Báo cáo khoa học: Irreversible cross-linking of heme to the distal tryptophan of stromal ascorbate peroxidase in response to rapid inactivation by H2O2 ppt

Irreversible cross-linking of heme to the distal tryptophan
of stromal ascorbate peroxidase in response to rapid
inactivation by H
2
O
2
Sakihito Kitajima
1
, Taise Shimaoka
2
, Miyo Kurioka
1
and Akiho Yokota
3
1 Graduate School of Science and Technology, Kyoto Institute of Technology, Japan
2 Research Institute of Innovative Technology for the Earth (RITE), Kyoto, Japan
3 Graduate School of Biological Science, Nara Institute of Science and Technology (NAIST), Nara, Japan
Ascorbate peroxidase (APX; EC 1.11.1.11) isoforms of
chloroplasts play a central role in scavenging reactive
oxygen species such as O
2
–Æ
and H
2
O
2
, which are gen-
erated in large amounts by photosystems when there is
an energy surplus. Chloroplasts of higher plants have
two APX isoforms, one localized in the stroma and
the other bound to the stromal side of the thylakoid

membrane [1]. APX first reacts with one molecule of
H
2
O
2
and forms a porphyrin-based (compound I) and
then a protein-based (decay product of compound I
[2]) radical intermediate. The intermediates are then
reduced back to the resting state through compound II
by interaction with two molecules of ascorbate.
Paradoxically, if an excess of H
2
O
2
is produced in
chloroplasts as a result of oxidative stress such as
drought or intense light, they are rapidly inactivated
because the reaction intermediate of the APX is attacked
by excess H
2
O
2
instead of being reduced by ascorbate
[1,3,4]. As a result of the inactivation of APX, H
2
O
2
con-
tinues to accumulate, resulting in cell damage. In vitro
experiments reveal that, under conditions of ascorbate

depletion, inacti vation of APX occurs w ithin m inutes [5,6].
Other APX isoforms are localized in the cytosol [7]
and microbodies [8] of plants. These other isoforms
and cytochrome c peroxidase (CCP) [9], a yeast homo-
log of APX, are much more tolerant to H
2
O
2
than
chloroplast APXs, although they have similar amino-
acid sequences and structures [10].
The aim of this study was to determine why chloro-
plast APXs are more sensitive to H
2
O
2
than other per-
oxidases. Using a chimera of stromal APX and red
algal H
2
O
2
-tolerant APX, we previously showed that a
unique loop structure near the catalytic site is involved
in the rapid inactivation of stromal APX [6]. In the
present study, we examined the structural change asso-
ciated with inactivation by H
2
O
2

.
Keywords
ascorbate peroxidase; chloroplast; cross-link;
hydrogen peroxide; inactivation
Correspondence
S. Kitajima, Graduate School of Science and
Technology, Kyoto Institute of Technology,
Sakyo-ku, Kyoto 606-8585, Japan
Fax: +81 75 724 7762
Tel: +81 75 724 7791
E-mail:
(Received 1 February 2007, revised 22
March 2007, accepted 16 April 2007)
doi:10.1111/j.1742-4658.2007.05829.x
Ascorbate peroxidase (APX) isoforms localized in the stroma and thyla-
koid membrane of chloroplasts play a central role in scavenging reactive
oxygen species generated by photosystems. These enzymes are inactivated
within minutes by H
2
O
2
when the reducing substrate, ascorbate, is deple-
ted. We found that, when the enzyme is inactivated by H
2
O
2
, a heme at
the catalytic site of a stromal APX isoform is irreversibly cross-linked to a
tryptophan residue facing the distal cavity. Mutation of this tryptophan to
phenylalanine abolished the cross-linking and increased the half-time for

inactivation from < 10 to 62 s. In contrast with H
2
O
2
-tolerant peroxidases,
rapid formation of the cross-link in APXs suggests that a radical in the
reaction intermediate tends to be located in the distal tryptophan so that
heme is easily cross-linked to it. This is the first report of a mutation that
improves the tolerance of chloroplast APXs to H
2
O
2
.
Abbreviations
APX, ascorbate peroxidase; CCP, cytochrome c peroxidase; tsAPX, stromal APX from tobacco.
FEBS Journal 274 (2007) 3013–3020 ª 2007 The Authors Journal compilation ª 2007 FEBS 3013
Results
Irreversible cross-linking of heme to apoprotein
in H
2
O
2
-inactivated stromal APX
We produced recombinant stromal APX from tobacco
(Nicotiana tabacum) (tsAPX) in Escherichia coli and
examined its elution on RP-HPLC before or after
treatment for 25 s with 20 mol H
2
O
2

