Tài liệu Báo cáo khoa học: A novel dicyclodextrinyl diselenide compound with glutathione peroxidase activity ppt

A novel dicyclodextrinyl diselenide compound with
glutathione peroxidase activity
Shao-Wu Lv
1
, Xiao-Guang Wang
1
, Ying Mu
1
, Tian-Zhu Zang
1
, Yue-Tong Ji
1
, Jun-Qiu Liu
2
,
Jia-Cong Shen
2
and Gui-Min Luo
1
1 Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, Changchun, China
2 Key Laboratory for Supramolecular Structure and Materials of the Ministry of Education, Jilin University, Changchun, China
Glutathione peroxidase (GPX; EC 1.11.1.9) is a well-
known selenoenzyme that catalyzes the reduction
of harmful hydroperoxides by glutathione (GSH)
(Scheme 1) and protects lipid membranes and other
cellular components against oxidative damage [1–4]. It
is related to many diseases and is regarded as one of
the most important antioxidant enzymes in living
organisms. GPX enzyme activity is sometimes
increased in disease, possibly as a compensatory mech-
anism to try to counteract the oxidative stress associ-

ated with the pathology, although it is also decreased
in other diseases [5–8]. Therefore, modulation of GPX
may be involved in many pathological conditions.
Because natural GPX has some shortcomings, such as
instability, antigenicity and poor availability, much
attention has been paid to its artificial imitation [9,10].
In synthetic approaches, an initial attempt is made
to synthesize organoselenium compounds in which the
interaction of Se–N in GPX catalysis is imitated by
inducing N or O in close proximity to selenium. One
way in which this is achieved is by binding the selen-
ium atom directly to a heteroatom such as nitrogen.
2-Phenyl-l,2-benzisoselenazol-3(2H)-one (Ebselen), the
first biologically active organoselenium compound,
represents an excellent example of a GPX mimic [9].
Another way to imitate the SeÆÆÆ N interaction in GPX is
if the selenium is not bound directly to the heteroatom
(N or O), but is located in close proximity to it, this
Keywords
cyclodextrins; enzyme mimics; glutathione
peroxidase; selenium; substrate binding
Correspondence
G M. Luo, Key Laboratory for Molecular
Enzymology and Engineering of the Ministry
of Education, Jilin University, 2519 Jiefang
Road, Changchun 130021, China
Fax: +86 431 8898 0440
Tel: +86 431 8849 8974
E-mail:
(Received 12 February 2007, revised

27 May 2007, accepted 31 May 2007)
doi:10.1111/j.1742-4658.2007.05913.x
A 6A,6A¢-dicyclohexylamine-6B,6B¢-diselenide-bis-b-cyclodextrin (6-CySeCD)
was designed and synthesized to imitate the antioxidant enzyme glutathione
peroxidase (GPX). In this novel GPX model, b-cyclodextrin provided a
hydrophobic environment for substrate binding within its cavity, and a
cyclohexylamine group was incorporated into cyclodextrin in proximity to
the catalytic selenium in order to increase the stability of the nucleophilic
intermediate selenolate. 6-CySeCD exhibits better GPX activity than 6,6¢-di-
selenide-bis-cyclodextrin (6-SeCD) and 2-phenyl-1,2-benzoisoselenazol-
3(2H)-one (Ebselen) in the reduction of H
2
O
2
, tert-butyl hydroperoxide and
cumenyl hydroperoxide by glutathione, respectively. A ping-pong mechanism
was observed in steady-state kinetic studies on 6-CySeCD-catalyzed reac-
tions. The enzymatic properties showed that there are two major factors for
improving the catalytic efficiency of GPX mimics. First, the substrate-bind-
ing site should match the size and shape of the substrate and second,
incorporation of an imido-group increases the stability of selenolate in the
catalytic cycle. More efficient antioxidant ability compared with 6-SeCD and
Ebselen was also seen in the ferrous sulfate ⁄ ascorbate-induced mitochondria
damage system, and this implies its prospective therapeutic application.
Abbreviations
BHT, 2,6-di-tert-butyl-4-methylphenol; b-CD, b-cyclodextrin; 6-CySeCD, 6A,6A¢-dicyclohexylamine-6B,6B¢-diselenide-bis-b-cyclodextrin;
CumOOH, cumenyl hydroperoxide; Ebselen, 2-phenyl-1,2-benzoisoselenazol-3(2H)-one; GPX, glutathione peroxidase; GSH, glutathione;
6-SeCD, 6,6¢-diselenide-bis-cyclodextrin; TBARS, thiobarbituric acid reactive substances; t-BuOOH, tert-butyl hydroperoxide.
3846 FEBS Journal 274 (2007) 3846–3854 ª 2007 The Authors Journal compilation ª 2007 FEBS
approach seems to help stabilize the selenolate and

