A selenium-containing single-chain abzyme with potent
antioxidant activity
Delin You
1
, Xiaojun Ren
1,2
, Yan Xue
1
, Guimin Luo
1
, Tongshu Yang
1
and Jiacong Shen
2
1
Key Laboratory of Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun, P. R. China;
2
Key Laboratory for Supramolecular Structure and Materials of Ministry of Education, Jilin University, Changchun, P. R. China
Reactive oxygen species (ROS) are products of normal
metabolic activities and are thought to be the cause of many
diseases. A selenium-containing single-chain abzyme 2F3
(Se-2F3-scFv) that imitates glutathione peroxidase has been
produced which has the capacity to remove ROS. To
evaluate the antioxidant ability of Se-2F3-scFv, we con-
structed a ferrous sulfate/ascorbate (Vc/Fe
2+
)-induced mito-
chondrial damage model system and investigated the
capacity of Se-2F3-scFv to protect mitochondria from oxi-
dative damage. Se-2F3-scFv markedly decreased mito-
chondrial swelling, inhibited lipid peroxidation, and

cells have evolved an enzymatic and nonenzymatic defense
system. The enzymatic antioxidant system is mainly
composed of glutathione peroxidase (GPX), catalase,
superoxide dismutase and thioredoxin peroxidase. The non-
enzymatic antioxidant system includes vitamin E, ascorbate,
glutathione (GSH) and uric acid. However, if the ROS
loading reaches a critical concentration, overwhelming the
antioxidative defense, oxidative damage to all cellular
components, such as DNA, proteins and lipids, eventually
occurs, resulting in ROS-mediated diseases [3–5]. Exam-
ples of such diseases are ischemia-reperfusion injury,
inflammation, age-related diseases, neuronal apoptosis,
cancer and cataract.
The individual antioxidant enzymes are located in
specific subcellular sites and reveal distinct substrate
specificity [6]. Superoxide dismutase is a metalloenzyme
that catalyzes the reduction of O
2
–•
to H
2
O
2
.H
2
O
2
produced by the reduction of O
2
–•

specifically in mouse epididymis, is interestingly selenium-
independent [17]. The mechanism by which cGPX cata-
lyzes the reduction of hydroperoxide has been extensively
investigated.
Because production of selenium-containing peroxidase
is extremely difficult by traditional genetic engineering,
attempts have been made to generate compounds that
imitate the enzymatic action of GPX. The strategies used
to generate GPX-like catalysts include chemical synthesis
of a model system and mutation of naturally occurring
enzyme by chemical or protein engineering [18–20]. Three
different strategies have been tested for chemically
synthesizing a GPX mimic: one in which the selenium
atom binds directly to a heteroatom such as nitrogen
Correspondence to G. Luo, Key Laboratory of Molecular
Enzymology and Engineering of Ministry of Education,
Jilin University, Changchun, P. R. China.
Fax: + 86 431 8923907, Tel.: + 86 431 8498974,
E-mail:
Abbreviations: ROS, reactive oxygen species; GSH, glutathione; GPX,
glutathione peroxidase; TBA, thiobarbituric acid; CCO, cytochrome c
oxidase; TBARS, thiobarbituric acid reactive substances.
(Received 20 April 2003, revised 6 July 2003,
accepted 22 August 2003)
Eur. J. Biochem. 270, 4326–4331 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03825.x
and generates the well-known GPX mimic, 2-phenyl-1,
2-benziososelenazol-3(2H)-one (Ebselen); a second in
which the selenium atom is not directly bound to the
heteroatom (N or O), but instead is located in close
proximity to it; and the third in which cyclodextrin is

