Investigation of solvent-dependent catalytic behaviour of hydrophobic guest artificial glutathione peroxidase using H2O2 and 3-carboxyl-4-nitrobenzenethiol as substrates

Article abstract of DOI:10.14233/ajchem.2015.18983

The investigation of the catalytic behaviour of a hydrophobic guest artificial glutathione peroxidase (GPx) (ADA-Te-OH) was carried out employing H₂O₂ and 3-carboxyl-4-nitrobenzenethiol (TNB) as substrates. The relation between the catalytic rate of ADA-Te-OH and the property of solvent used in the determination of catalytic activity was revealed. Typically, the co-solvents including ethanol, DMSO, DMF and CH3CN were employed in the determination of catalytic rates. It indicated that ADA-Te-OH exhibited the typical solventdependent catalytic behaviour. Especially, higher catalytic rate was observed when polar protic solvent (ethanol) was used compared with other co-solvents. It suggested that polar protic solvent was the appropriate co-solvent for the assay of catalytic activity of hydrophobic artificial glutathione peroxidase. Additionally, the strong polarity of polar aprotic solvent plays an important role in the enhancement of glutathione peroxidase catalytic activity. This study embodies well understanding of the catalytic behaviour of hydrophobic guest artificial glutathione peroxidase.

Full text of DOI:10.14233/ajchem.2015.18983

Asian Journal of Chemistry; Vol. 27, No. 10 (2015), 3799-3802
A
SIAN
J
OURNAL OF HEMISTRY
C
http://dx.doi.org/10.14233/ajchem.2015.18983
Investigation of Solvent-Dependent Catalytic Behaviour of Hydrophobic Guest Artificial
Glutathione Peroxidase Using H2O2 and 3-Carboxyl-4-nitrobenzenethiol as Substrates
*
*
R.R. ZHANG, Y.Z. YIN , S.F. JIAO , Y.Y. ZHENG, S.M. ZHONG, J.R. GAN, H.X. LI, J.H. HUANG and Z.W. ZHAN
Guangxi Colleges and Universities Key Laboratory of Beibu Gulf Oil and Natural Gas Resource Effective Utilization, Qinzhou University,
No. 89, Xihuan Nanlu, Qinzhou 535000, P.R. China
*Corresponding authors: Tel: +86 7772807716; E-mail: yinyanzhen2014@163.com; jiaoshufei2013@163.com
Received: 5 January 2015;
Accepted: 5 February 2015;
Published online: 22 June 2015;
AJC-17350
The investigation of the catalytic behaviour of a hydrophobic guest artificial glutathione peroxidase (GPx) (ADA-Te-OH) was carried out
employing H2O2 and 3-carboxyl-4-nitrobenzenethiol (TNB) as substrates. The relation between the catalytic rate of ADA-Te-OH and the
property of solvent used in the determination of catalytic activity was revealed. Typically, the co-solvents including ethanol, DMSO,
DMF and CH3CN were employed in the determination of catalytic rates. It indicated that ADA-Te-OH exhibited the typical solvent-
dependent catalytic behaviour. Especially, higher catalytic rate was observed when polar protic solvent (ethanol) was used compared with
other co-solvents. It suggested that polar protic solvent was the appropriate co-solvent for the assay of catalytic activity of hydrophobic
artificial glutathione peroxidase. Additionally, the strong polarity of polar aprotic solvent plays an important role in the enhancement of
glutathione peroxidase catalytic activity. This study embodies well understanding of the catalytic behaviour of hydrophobic guest artificial
glutathione peroxidase.
Keywords: Artificial enzymes, Catalytic behaviour, Enzyme activity, Glutathione peroxidase.
peroxidase and the determination of the catalytic activity of
small molecule artificial glutathione peroxidases are carried
out in solvent mixture. However, up to now, the investigation
of relation between the property of solvent mixture and the
catalytic rate of artificial glutathione peroxidase was less
reported, which has largely limited the further development
of novel supramolecular self-assembled artificial glutathione
peroxidase. Therefore, the elucidation of relation between the
catalytic rate of artificial glutathione peroxidase and the
property of solvent mixture is still a challenge.
Therefore, to achieve such a significant goal, a hydrophobic
guest artificial glutathione peroxidase (ADA-Te-OH) was
employed. And the catalytic behaviour of ADA-Te-OH was
detailed investigated using H2O2 and 3-carboxyl-4-nitrobenzene-
thiol as substrates. These substrates have been proved to be more
excellent and appropriate substrates for the determination of
catalytic activity of glutathione peroxidase4,5,9. Herein, this
method highlights the further development of novel supramole-
cular self-assembled artificial glutathione peroxidase using
hydrophobic glutathione peroxidase as building block.
INTRODUCTION
Glutathione peroxidase (GPx, Ec.1.11.1.9) functions to
protect various living organism from aerobic oxidative stresses
by catalyzing the reduction of reactive oxygen species using
glutathione (GSH) as reducing substrate, which is an important
selenium-containing enzyme of the family of the antioxidative
enzyme system1. Commonly, glutathione peroxidase can clear
the overproduced reactive oxygen species (ROS) that lead to
many human oxidative stress-related diseases2,3. Owing to its
biologically crucial role, some artificial glutathione peroxi-
dases have been designed based on macromolecular scaffolds
in our group4,5.
Among various artificial glutathione peroxidases with
antioxidative catalytic ability constructed during the past
decades, artificial glutathione peroxidases based on small
molecules scaffolds have attracted more attentions. It is because
that the accurately catalytic elements of glutathione peroxidase
with designable structure can be anchored to small molecule
artificial glutathione peroxidases6-8. Recently, employing the
small molecule artificial glutathione peroxidases as building
blocks, self-assembled supramolecular artificial glutathione
peroxidases are constructed9. Generally, both the construction
of the supramolecular self-assembled artificial glutathione
EXPERIMENTAL
Hydrogen peroxide, 3-carboxyl-4-nitrobenzenethiol (TNB),
NaH2PO4, Na2HPO4, ethanol were purchased from J&K Scientific