, which reduced
the activity by  80% (Fig. 1). Separate peaks for
heme (18.2 min) and apoprotein (25.4 min) were
observed for untreated tsAPX (Fig. 2A), whereas signi-
ficant amounts of heme were coeluted with the apo-
protein (broad peaks at 26.4 and 27.0 min) when
tsAPX was treated with H
2
O
2
(Fig. 2B). MALDI-
TOF-MS of the H
2
O
2
-treated enzyme revealed a peak
for the apoprotein ( 32 720 m ⁄ z) and a second peak
( 33 360 m ⁄ z) roughly corresponding to apoprotein
plus heme (616.48 Da) and oxygen (Fig. 2E). The mass
measurement error in these experiments was less than
250 p.p.m. Although part of tsAPX was polymerized
by treatment with H
2
O
2
, detectable by SDS ⁄ PAGE
(data not shown) as indicated in CCP [11], the poly-
mers were not detectable by MALDI-TOF-MS because
of low sensitivity in the higher mass range. These
results indicate that, when inactivated by H

2
O
2
, heme
is irreversibly cross-linked to the APX apoprotein.
Identification of the heme-binding amino-acid
residue in H
2
O
2
-inactivated stromal APX
To determine which amino-acid residue in the apopro-
tein was cross-linked to heme, the inactivated tsAPX
was digested with trypsin and subjected to RP-HPLC.
We found at least four peaks that absorbed at 400 nm
(38.0, 38.9, 40.5 and 50.9 min; Fig. 3A). Because the
retention time for the product eluted at 50.9 min was
0
20
40
60
80
100
remaining activity (%)
0 30 60 90 120 150 180
time (sec)
tsAPXW35F, – H
2
O
2

tsAPXW35F, + H
2
O
2
tsAPX, – H
2
O
2
tsAPX, + H
2
O
2
Fig. 1. Remaining activities of tsAPX and tsAPXW35F treated with
H
2
O
2
. APXs were treated with or without 20 mol H
2
O
2
in O
2
-free
50 m
M sodium phosphate, pH 7.0, at 25 °C. The concentration of
treated and untreated APXs was 1.9 l
M. Results are mean ± SD
from five measurements.
Fig. 2. Inactivation and cross-linking of APXs by H

2
O
2
. APXs were
untreated (A,C) or treated (B,D) with 20 mol H
2
O
2
in O
2
-free 50 mM
sodium phosphate, pH 7.0, at 25 °C. Enzymes were separated by
HPLC on a C4 reversed-phase column. Protein and heme were
detected at 220 nm (thin line) and 400 nm (thick line), respectively.
(A) 2.0 l
M untreated tsAPX; (B) 1.9 lM tsAPX treated with H
2
O
2
for
25 s; (C) 2.1 l
M untreated tsAPXW35F; (D) 2.1 lM tsAPXW35F
treated with H
2
O
2
for 120 s; (E) MALDI-TOF-MS spectra of tsAPX
treated with or without 5 mol H
2
O

