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
Zhang