enhance the GPX-like activity of diselenides [11].
Although some GPX mimics show some increased
activity, most show only limited catalytic enhancement.
Based on an understanding of the structure of GPX,
its mode of molecular recognition and catalysis, as well
as previous studies [12–14], we believe that generation
of specific binding ability for the thiol substrate and
correct incorporation of the functional selenium group
should be critical in the construction of an effective
GPX model. Previous studies by our group in prepar-
ing GPX models using a mAb technique [15,16], bio-
imprinting [17] and the chemical modification of native
enzymes [18] have supported this hypothesis. Recently,
we developed some GPX mimics in which the b-cyclo-
dextrin (b-CD) cavity provided a hydrophobic environ-
ment for substrate binding [19–23]. For example,
6,6’-diseleno-bis-cyclodextrin (6-SeCD) activity for the
reduction of hydrogen peroxide (H
2
O
2
) by GSH is 4.3
times that of Ebselen because of the role of the hydro-
phobic cavity of b-CD in binding substrate.
In this study we designed and synthesized a new GPX
mimic, 6A,6A¢-dicyclohexylamine-6B,6B¢-diselenide-
bis-b-cyclodextrin (6-CySeCD), in which the cyclo-
hexylamine group was incorporated in the proximity of
the selenium atom and the b-CD cavity provided a
hydrophobic environment for substrate binding.

6-CySeCD showed higher GPX activity than 6-SeCD
for the reduction of H
2
O
2
, tert-butyl hydroperoxide
(t-BuOOH) and cumenyl hydroperoxide (CumOOH)
by GSH, indicating that incorporation of the imido-
group in the proximity of the selenium atom may
increase the stability of the nucleophilic intermediate
selenolate and enhance GPX-like activity in selenium-
containing GPX mimics. We also studied the catalytic
mechanism using steady-state kinetics of 6-CySeCD
catalysis and investigated the antioxidant ability of
6-CySeCD using a mitochondria injury system.
Results and Discussion
Synthesis and characterization of 6-CySeCD
The synthetic routes of 6-CySeCD are shown in
Scheme 2. 6-CySeCD was analyzed using elemental
analysis, found (calculated for C
96
H
160
O
66
N
2
Se
2
Æ6H

2
O)
%: C, 43.67 (43.28); H, 6.49 (6.31); N, 1.06 (1.05). IR
(KBr): 3376(-OH), 2926(CH,CH
2
), 1644(-OH), 1568,
1467(-NH-), 1158, 1079, 1031(-O-), 948, 840, 755, 709,
579 cm
)1
.
13
C NMR (400 MHz, D
2
O) d(p.p.m.):
102.5(C1), 81.9(C4), 73.3(C3), 72.7(C5), 72.0(C2),
60.3(C6), 59.2(C6¢), 52.3(C7), 35.1(C8), 28.5(C10),
25.7(C9).
The content and valence of selenium in 6-CySeCD
were measured by X-ray photoelectron spectroscopy.
The Se3d electronic-binding energy of 6-CySeCD
is 54.9 eV, which approaches the binding energy of
SeCys (55.1 eV), indicating that the selenium in
6-CySeCD is present in the form of )1 valence (diseleni-
um bridge, -Se-Se-). The experiment also gave the C ⁄ Se
ratio, which is 48.3 : 1 (calculated 48 : 1), indicating
2 mol of selenium per mol of mimic. Thus, the structure
of 6-CySeCD should be as shown in Scheme 3.
GPX activity of 6-CySeCD
The initial reaction rate for the reduction of hydro-
peroxides by GSH was determined by observing the

