2PO4, Na2HPO4,
methanol were purchased from J&K scientific Ltd and
were used without further purification. 3-Carboxyl-4-nitro-
benzenethiol (TNB) was synthesized from 5,5'-dithiobis(2-
nitrobenzoic acid) as described previously10. ADA-Te-ADA
was used as endowed from liu's group. The structure ofADA-
Te-ADA was determined by Bruker 300 MHz spectrometer
using a TMS proton signal as the internal standard (1H NMR
(300 MHz, CDCl3) δ (ppm) 4.09 (t, 2 H, -COOCH2), 2.66 (t, 2
H, -TeCH2), 2.07 (m, 2 H, -CH2-), 2.01 (s, 3 H, adamantane),
1.88 (s, 6 H, adamantane),1.71 (s, 6 H, adamantane)). UV-
visible spectra were obtained using a pgeneral T6 UV-visible

2312 Yin et al.
Asian J. Chem.
spectrophotometer. The buffer pH values were determined with
a METTLER TOLEDO 320 pH meter.
phobic groups presented in ADA-Te-ADA, such as adaman-
tane, -TeCH2-, -CH2- and so on. The solubility of ADA-Te-
ADA in water was poor. Therefore, the catalytic property of
ADA-Te-ADA was investigated using ethanol, DMSO, DMF,
CH3CN, as co-solvent, respectively. Typically, the catalytic
activity of ADA-Te-ADA for the reduction of cumene hydro-
peroxide by 3-carboxyl-4-nitro-benzenethiol was evaluated
according to the modified method reported by Wu and Hilvert11
using 3-carboxyl-4-nitro-benzenethiol as a glutathione (GSH)
alternative (Fig. 1). The absorption decrease of 3-carboxyl-4-
nitro-benzenethiol at 410 nm (ε410 = 13600 M–1 cm–1, pH = 7)
was monitored. Typically, the curves of the absorption decrease
vs. time in the catalytic reduction using ethanol as co-solvent
were depicted in Fig. 2. Compared with curve a of the absor-
ption decrease without catalyst, the traditional small molecule
artificial GPx PhSeSePh slightly enhanced the absorption
decrease (curve c, υ0 = 0.019 µM min-1). However, whenADA-
Te-ADA was used under the same condition, a remarkable
rate enhancement (curve c, υ0 = 4.16 µM min-1) was observed.
This observation proved that ADA-Te-ADA exhibited more
excellent catalytic ability than traditional PhSeSePh.
Additionally, the highest catalytic rates were observed when
different co-solvents were used, which were given in Table-1.
Determination of GPx catalytic rate influenced by
co-solvent: Herein, the solvent mixture consisted of PBS and
co-solvent was employed as assay solution to determine the
GPx catalytic rate. The ratio of PBS to co-solvent was fixed to
9:1; 8:2; 7:3; 6:4; 5:5; 4:6; 3:7; 2:8; 1:9, respectively. Typically,
GPx catalytic rate influenced by increasing added ethanol was
investigated. The plotting of the catalytic reaction rate against
the volume ratio of PBS to co-solvent was given in Fig. 3. Fig.
3a showed that the catalytic reaction rate of ADA-Te-ADA
increased to some extent with ethanol increasing added. The
highest value (4.16 µM min-1) was obtained when the volume
ratio was 6:4. However, the catalytic reaction rate largely decre-
ased when the volume ratio increased further. Additionally,
the similarly catalytic behaviours were also observed when
DMSO, DMF and CH3CN were used as co-solvent based on
Fig. 3 b, c, d.
Determination of GPx activity in solvent mixture of
PBS and ethanol: The catalytic activity was assayed according
to a modified method reported by Wu and Hilvert11. The typical
assay process was shown as follows: The reaction was carried
out at 25 °C in a 1 mL quartz cuvette, 700 µL solvent mixture
of PBS and ethanol and 100 µL of the catalyst (ADA-Te-ADA)
(0.025 mM) were added and then 100 µL of the 3-carboxyl-4-
nitro-benzenethiol solution(1 mM) was added. The mixture
in the quartz cuvette was pre-incubated at the 25 °C for 3 min.