O
2
were determined to be 8.27 and 47.2 °C,
respectively, similar to those of native GPX [29]. In this
study, we constructed a biological model of ROS-induced
mitochondrial damage to study the ability of Se-2F3-
scFv to protect mitochondria from oxidative damage. We
found it to be a potent antioxidant.
Materials and methods
Materials
GSH was obtained from Aldrich. Ebselen, glutathione
reductase (type III baker’s yeast) and NADPH (tetrasodium
salt) were obtained from Sigma. Thiobarbituric acid (TBA)
was obtained from Shanghai Second Reagent Plant,
Shanghai, China. Cytochrome c was obtained from Tianjin
Biochemical Plant (Tianjin, China). Hepes was from Fluka.
All other chemicals were of analytical grade.
Generation of Se-2F3-scFv
The expression vector pTMFscFv containing target genes
was constructed as described previously and transformed
into bacterial cells BL21 (coden plus). After isopropyl thio-
b-
D
-galactoside induction, the expressed amount of 2F3-
scFv proteins was 25–30% of total bacterial proteins. The
2F3-scFv proteins were purified and refolded into the active
form. Incorporation of selenium into 2F3-scFv protein by
chemical mutation resulted in the selenoenzyme Se-2F3-
scFv. The GPX activity of Se-2F3-scFv was determined by
the coupled coenzyme system. One unit of activity is defined

GSH,
25 m
M
Hepes/NaOH, pH 7.4) were subjected, in the
absence and presence of Se-2F3-scFv, to oxidative stress
generated by 50 l
M
Fe
2+
plus 2 m
M
ascorbate at 37 °C.
Damage experiments were carried out without Se-2F3-scFv
protein and known as the damage group; experiments
carried out without Se-2F3-scFv, ascorbate, and Fe
2+
were
known as the control group [32].
Measurement of lipid peroxidation
LipidperoxidationintheVc/Fe
2+
-induced mitochondrial
damage system was analyzed by the TBA assay. In this
assay, TBA reacts with malonaldehyde and/or other
carbonyl by-products of free-radical-mediated lipid per-
oxidation to give 2 : 1 (mol/mol) colored conjugates [33].
Before and during incubation with the different concentra-
tions of Se-scFv-2F3, a 1.0 mL aliquot was taken and
vortex-mixed with 1 mL 75% (w/v) trichloroacetic acid and
1 mL 0.5% (w/v) TBA in water. The assay mixtures were

potassium phosphate buffer, pH 7.4, contain-
ing 125 m
M
KCl, 1 m
M
MgCl
2
,and5m
M
glutamate. Then
it was suspended in a small amount of 100 m
M
potassium
phosphate buffer, pH 7.0, and an aliquot was taken for
assay of CCO activity [35]. The CCO activity was measured
in 2 mL of the reaction system, in which the cytochrome c
concentration was 15 l
M
. The absorbance was decreased
Ó FEBS 2003 Se-containing abzyme with potent antioxidant activity (Eur. J. Biochem. 270) 4327
with oxidation of cytochrome c in the sample cell, into
which 5 lL10m
M
K
3
Fe(CN)
6
was added to oxidize
cytochrome c thoroughly when the reaction was completed.
The absorbance intensity at this time was recorded as A

Se-2F3. This activity is 2870 times that of the well-studied
GPX mimic Ebselen (PZ51). These results are similar to
previous reports [28,29].
Inhibition of lipid peroxidation by Se-2F3-scFv
The polyunsaturated fatty acid in mitochondrial membrane
is readily attacked by ROS, especially
•
OH produced by the
Fenton reaction, producing TBARS. TBARS therefore was
used to measure the extent of lipid peroxidation. TBA reacts
with malonaldehyde and/or other carbonyl by-products of
free-radical-mediated lipid peroxidation to give 2 : 1 (mol/
mol) colored conjugates [33], which have an A
532
value.
Bovine heart mitochondria exposed to (Fe
2+
plus
ascorbate)-induced oxidative stress are peroxidized in a
time-dependent manner as indicated by the formation of
TBARS from membrane lipids. Over 50 min, the amount of
TBARS accumulated in the damage group was between
2.40 ± 0.02 and 3.14 ± 0.03 nmol per mg protein and for
the control group it was between 2.03 ± 0.02 and 2.32 ±
0.02 nmol per mg protein. The increased TBARS in the
damage group was 2.2-fold higher than that in the control
group.
Figure 1 shows that Se-2F3-scFv effectively protects
membrane lipids from Fe
2+

integrity.
The A
520
for the control group remained basically
constant, whereas that for the damage group decreased
considerably with time, indicating that the Fe
2+
/Vc-induced
damage resulted in extensive mitochondrial swelling. The
reason for the swelling is that H
2
O
2
produced by Fe
2+
/Vc is
converted into
•
OH by the Fenton reaction, which initiates
Table 1. GPX activity of Se-2F3-scFv, Se-2F3, Ebselen and native GPX
from rabbit liver. GPX activity was assayed by the coupled coenzyme
system. Reactions were carried out in 50 m
M
potassium phosphate
buffer, pH 7.0, at 37 °C, 1 m
M
GSH, 0.5 m
M
H
2