3800 Zhang et al.
Asian J. Chem.
Ltd. and were used without further purification. ADA-Te-OH
was synthesized according to the previous reported10. The
structure of ADA-Te-OH was determined as this. (1H NMR (300
MHz, CDCl3) δ (ppm) 4.09 (t, 2 H), 3.71 (t, 2 H), 2.72 (t, 2 H),
2.66 (t, 2 H), 2.07 (m, 2 H), 2.00 (s, 3 H), 1.88 (s, 6 H), 1.71 (s,
6 H)). UV-vis spectra were obtained using a Shimadzu 2600
UV-visible-NIR spectrophotometer. The buffer pH values were
determined with a METTLER TOLEDO 320 pH meter.
Determination of glutathione peroxidase activity in
solvent mixture of PBS and co-solvents: The catalytic activity
was assayed according to a modified method reported by
Hilvert and Wu11. The typical assay process of glutathione
peroxidase activity in solvent mixture of PBS and ethanol 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-OH)(0.025 mM) were
added and then 100 µL of the 3-carboxyl-4-nitrobenzenethiol
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 H2O2 (2 mM) and
the absorption decrease of 3-carboxyl-4-nitrobenzenethiol at
410 nm (ε410 = 13600 M–1 cm–1. pH = 7.0) was monitored by a
Shimadzu 2600 UV-visible-NIR spectrophotometer.Appropriate
control of the non-enzymatic reaction was performed and was
subtracted from the catalyzed reaction. The glutathione
peroxidase activities in solvent mixture of PBS and other co-
solvents were assayed similarly except ethanol was replaced
by other co-solvents.
ability than traditional PhSeSePh. Additionally, the highest
catalytic rates were observed when different co-solvents were
used and they were given in Table-1.
Fig. 1. Determination of glutathione peroxidase catalytic rates of ADA-Te-
OH for the reduction of H2O2 using 3-carboxyl-4-nitrobenzenethiol
as substrate
TABLE-1
INITIAL RATES (ν0) AND ACTIVITIES FOR THE REDUCTION
OF H2O2 (2 mM) BY 3-CARBOXYL-4-NITROBENZENETHIOL
(1 mM) IN THE PRESENCE OF THE ADA-Te-OH
(0.025 mM) AT pH 7.0 AND 25 °C
ν0 (mM min–1)a
3.68 0.22
2.59 0.18
1.72 0.13
0.95 0.08
Co-solvent
PBS:Co-solvent (v:v)
Ethanol
DMSO
DMF
6:4
6:4
5:5
6:4
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
Determination of glutathione peroxidase catalytic rates
influenced by co-solvents: Typically, the volume ratios of
PBS:ethanol used in the determination of the glutathione
peroxidase catalytic rate were shown as follows: 9:1; 8:2; 7:3;
6:4; 5:5; 4:6; 3:7; 2:8. The catalytic activities influenced by
other co-solvents were assayed similarly except ethanol was
replaced by other co-solvents.
Determination of glutathione peroxidase catalytic rate
influenced by co-solvent: Herein, the solvent mixture con-
sisted of PBS and co-solvent was used as assay solution to
determine the glutathione peroxidase 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, respectively.Vividly, by plotting the catalytic reaction
rate against the volume ratio of PBS to co-solvent, Fig. 2 was
given. Typically, glutathione peroxidase catalytic rate
influenced by increasing added ethanol was investigated and
shown in Fig. 2a. It is noted that the catalytic rate of ADA-Te-
OH increased to some extent with ethanol increasing added.
And the highest value (3.68 µM×min–1) was obtained when
the volume ratio was 6:4. However, the catalytic reaction rate
largely went down when the volume ratio increased further.
Additionally, the similar catalytic behaviours were also
observed when DMSO, DMF and CH3CN were used as co-
solvents based on Fig. 2b-d.
Considering that ADA-Te-OH consisted of several hydro-
phobic groups, we speculated that the interesting phenomena
of catalytic rate increasing to some extent with the volume
ratio going up was derived from the change of solubility of
ADA-Te-OH in solvent mixture. Therefore, the better solu-
bility of ADA-Te-OH was favourable for the homogeneous
phase system consisted ADA-Te-OH and substrates. And the
highest value was exhibited when the appropriate solubility
of ADA-Te-OH and substrates was achieved. Furthermore,
the possible reason for the decreased catalytic reaction rate
might be endowed from the decreasing of PBS. It was noted
that the polar environment endowed from PBS played an
important role in maintaining the high catalytic rate and were
RESULTS AND DISCUSSION
Determination of glutathione peroxidase catalytic
activity of ADA-Te-OH: Herein, ADA-Te-OH was selected
as the typical hydrophobic artificial glutathione peroxidase
to reveal the relation between the catalytic rate of artificial
glutathione peroxidase and the property of solvent mixture.
Fig. 1 showed that the several hydrophobic groups presented
in ADA-Te-OH, such as adamantane, -TeCH2-, -CH2-, etc.
Therefore, the solubility of ADA-Te-OH in water was poor.
So the catalytic property of ADA-Te-OH was investigated using
ethanol, DMSO, DMF, CH3CN, as co-solvents, respectively.
Typically, the catalytic activity of ADA-Te-OH for the
reduction of H2O2 by 3-carboxyl-4-nitro-benzenethiol was
evaluated according to the modified method reported by Hilvert
and Wu11 using 3-carboxyl-4-nitrobenzenethiol as an alter-
native of glutathione (Fig. 1). Compared with the catalytic
rate of the traditional small molecule artificial glutathione
peroxidase PhSeSePh (ν0 = 0.019 µM min–1), a remarkable
rate enhancement was observed under the conditions of
different solvent mixture when ADA-Te-OH was functioned
as artificial glutathione peroxidase (Table-1). This observation
suggested that ADA-Te-OH exhibited more excellent catalytic