change in NADPH absorption at 340 nm (Eqns 1,2).
Scheme 2. Synthetic route of 6-CySeCD.
Scheme 1. Catalytic cycle for GPX.
S W. Lv et al. 6-CySeCD displays GPX activity
FEBS Journal 274 (2007) 3846–3854 ª 2007 The Authors Journal compilation ª 2007 FEBS 3847
The GPX activities of 6-CySeCD and other GPX mim-
ics catalyzed the reduction of hydroperoxides by GSH
are listed in Table 1.
ROOH + 2GSH À!
GPX
ROH þ GSSG þ H
2
O ð1Þ
GSSG þ NADPH þ H
þ
ÀÀÀÀÀÀÀÀ!
GSH reductase
2GSH þ NADP
þ
ð2Þ
The GPX activities of 6-CySeCD and 6-SeCD for the
reduction of H
2
O
2
by GSH were 7.9 and 4.2 min
)1
,
respectively, indicating that 6-CySeCD and 6-SeCD
display higher GPX activity than Ebselen. This result

is not surprising, because b-CD shows good substrate
binding compared with Ebselen [15]. When the sub-
strates were H
2
O
2
, t-BuOOH and CumOOH, we found
that the GPX activities with 6-CySeCD for the reduc-
tion of H
2
O
2
, t-BuOOH and CumOOH by GSH were
higher than with 6-SeCD.
In the investigation of GPX mimics, Wilson’s disele-
nides are successful [11]. As shown in Scheme 4, there
are two processes (oxidation and reduction with thiols)
in the mechanism, and the N near the selenium moiety
apparently helps stabilize the selenolate and enhance
the GPX-like activity of diselenides. In this study, a
cyclohexylamine group was incorporated in the prox-
imity of the active selenium atom in the 6-CySeCD
molecule and the GPX activity for 6-CySeCD was
higher than for 6-SeCD.
Kinetics of 6-CySeCD
Steady-state kinetics was observed for substrates H
2
O
2
and GSH. The initial velocities for reduction of H

2
O
2
by GSH were determined as a function of the substrate
Table 1. Comparison between GPX activities of the 6-CySeCD-cata-
lyzed reduction of hydroperoxides by GSH and other species. One
unit of enzyme activity is defined as amount of mimic that utilizes
of 1 lmol of NADPH per minute. All data are presented as
means ± SD.
Mimics Hydroperoxide Activity (min
)1
)
Ebselen H
2
O
2
0.99
b
6-SeCD H
2
O
2
4.20 ± 0.15
b
t-BuOOH 6.3 ± 0.2
CumOOH 10.7 ± 0.4
6-CySeCD
a
H
2

O
2
7.9 ± 0.4
t-BuOOH 12.3 ± 0.3
CumOOH 18.3 ± 0.5
a
Reactions were carried out in 50 mM potassium phosphate buffer,
pH 7.0, at 37
°
C, 1 mM GSH, 0.5 mM hydroperoxide.
b
Obtained
from Liu et al. [19].
Scheme 3. Structures of 6-CySeCD.
Scheme 4. Catalytic mechanism proposed
by Wilson et al. [11].
6-CySeCD displays GPX activity S W. Lv et al.
3848 FEBS Journal 274 (2007) 3846–3854 ª 2007 The Authors Journal compilation ª 2007 FEBS
concentration at 37 °C and pH 7.0, by varying one
substrate concentration while another was fixed. The
relevant steady-state equation (Eqn 3) for the mimic
reaction is
m
0
=½E
0
¼
k
max
½GSHÁ½H

2
O
2

ðKH
2
O
2
½GSHþK
GSH
Á½H
2
O
2
þ½GSHÁ½H
2
O
2
Þ
ð3Þ
Where v
0
is the initial reaction rate, [E]
0
is the initial
enzyme mimic concentration, k
max
is a pseudo-first-
order rate constant K
H