Finally, the reaction was initiated by the addition of 100 mL
of cumene hydroperoxide (2 mM) and the absorption decrease
of 3-carboxyl-4-nitro-benzenethiol at 410 nm (ε410 = 13600
M-1cm-1. pH = 7) was monitored using a Pgeneral T6 UV-visible
spectrophotometer. Appropriate control of the non-enzymatic
reaction was performed and was subtracted from the catalyzed
reaction. Each initial rate was measured at least 5 times and
calculated from the first 5-10 % of the reaction.
Determination of GPx activity in solvent mixture of
PBS and other co-solvent: The catalytic activity was assayed
according to the process mentioned above except ethanol was
replaced by other co-solvent.
Determination of the GPx catalytic rate influenced by
ethanol: Typically, the volume ratios of PBS:ethanol used in
the determination of the GPx catalytic rate were shown as
follows: 9:1; 8:2; 7:3; 6:4; 5:5; 4:6; 3:7; 2:8; 1:9.
Determination of the GPx catalytic rate influenced by
other co-solvent: The catalytic activity was assayed according
to the process mentioned above except ethanol was replaced
by other co-solvent.
RESULTS AND DISCUSSION
Determination of GPx catalytic activity of ADA-Te-
ADA: Herein, as shown in Fig.1, ADA-Te-ADA was selected
as the typical hydrophobic artificial GPx. The catalytic
behaviour of it was investigated. The structure of ADA-Te-
ADA was illustrated in Fig.1. It was clearly that several hydro-
O
O
O
Te
O
CH3
O2N
SH
OOH
+
(ADA-Te-ADA)
CH3
(CUOOH)
HOOC
(TNB)
CH3
CH3
O2N
S
S
NO2
+ H2O
OH
+
HOOC
CH2OOH
(DTNB)
Fig. 1. Determination of GPx catalytic rates of ADA-Te-ADA for the reduction of cumene hydroperoxide (CUOOH) using 3-carboxyl-4-nitro-benzenethiol
(TNB) as substrates

Vol. 26, No. 8 (2014)
Solvent-Dependent Catalytic Behaviour of Hydrophobic Guest Artificial Glutathione Peroxidase 2313
5
2.2
a
(a)
2.0
1.8
1.6
1.4
1.2
1.0
4
b
3
2
1
0
c
0.8
0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
CH3CH2OH
20
40
60
80
Time (s)
4
Fig. 2. Plots of absorbance vs. time in the catalytic reduction of cumene
hydroperoxide (2 mM) by 3-carboxyl-4-nitro-benzenethiol (1 mM)
at pH 7 and 25 °C using ethanol as co-solvent (PBS: ethanol, v:v,
6:4) (a) No catalyst, (b) 0.025 mM PhSeSePh, (c) ADA-Te-ADA
(0.025 mM)
(b)
3
2
1
0
TABLE-1
INITIAL RATES (υ0) AND ACTIVITIES FOR THE
REDUCTION OF CUMENE HYDROPEROXIDE (2 mM) BY
3-CARBOXYL-4-NITRO-BENZENETHIOL (1 mM) IN THE
PRESENCE OF THE ADA-Te-ADA (0.025 mM) AT pH 7 AND 25 °C
υ0 (mM min-1)a
4.16 0.21
3.77 0.26
3.27 0.14
3.02 0.17
Co-solvent
PBS: co-solvent (v:v)
Ethanol
DMSO
DMF
6:4
7:3
7:3
6:4
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
DMSO
CH3CN
aInitial rate of reaction was corrected for the spontaneous oxidation. And
the concentration of catalyst is 0.025 mM and assuming one molecule
catalytic center (tellurium moiety) as one active site of enzyme
3
(c)
As ADA-Te-ADA consisted of several hydrophobic
groups, we speculated that the interesting phenomena of cata-
lytic rate increasing to some extent with the volume ratio going
up was driving from the change of solubility ofADA-Te-ADA
in solvent mixture. So the better solubility of ADA-Te-ADA
was favorable for the homogeneous phase system consisted
ADA-Te-ADA and substrates. And the highest value was exhi-
bited when the appropriate solubility of ADA-Te-ADA and
substrates was endowed. Furthermore, the possible reason for
the decreased catalytic reaction rate might endow from the
hydrophobic driving force. It is noted from previous report
that the hydrophobic driving force might result in the confor-
mation change of hydrophobic dendrimer-based artificial
GPx8. Such conformation change could alter the substrate
selectivity of artificial GPx. Similarly, ADA-Te-ADA and
substrate cumene hydroperoxide were hydrophobic molecules.