2
for 2 min. The mass measure-
ment error was less than 250 p.p.m. mAU, Milliabsorbance units.
H
2
O
2
-mediated inactivation of stromal APX S. Kitajima et al.
3014 FEBS Journal 274 (2007) 3013–3020 ª 2007 The Authors Journal compilation ª 2007 FEBS
identical with that for a commercial sample of hemin
(data not shown), we concluded that it represented a
free heme species. The mass spectrum of the most
abundant peak (38.0 min; Fig. 3A, inset) had a
[M +H]
+
ion at 1892.8 m ⁄ z, and its MS ⁄ MS spec-
trum indicated that it was a peptide with the sequence
HDAGTYNK (Fig. 3B). This matches a predicted
tryptic peptide corresponding to residues 33–43
(LGWHDAGTYNK) (Fig. 4A), although Leu33,
Gly34, and Trp35 could not be assigned in the MS ⁄ MS
spectrum. Of these three residues, Trp35 is the only
reactive residue and therefore the most likely to cross-
link to the heme. The 1892.8 m ⁄ z value obtained was
15.7 higher than the sum of the calculated masses for
the protonated peptide LGWHDAGTYNK (1260.67)
plus heme (616.48), suggesting incorporation of an oxy-
gen atom. We could not identify the two other minor
peaks (38.9 and 40.5 min). The side chain of Trp35
faces the distal cavity formed by the heme and N-ter-

minal half of the apoprotein of the catalytic site
(Fig. 4B). Because the indole ring of Trp35 is 3.18 A
˚
from the porphyrin ring in tsAPX (the distance
between nitrogen of the indole ring and C6 of the por-
phyrin ring) [10], the heme must move toward Trp35 to
form a covalent bond. In active cytosolic APX, ascor-
bate binds to the c-meso edge of heme (a propionated
side of heme) [12]. The loss of ascorbate-oxidizing
0
100
200
300
400
500
600
700
800
15 20 25 30 35 40 45 50 55
time (min)
mAU (220 nm)
0
100
200
300
400
500
600
mAU (400 nm)
A

38.0 min
38.9 min
40.5 min
50.9 min
0
100
200
300
400
500
600
700
240 280 320 360 400 440
nm
mAU
38.0 min
LGW(O-heme) (987.3)LGW(O-heme) (987.3)LGW(O-heme) (987.3)
B
LGW(O-heme) (987.3)
Fig. 3. Identification of the amino-acid resi-
due cross-linked to heme in H
2
O
2
-treated
tsAPX. tsAPX was treated with 20 mol
H
2
O
2

at 25 °C for 20 s and then digested
with trypsin. (A) C18 RP-HPLC. Peptide and
heme were detected at 220 nm (thin line)
and 400 nm (thick line), respectively. Inset,
UV ⁄ Vis spectra of the product eluted at
38.0 min. (B) MS ⁄ MS spectrum of the
product eluted at 38.0 min. The peptide
sequences obtained from b and y fragment
ions are indicated. mAU, Milliabsorbance
units.
Distal side
Proximal side
Trp35
Distal side
Proximal side
Trp35
A
B
Fig. 4. Deduced amino-acid sequence of tsAPX (A) and structure of
its catalytic site [10] (B). The sequence of the tryptic peptide identi-
fied from the MS ⁄ MS spectrum in Fig. 3B is underlined.
S. Kitajima et al. H
2
O
2
-mediated inactivation of stromal APX
FEBS Journal 274 (2007) 3013–3020 ª 2007 The Authors Journal compilation ª 2007 FEBS 3015
activity in the cross-linked form of tsAPX may there-
fore be due to the repositioning of heme, preventing it
from interacting with ascorbate.

Effect of Trp35 mutation
To investigate the role of Trp35 in the cross-link and
the inactivation of APX by H
2
O
2
, we created a mutant
form of tsAPX in which Trp35 was changed to phenyl-
alanine (tsAPXW35F). We found that tsAPX in
O
2
-free 50 mm sodium phosphate, pH 7.0, had a
Soret band at 404 nm (e
404
¼ 105 mm
)1
Æcm
)1
) and
a shoulder around 380 nm (Fig. 5A). This is similar
to the spectrum for yeast CCP, which has a five-
coordinated high-spin ferric heme [13]. The spectrum
of tsAPXW35F, however, lacked the shoulder around
380 nm (Fig. 5B) and had a more intense Soret band
(e
405
¼ 122 mm
)1
Æcm
)1