2
O
2
and K
GSH
are the Michaelis–
Menten constants (K
m
) for H
2
O
2
and GSH, respectively.
Double reciprocal plots of the initial velocity versus the
concentration of substrates gave a family of parallel
lines (Fig. 1), indicating that the reaction mechanism is
a ping-pong mechanism. This result demonstrated that
the GPX mimic, 6-CySeCD, has the same catalytic
mechanism as native GPX. From the steady-state equa-
tion, the kinetic parameters were obtained (Table 2).
During investigation of the reduction of peroxides, it
is natural to consider the possibility of free radical
reactions. Bell and Hilvert [24] used a radical trap, 2,6-
di-tert-butyl-1-4-methylphenol (BHT), to inhibit the
reduction of t-BuOOH by a thioyl compound in the
presence of a GPX mimic, selenosubtilisin, and showed
that the enzyme-catalyzed reduction of hydroperoxide
proceeds via a nonradical mechanism, although the
spontaneous reduction of hydroperoxide by GSH
involves the production of free radicals. The same

results were found for the 6-CySeCD-catalyzed reduc-
tion of H
2
O
2
by GSH. BHT inhibited the spontaneous
reaction, but not the 6-CySeCD-catalyzed reduction
(Fig. 2). This suggested that 6-CySeCD also catalyzes
the reduction of hydroperoxide by GSH via a nonradi-
cal mechanism.
Protection of mitochondria against oxidative
damage by 6-CySeCD
The swelling and shrinking of mitochondria are nor-
mal physiological phenomena during respiration. How-
ever, abnormal swelling disrupts the mitochondrial
membrane resulting in cell death. Mitochondrial swell-
ing therefore characterizes its integrity. Figure 3A
shows that the mitochondrial swelling is greatly
increased by ferrous sulfate ⁄ ascorbate-induced mito-
chondrial damage and the swelling is decreased by
addition of 6-CySeCD.
The absorbance at 520 nm for the control group
was basically constant, whereas that for the damage
group decreasede considerably over time, indicating
that mitochondrial swelling was increased. However,
the swelling in the protection group, which contained a
certain concentration of 6-CySeCD, was apparently
decreased compared with the damage group, and
the mitochondrial swelling decreased with increasing
Fig. 1. Double-reciprocal plots for the reduction of H

2
O
2
by GSH
catalyzed by 5 l
M 6-CySeCD. (A) [E]
0
⁄ v
0
versus 1 ⁄ [H
2
O
2
](mM
)1
)at
[GSH] 0.5 (j), 1 (d) and 3 m
M (.). (B) [E]
0
⁄ v
0
versus 1 ⁄ [GSH]
(m
M
)1
)at[H
2
O
2
] 0.5 (d), 1 (m) and 2 mM (.).

Table 2. Kinetic parameters of the 6-CySeCD. Reactions were carried out in 50 mM potassium phosphate buffer, pH 7.0, at 37 °C,
0.5–3.0 m
M GSH, 0.5–2.0 mM H
2
O
2
. All data are presented as means ± SD.
GPX mimic k
max
(min
-1
) K
GSH
(mM) k
max
⁄ K
GSH
(M
)1Æ
min
-1
) K
H2O2
(lM) k
max
⁄ K
H2O2
(M
)1Æ
min

)1
)
6-CySeCD 18.3 ± 0.5 1.63 ± 0.14 (1.12 ± 0.22) · 10
3
547 ± 13 (3.35 ± 0.17) · 10
4
S W. Lv et al. 6-CySeCD displays GPX activity
FEBS Journal 274 (2007) 3846–3854 ª 2007 The Authors Journal compilation ª 2007 FEBS 3849
6-CySeCD concentration. The ability of GPX mimics
(6-CySeCD, 6-SeCD and Ebselen) to protect mito-
chondria differed, as shown in Fig. 3B, and 6-CySeCD
was the best among them. This is in agreement with
ability of these GPX mimics to remove H
2
O
2
.
In this study, we used thiobarbituric acid reactive
substances (TBARS) as a marker for lipid per-
oxidation, and 6-CySeCD, 6-SeCD and Ebselen as
antioxidants in ferrous sulfate ⁄ ascorbate-induced mito-
chondrial damage to determine the levels of lipid per-
oxidation. Figure 4A shows the extent of protection
afforded by 6-CySeCD. The amount of TBARS
seen during mitochondrial damage was reduced
considerably in the presence of 6-CySeCD, and the
amount of TBARS decreased with increasing 6-Cy-
SeCD concentration. When the 6-CySeCD concentra-
tion was 20 lm and mitochondria were damaged for
50 min, the TBARS content was only 24% of that in