Vol. 27, No. 10 (2015)
Solvent-Dependent Catalytic Behaviour of Hydrophobic Guest Artificial Glutathione Peroxidase 3801
2.8
2.6
2.4
4.0
3.6
3.2
2.8
2.4
2.0
1.6
1.2
0.8
0.4
(a)
(b)
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
CH3CH2OH
DMSO
1.1
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
(c)
(d)
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.1
0.2
0.3
0.4
DMF
Fig. 2. Plots of catalytic rates ν0 against the volume ratios of co-solvent: (a) ethanol; (b) DMSO; (c) DMF; (d) CH3CN
0.5
0.6
0.7
0.8
CH3CN
favourable for the accomplishment of catalytic process8. Thus,
it is concluded that the decreased catalytic reaction rate might
be endowed from the decreasing of PBS. So we can draw a
conclusion that only co-solvent added with appropriated ratio
was favourable for the enhancement of glutathione peroxidase
catalytic ability.
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
Solvent-dependent catalytic behaviour ofADA-Te-OH:
Moreover, it was shown in Fig. 3 that the sequence of the
highest initial rates obtained using different co-solvents was
like this:A(ethanol) > B(DMSO) > C(DMF) > D(CH3CN).As
it is known that among the four co-solvents, ethanol was polar
protic solvent. DMSO, DMF and CH3CN were polar aprotic
solvent. Fig. 3 concluded that ethanol was the most suitable
co-solvent for the enhancement of catalytic activity as the polar
protic solvent. Additionally, among the three polar aprotic
solvents, the polarity sequence was as follow: DMSO > DMF
> CH3CN. Obviously, the sequence of the highest initial rates
related to the three polar aprotic solvents was in accordance
with polarity sequence. It suggested that the strong polarity of
polar aprotic solvent was favourable for the enhancement of
glutathione peroxidase catalytic activity. Therefore, a conclu-
sion has been drawn that polar protic solvent is the suitable
co-solvent for the enhancement of catalytic activity. And the
A
B
C
DMF
D
CH3CH2OH
DMSO
CH3CN
Fig. 3. Highest initial rates (ν0) obtained using different co-solvents (A)
ethanol; (B) DMSO; (C) DMF; (D) CH3CN
strong polarity of polar aprotic solvent plays an important
role in the enhancement of glutathione peroxidase catalytic
activity. This conclusion might function as the basement for
the understanding of the catalytic behaviour of hydrophobic
guest artificial glutathione peroxidase.

3802 Zhang et al.
Asian J. Chem.
Conclusion
the Natural Science Foundation of Guangxi Province (No.
2013GXNSFBA019043).
Herein, the investigation of the relation between the
catalytic rate of ADA-Te-OH and the property of solvent was
carried out. It was proved that ADA-Te-OH exhibited the
typical solvent-dependent catalytic behaviour when different
volume ratios of co-solvents were respectively added. More-
over, 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 glutathione
peroxidase. And the strong polarity of polar aprotic solvent
plays an important role in the enhancement of glutathione
peroxidase catalytic activity.
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