), which is typical of a six-
coordinated ferric heme, as found in the CCP mutant
[13,14]. Spectra for these two APXs in the visible
region were similar, and both had two charge-transfer
bands (Fig. 5A,B), which is characteristic of high-spin
heme species. This is slightly different from a similar
mutant of cytosolic APX from soybean reported by
Badyal et al. [15]; specifically, when the corresponding
tryptophan was changed to alanine in soybean cytoso-
lic APX, a peak appeared at 564 nm, which is charac-
teristic of low-spin heme species. Furthermore, the
spectra for the six-coordinated low-spin ferric forms,
prepared by treatment with KCN, were similar for
tsAPX and tsAPXW35F (Fig. 5A,B). Finally, the K
m
and k
cat
values for tsAPXW35F were only slightly dif-
ferent from those for tsAPX (Table 1). These results
suggest that, except for a difference in the coordination
of the distal side of the heme ferric atom, the W35F
mutation did not cause a significant change in the
structure of tsAPX.
Next, we examined the effects of the mutation on
the interaction of tsAPX with excess H
2
O
2
. In con-
trast with tsAPX, a cross-link was not observed in

tsAPXW35F, even when it was treated with 20 mol
H
2
O
2
for 120 s (Fig. 2C,D). This agrees with the
MS ⁄ MS results showing that Trp35 is the most likely
site for cross-linking to heme. This also indicates that
the tryptic peptides of two unidentified minor peaks
eluted at 38.9 and 40.5 m (Fig. 3A) are probably parti-
ally digested products containing Trp35 cross-linked to
heme.
In the presence of 20 mol H
2
O
2
, tsAPXW35F had a
half-time of inactivation of 62 s, which is more than
6.2-fold longer than for tsAPX (< 10 s) (Fig. 1). We
obtained similar results when we included excess BSA
in the reaction to exclude the possible effect of con-
taminating apoprotein (data not shown). These results
strongly support the idea that the formation of the
cross-link is at least part of the reason for the rapid
inactivation of tsAPX.
In these experiments, tsAPXW35F was inactivated
in 3 min by H
2
O
2

. Whether this reflects other aspects
of the inactivation mechanism for tsAPX is not clear;
it is possible that tsAPXW35F is inactivated by a
distinct mechanism because the heme can no longer
be cross-linked to the enzyme. The possibility of a
distinct mechanism of inactivation is supported by the
difference in the spectral changes for H
2
O
2
-treated
tsAPXW35F and tsAPX (Fig. 6A,B).
Discussion
In these studies, we showed that heme cross-links to
the distal tryptophan in tsAPX within minutes when
Fig. 5. Absorption spectra of tsAPX (A) and tsAPXW35F (B) treated
with or without 0.2 m
M KCN in O
2
-free 50 mM sodium phosphate,
pH 7.0.
Table 1. Steady-state kinetic parameters of APXs.
K
m(Asc)
a
(lM)
K
mðH
2
O

2
Þ
b
(lM)
k
cat
(s
)1
Æheme
)1
)
tsAPX
c
395 ± 27 21.7 ± 1 2510 ± 90
tsAPXW35F 170 ± 7 93.9 ± 0.9 3410 ± 130
a
K
m
for ascorbate;
b
K
m
for H
2
O
2
;
c
Kitajima et al.[6].
H

2
O
2
-mediated inactivation of stromal APX S. Kitajima et al.
3016 FEBS Journal 274 (2007) 3013–3020 ª 2007 The Authors Journal compilation ª 2007 FEBS
treated with H
2
O
2
in the absence of ascorbate. On the
basis of these results, we propose that the rapid inacti-
vation of tsAPX is at least partly due to repositioning
of heme caused by cross-linking between heme and the
distal tryptophan resulting from reaction with H
2
O
2
.
Recently, Pipirou et al. [16] reported that part of the
heme molecule is cross-linked to the distal tryptophan
in the cytosolic APX isoform when it reacts with
excess H
2
O
2
in the absence of ascorbate. They pro-
posed that a vinyl group of heme is bound to C1 of
the distal tryptophan and hydroxylated. The cross-link
between heme and tryptophan may occur in a similar
way in tsAPX. In a H