the damage group without 6-CySeCD, indicating that
76% of TBARS production was inhibited. In order to
gauge the ability of the three GPX mimics (6-CySeCD,
6-SeCD, and Ebselen) to inhibit TBARS production,
their antioxidant activities were compared under iden-
tical conditions. As shown in Fig. 4B, the ability of
6-CySeCD to decrease the accumulation of TBARS
was greater than that of 6-SeCD and Ebselen. In addi-
tion, we also tested the effect of 20 lm 6-CySeCD in
the absence of damage (data no shown) and the result
shows that 20 lm 6-CySeCD did not have any effect
Fig. 2. Plots of v
0
versus [H
2
O
2
] for 1 mM GSH in 50 mM potas-
sium phosphate buffer, pH 7.4, and 37 °C, at [BHT] ¼ 0 l
M (a) and
50 l
M (b). (A) [6-CySeCD], 0 lM; (B) [6-CySeCD], 5 lM.
Fig. 3. (A) Effect of concentration of 6-CySeCD on the swelling of
mitochondria. (a) Control; (b) damage + 20 l
M 6-CySeCD; (c) dam-
age + 10 l
M 6-CySeCD; (d) damage + 4 lM of 6-CySeCD; (e) dam-
age. (B) Effect of different GPX mimics on mitochondrial swelling.
(a) Control; (b) damage + 10 l
M 6-CySeCD; (c) damage + 10 lM

6-SeCD; (d) damage + 10 lM of Ebselen; (e) damage.
6-CySeCD displays GPX activity S W. Lv et al.
3850 FEBS Journal 274 (2007) 3846–3854 ª 2007 The Authors Journal compilation ª 2007 FEBS
on mitochondrial swelling and lipid peroxidaton in the
absence of damage.
Exposing mitochondria in vitro to redox active xeno-
biotics may simulate oxidative damage of mitochondria
in vivo. The reactions for ferrous sulfate⁄ ascorbate-
inducing mitochondrial damage can be proposed as
follows:
Ascorbic acid + O
2
! dehydroascorbic acid þ H
2
O
2
ð4Þ
Fe
2þ
þ H
2
O
2
! Fe
3þ
þ OH
À
þ
Á
OH ð5Þ

Ascorbic acid þ 2Fe
3þ
! dehydroascorbic acid þ 2Fe
2þ
þ 2H
þ
ð6Þ
where H
2
O
2
was produced by oxidation of ascorbic
acid to dehydroascorbic acid (Eqn 4) [22], in addition,
mitochondria can produce superoxide by Fe(II), which
could be dismutated by mitochondrial superoxide
dismutase to hydrogen peroxide. A hydroxyl radical
was produced via the Fenton reaction (Eqns 5,6)
[25–27]. The biological molecules in mitochondria are
easily attacked by hydroxyl radicals, when changes in
composition, morphology, structure, integrity, and
function of the mitochondria take place. GPX mimics
can scavenge hydroperoxides and block hydroxyl
radical production, therefore protecting mitochondria
against oxidative damage.
In the ferrous sulfate ⁄ ascorbate-induced mitochond-
rial damage model system, swelling and TBARS con-
tent were chosen according to the standard, which was
used to determine the injury and extent of protection
in mitochondria. 6-CySeCD reduced the mitochondrial
swelling during damage and decreased the maximal