So PBS was poor solvent for the hydrophobic artificial enzyme
ADA-Te-ADA and the hydrophobic substrate cumene hydro-
peroxide. Therefore, the solvent mixture with more PBS might
drive ADA-Te-ADA and cumene hydroperoxide matched
closely in the catalytic reaction. When more co-solvent was
added and PBS was less, the match of ADA-Te-ADA and
cumene hydroperoxide could not be achieved appropriately
as the hydrophobic driving force from PBS was poor although
theADA-Te-ADA dissolved well. So it is concluded that only
2
1
0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
DMF
3
2
1
0
(d)
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
CH3CN
Fig. 3. Plots of catalytic reaction rates υ0 against the volume ratios of co-
solvent: (a) ethanol; (b) DMSO; (c) DMF; (d) CH3CN

2314 Yin et al.
Asian J. Chem.
co-solvent added with appropriated ratio was favourable for
the enhancement of GPx catalytic ability.
Conclusion
Herein, a hydrophobic guest molecule, ADA-Te-ADA,
was selected as the hydrophobic artificial GPx.And the relation
between the catalytic rate of it and the property of solvent was
investigated. It was proved that ADA-Te-ADA exhibited the
typical solvent-dependent catalytic behaviour when different
volume ratios of co-solvents were respectively added. More-
over, the higher catalytic rate was observed when polar protic
solvent (ethanol) was used compared with other co-solvents,
which suggested that polar protic solvent was the appropriate
co-solvent for the assay of hydrophobic artificial GPx. And
the strong polarity of polar aprotic solvent plays an important
role in the enhancement of GPx catalytic activity.
Discussion of the solvent-dependent catalytic beha-
viour of ADA-Te-ADA: Moreover to illustrate and compare
the highest initial rates (υ0) obtained when various co-solvent
with appropriate ratio was used (Fig. 4). It was shown that the
sequence of the highest initial rates obtained using co-solvents
was like this:A(ethanol) > B(DMSO) > C(DMF) > D(CH3CN).
It is known that among the four co-solvents, ethanol was polar
protic solvent. DMSO, DMF and CH3CN were polar aprotic
solvent. Combining the sequence of the highest initial rates
and the properties of four co-solvents, it is concluded that the
ethanol was the most suitable co-solvent for the enhancement
of catalytic activity as the polar protic solvent.Among the three
polar aprotic solvents, the polarity sequence was as follows:
DMSO > DMF > CH3CN.As shown in Fig. 4, the sequence of
the highest initial rates of the three polar aprotic solvents was
in accordance with polarity sequence. It is suggested that the
strong polarity of polar aprotic solvent was favorable for the
enhancement of GPx catalytic activity. Therefore, we can draw
a conclusion that polar protic solvent is the suitable co-solvent
for the enhancement of catalytic activity.And the strong polarity
of polar aprotic solvent plays an important role in the enhance-
ment of GPx catalytic activity. This conclusion might function
as the basement for the understanding of the catalytic behaviour
of hydrophobic guest artificial GPx.
ACKNOWLEDGEMENTS
This research was supported by Guangxi Experiment
Centre of Science and Technology (No. LGZXKF201106).
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A
CH3CH2OH
B
DMSO
D
CH3CN
C
DMF
Fig. 4. The highest initial rates (υ0) obtained using various co-solvent; (a)
ethanol; (b) DMSO; (c) DMF; (d) CH3CN