2
O
2
-tolerant APX isoform from
red algae [6,17], excess H
2
O
2
also causes cross-linking
between heme and the apoprotein, but the ratio of
cross-linked heme to total heme was lower than in
tsAPX (data not shown). In CCP of yeast, the distal
tryptophan is also conserved, but it has not been
reported to cross-link to heme. Thus, H
2
O
2
-mediated
cross-linking in perxoxidases other than tsAPX, if it
occurs, may be much slower.
Why the heme rapidly forms a cross-link in tsAPX
is uncertain. In CCP, a proximal tryptophan residue
distant from the porphyrin is a major radical site in
its reaction intermediate [18]. In addition, when its
reducing substrate, cytochrome c, is absent, the rad-
ical is transferred to, oxidizes, and disrupts trypto-
phan and tyrosine residues distant from the heme
[19–21]. Also, in cytosolic APX of pea [2] and in
fungal lignin peroxidase [22], when the reducing sub-
strate is absent, the radical is thought to transfer

from porphyrin to tryptophans far from heme,
resulting in their hydroxylation. The relocation of
the radical means that these amino-acid residues
directly or indirectly donate electrons as endogenous
reducing substrates to the porphyrin radical, protect-
ing the enzyme from over-oxidation by excess H
2
O
2
.
The cross-linking of Trp35 to heme of tsAPX thus
suggests that the radical in the reaction intermediate
is located in porphyrin and Trp35, but that reloca-
tion to residues distant from heme, if it occurs, is
much slower than in other peroxidases. As a result,
the cross-linking of heme may occur readily in
tsAPX.
In bifunctional catalase–peroxidase, a bacterial
homolog of APX, a radical is transferred from por-
phyrin to another tryptophan residue that is connec-
ted to a propionate side chain of porphyrin by a
hydrogen-bonding network through two water mole-
cules [23]. The interaction of the propionate side
chain with the protein is different for tsAPX and
cytosolic APX, because of a unique 16-amino-acid
stretch [10] that confers higher sensitivity to H
2
O
2
[6].

A change in the interaction of the propionate with
amino-acid residues may therefore influence transfer
of the radical in the reaction intermediate of tsAPX.
Theoretically, the propionate side chain is also
involved in electron transfer from amino-acid residues
to the porphyrin [24].
The tendency of the radical to remain near the heme
may allow a more rapid catalytic turnover, although at
the expense of tolerance to H
2
O
2
. This might have
been evolutionary pressure on the chloroplast APXs.
In fact, the specific activities of chloroplast APXs
reported to date are much higher than those of the
cytosolic APXs [25–30].
In conclusion, we have shown that the rapid inacti-
vation of tsAPX is at least partly due to cross-linking
between heme and the distal tryptophan as a result of
reaction with H
2
O
2
. Given the amino-acid sequence
similarity between stromal and thylakoid-bound APXs
(reviewed in [31]), the inactivation mechanism pro-
posed here should also be relevant for thylakoid-bound
APX.
Absorbance

Absorbance
Absorbance
Absorbance
0.005
0.01
0.015
0.02
0.025
0.03
0.05
0.1
0.15
0.2
0.25
A
0
0.05
0.1
0.15
0.2
0
0.005
0.01
0.015
0.02
00
B
untreated
untreated
300 350 400 450 500 550 600 650 700

nm
untreated
untreated
Fig. 6. Spectral change in tsAPX and tsAPXW35F treated with
20 mol H
2
O
2
. APXs were solubilized in O
2
-free 50 mM sodium
phosphate, pH 7.0, and then treated with H
2
O
2
. (A) tsAPX (2.1 lM)
before treatment and 3, 9, 18, 60, and 120 s after addition of
H
2
O
2
. (B) tsAPXW35F (1.9 lM) before treatment and 3, 60, 120,
180, and 300 s after the addition of H
2
O
2
. Spectral changes were
monitored at 22 °C using a photodiode array spectrophotometer.
S. Kitajima et al. H
2