TBARS content. Mitochondrial swelling and the
amount of TBARS were decreased in a dose-dependent
manner by 6-CySeCD. The inhibited TBARS content
and decreased mitochondrial swelling can be explained
by 6-CySeCD acting as a GPX mimic, which effect-
ively scavenged hydroperoxide and protected mito-
chondria against oxidative damage.
Conclusion
We developed a novel GPX mimic, 6-CySeCD. In this
enzyme model, the cavity of b-CD supplied a hydro-
phobic environment for substrate binding, and mimic
activity was increased greatly by the incorporation of a
cyclohexylamine group in the proximity of the active
selenium atom. Compared with Ebselen and 6-SeCD,
6-CySeCD is a better GPX mimic, as evidenced by its
enzymatic properties and protection of mitochondria.
These studies show that there are two key factors for
improving catalytic efficiency of GPX mimics. First,
the substrate-binding site should match the size and
shape of the substrates, and second, incorporation of
an imido-group increases the stability of transition
state selenolate in the catalytic cycle. We believe that
this is significant when designing mimics with high cat-
alytic efficiency.
Experimental procedures
Apparatus and materials
Structural characterization of 6-CySeCD was performed
with a IFS-FT66V infrared spectrometer (Bruker, Bremen,
Germany), a Varian Unity-400 NMR spectrometer (Varian
Fig. 4. (A) Dependence of extent of TBARS accumulation on the con-

centration of 6-CySeCD. (a) Control; (b) damage + 20 l
M 6-CySeCD;
(c) damage + 10 l
M 6-CySeCD; (d) damage + 4 lM 6-CySeCD; (e)
damage. (B) Effect of different GPX mimics on TBARS accumulated
during mitochondrial damage. (a) Control; (b) damage + 10 l
M
6-CySeCD; (c) damage + 10 lM 6-SeCD; (d) damage + 10 lM Ebse-
len; (e) damage. Relative TBARS content calculated based on
amount of TBARS for 50 min with damage group ¼ 1.
S W. Lv et al. 6-CySeCD displays GPX activity
FEBS Journal 274 (2007) 3846–3854 ª 2007 The Authors Journal compilation ª 2007 FEBS 3851
Inc, Palo Alto, CA) and a Perkin-Elmer 240 DS elemental
analyzer (Wellesley, MA). The content and valence of
selenium in the 6-CySeCD were determined by using an
ESCALAB MKII X-ray photoelectron spectrometer
(VG Scientific, Sussex, UK). Spectrometric measurements
were carried out by using a Shimadzu UV-2550 spectropho-
tometer (Kyoto, Japan).
b-CD was purchased from Shanghai Sanpu Chemical
Plant (Shanghai, China), recrystallized three times from
water and dried for 12 h at 120 °C in vacuum. Sodium
borohydride, selenium, BHT and 1,3-benzene-disulfonyl
chloride were obtained from Sigma (St Louis, MO). GSH,
glutathione reductase, t-BuOOH, CumOOH, and NADPH
were also obtained from Sigma. Sephadex G-25 was pur-
chased from Pharmacia (Uppsala, Sweden). All the other
materials were of analytical grade and obtained from
Beijing Chemical Plant (Beijing, China).
Synthesis of 6-CySeCD

Two grams of 6A,6B-diiodo-6A,6B-dideoxy-b-cyclodextrin
[28] was dissolved in 30 mL of dry dimethylformamide, and
185 lL of cyclohexylamine was added. The mixture was
stirred at 45 °C for 4 h, dimethylformamide was evaporated
under reduced pressure. The residue was dissolved in
45 mL potassium phosphate buffer (50 mm, pH 7.0) and
30 mL dimethylformamide (cosolvent). Then 7 mL of 1 m
sodium hydroselenide (NaHSe), prepared according to the
procedure of Klayman and Griffin [29] was added under
the pure nitrogen. The mixture was kept under nitrogen for
36 h at 60 °C, oxidized in air and finally purified by centrif-
ugation and Sephadex G-25 column (U 2 · A60 cm) chro-
matography (k ¼ 254 nm) with distilled water as the eluent.
The resulting solution was freeze-dried and the lyophilized
powder was washed with ethyl ether three times and dried
under vacuum to obtain a light yellow, pure sample with
34% yield. The structure of 6-CySeCD was analyzed by
means of elemental analysis, IR,
13
C NMR. The content
and valence of selenium in the 6-CySeCD was determined
by X-ray photoelectron spectroscopy. The energy of the
exciting X-ray was 1253.6 eV (Mg, Ka). C
1s
¼ 285.0 eV
served as standard. Scans were performed 12 times.
Determination of GPX-like activity and kinetics
Catalytic activities were determined by the method of Wil-
son et al. [11]. The reaction was carried out at 37 °Cin
700 lL of solution containing 50 mm, pH 7.0, potassium