M
) of Se-2F3-scFv protein were added, the mito-
chondrial swelling was apparently inhibited compared with
the damage group, and this was dependent on Se-2F3-scFv
concentration. Furthermore, the protection afforded by
Se-2F3-scFv was much greater than that by Ebselen at
8.00 l
M
(Fig. 2).
Protection of CCO activity in damaged mitochondria
CCO is one of the key redox enzymes in the electron-
transport chain of mitochondria and is also the marker
enzyme of mitochondria. The integrity of the mitochondrial
membrane is important for enzyme activity. Mitochondria
exposed to Fe
2+
/Vc-induced oxidative stress are peroxi-
dized, producing TBARS. The integrity of the mitochondria
therefore is destroyed, resulting in a decrease in CCO
activity. Over 60 min, CCO activity in the damage group
decreased from 0.356 ± 0.012 to 0.208 ± 0.010 U per mg
protein, i.e. by % 41.6%. Figure 3 shows that CCO
protection increased with increasing Se-2F3-scFv concen-
tration. When the Se-2F3-scFv concentration was 4.15 l
M
,
over 60 min, 90.2 ± 2.0% of CCO activity was retained;
for 8.00 l
M
Ebselen only 71 ± 2.5% of CCO activity was

O
2
and t-BuOOH
[41,42]. Another approach is to use a ROS-producing
system such as Fe
2+
/Vc or XO/HX [43,44]. The reactions for
Fe
2+
/Vc-induced mitochondrial damage are proposed to be
as follows:
Ascorbic acid þ 2Fe
3þ
! dehydroascorbic acid
þ 2Fe
2þ
þ 2H
þ
ð1Þ
Fe
2þ
þ H
2
O
2
! Fe
3þ
þ OH
À
þ

50mintoascorbate(2m
M
)/Fe
2+
(50 l
M
) in the presence of various
concentrations of Se-2F3-scFv at 37 °C. The extent of the swelling
was measured as described in Materials and methods. Data are
means ± SD (n ¼ three separate experiments).
Fig. 3. Effect of Se-2F3-scFv on CCO activity of mitochondria. Bovine
heart mitochondria were incubated for 60 min to ascorbate (2 m
M
)/
Fe
2+
(50 l
M
) in the presence of various concentrations of Se-2F3-scFv
at 37 °C. CCO activity was measured as described in Materials and
methods. Data are means ± SD (n ¼ three separate experiments).
Ó FEBS 2003 Se-containing abzyme with potent antioxidant activity (Eur. J. Biochem. 270) 4329
LOOH þ Fe
2þ
! LOO

þ Fe
3þ
ð6Þ
where L represents lipid compounds. Unsaturated

2
and LOOH would be an alternat-
ive approach to protecting cells from oxidative damage.
In many mitochondria, catalase is lacking [46]. Thus,
GPXs, including cGPX and PHGPX, play an import-
ant role in scavenging hydroperoxides. GPX mimics
with high activity can efficiently scavenge hydroper-
oxides, block lipid peroxidation, and protect mito-
chondria from oxidative damage (Eqn 7). In the living
organism, oxidized GSH (GSSG) produced in the first
step (Eqn 7) would be reduced to be GSH by GSH
reductase (Eqn 8).
ROOH þ 2GSH ÀÀÀÀ!
GPX
ROH þ GSSG þ H
2
O
2
ð7Þ
NADPH þ H
þ
þ GSSG ÀÀÀÀÀÀÀÀÀÀÀÀÀÀ!
Glutathione reductase
NADP
þ
þ2GSH ð8Þ
Se-2F3-scFv exhibited high GPX activity, efficiently
catalyzed the reduction of hydroperoxides by GSH, and
blocked lipid peroxidation. In the Fe
2+

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