phosphate buffer, 1 mm EDTA, 1 mm sodium azide, 1 mm
GSH, 0.25 mm NADPH, 1 unit glutathione reductase,
5 lm 6-SeCD and 6-CySeCD. The reaction was initiated by
addition of 0.5 mm hydroperoxide. Organic hydroperoxides
(t-BuOOH, CumOOH) were dissolved in 0.2% (v ⁄ v) Tri-
ton X-100, which was the cosolvent and did not affect the
GPX-like activity assay. Activity was determined by the
decrease of NADPH absorption at 340 nm (e
NADPH
¼
6220 m
)1
cm
)1
). Background absorption of the noncatalytic
reaction was run without mimic and was subtracted. The
activity unit of enzyme mimic was defined as the amount of
enzyme mimic, which utilizes 1 lmol NADPH per min.
The assay of 6-CySeCD kinetics was similar to that for
native GPX [30]. Initial reduction rates of H
2
O
2
by GSH
were determined by observing the change in NADPH
absorption at 340 nm at 37 °C and pH 7.0, varying one
substrate concentration while another is fixed. All kinetic
experiments were performed at 37 °Cin700lL of reaction
solution containing 0.5–3.0 mm GSH, 0.5–2.0 mm H
2

O
2
,
50 mm potassium phosphate buffer (pH 7.0), 1 mm EDTA,
0.25 mm NAPDH, 1 unit GSH reductase and 5 lm
6-CySeCD. Background absorption of the noncatalytic
reaction was run without mimic and was subtracted.
Kinetic data were analyzed by double-reciprocal plotting.
Preparation of mitochondria
Bovine heart mitochondria were isolated from fresh bovine
heart [31] and suspended in 0.25 m sucrose, 10 mm EDTA
and 25 mm Hepes-NaOH buffer, pH 7.4, and maintained
on ice. The concentration of the mitochondrial protein was
determined by Coomassie Brilliant Blue [32] using BSA as
the standard.
Ferrous sulfate
⁄
ascorbate-induced mitochondrial
damage
Mitochondria (0.5 mg proteinÆmL
)1
) suspended in medium
(0.125 m KCl, 1 mm MgCl
2
,5mm glutamate, 1 mm GSH,
10 mm potassium phosphate buffer, pH 7.4) were subjected,
in the absence and presence of the mimic, to oxidative
stress generated by 0.5 mm ascorbate plus 12.5 lm ferrous
sulfate at 37 °C. Damage experiments were carried out
without mimic and known as the damage group; the experi-

ment carried out without the mimic, ascorbate, and ferrous
sulfate was known as the control group.
Biological analysis of mimics against
mitochondrial damage
Mitochondrial swelling was assayed as described by Hunter
et al. [33]. Mitochondrial swelling was measured by the
decrease in the turbidity of the reaction mixture at 520 nm.
The decrease in absorbance indicated an increase in mito-
chondrial swelling and a decrease in mitochondria integrity.
TBARS content in ferrous sulfate ⁄ ascorbate-treated
mitochondria was analyzed by thiobarbituric acid assay
[34]. In this assay, thiobarbituric acid reacts with malonal-
dehyde and ⁄ or other carbonyl by-products of free-radical-
mediated lipid peroxidation to give 2 : 1 (mol ⁄ mol) colored
conjugates, which have an A
532
value.
6-CySeCD displays GPX activity S W. Lv et al.
3852 FEBS Journal 274 (2007) 3846–3854 ª 2007 The Authors Journal compilation ª 2007 FEBS
Acknowledgements
This research was supported by Natural Science Foun-
dation of China (Project no. 20572035 and 20534030)
and Jilin University, Changchun, China.
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