O
2
-mediated inactivation of stromal APX
FEBS Journal 274 (2007) 3013–3020 ª 2007 The Authors Journal compilation ª 2007 FEBS 3017
Experimental procedures
Preparation of recombinant APXs
Expression plasmids for tsAPX were constructed as des-
cribed previously [6]. Trp35 was mutated by PCR-mediated
site-directed mutagenesis. The expression plasmids encoded
APXs corresponding to residues 92–386 of accession num-
ber AB022274, with methionine and glycine residues at the
N-terminus, which are not present in the native enzyme.
Recombinant APXs produced in E. coli BL21(DE3) were
purified by sequential steps of chromatography on HiPrep
16 ⁄ 10 DEAE FF (Amersham Bioscience, Piscataway, NJ,
USA), HiLoad 16 ⁄ 10 Phenyl Sepharose HP (Amersham
Bioscience), and HiLoad 16 ⁄ 60 Superdex 75 pg (Amersham
Bioscience) as previously described [6]. Purified tsAPX and
tsAPXW35F appeared as single bands when separated by
SDS ⁄ PAGE (data not shown).
O
2
-free APX solution was prepared by passing APX in
10 mm potassium phosphate, pH 7.0, 1 mm EDTA, 1 mm
ascorbate, and 0.15 m KCl through two Sephadex G25 col-
umns (NAP5 and PD10 columns; Amersham Bioscience)
and elution with 50 mm sodium phosphate, pH 7.0, that
had been degassed by bubbling with N
2
gas. Before analy-

sis, the concentration of APX was determined from the
absorption of heme.
The absorption coefficients of the Soret peak for tsAPX
and tsAPXW35F were 105 [6] and 122 mm
)1
Æcm
)2
, respect-
ively. The value for tsAPXW35 was determined according
to the heme content and UV ⁄ Vis absorption spectra. Heme
contents were determined by the pyridine hemochromogen
method [32] with horseradish peroxidase (Nacalai tesque,
Kyoto, Japan) as a standard (e ¼ 100 mm
)1
Æcm
)1
at 403
nm [33]). The heme contents per tsAPX and tsAPXW35F
molecule were  70% and 80%, respectively, indicating that
 30% and 20% were the apoenzyme.
Enzyme assay
APX activity was measured as described previously [6],
except that the reaction mixture was supplemented with
0.01 mgÆmL
)1
BSA for the experiment in Fig. 1E. The K
m
values for ascorbate and H
2
O

2
and the k
cat
values were
determined as described previously [6].
HPLC and MS
HPLC analysis was performed using an LC-VP HPLC
system (Shimadzu, Kyoto, Japan) equipped with a SPD-
M10AVP photodiode array UV-Vis detector (Shimadzu).
The column was maintained at 40 °C. For treatment of
APXs with H
2
O
2
, 20 mol H
2
O
2
was manually added to
3.5 mL O
2
-free APX solution with stirring at 25 °C. The
reaction was terminated by adding 0.5 mm ascorbic acid.
H
2
O
2
and ascorbate were removed by passing the sample
through an Econopack 10DG column (Bio-Rad, Hercules,
CA, USA) that had been equilibrated with 50 mm sodium

phosphate, pH 7.0. For analysis of the heme–apoprotein
cross-link, APX was denatured by adding a half volume of
8 : 3 HCl ⁄ acetic acid before injection on to a C4 reversed-
phase column (4.6 · 250 mm; 5 lm; Vydac, Hesperia, CA,
USA). Protein and heme were separated by delivery of
35% acetonitrile and 0.1% trifluoroacetic acid for 14 min,
followed by a linear gradient of 35–45% acetonitrile over
15 min.
For MS of undigested APX, acid-denatured APX sample
was washed with a ZipTip C4 microcolumn (Millipore,
Bedford, MA, USA), and MALDI-TOF analysis was per-
formed on a Reflex III mass spectrometer (Bruker Dalto-
nics, Bremen, Germany) in linear mode using sinapic acid
(Fluka, Buchs, Switzerland) as the matrix. MALDI spectra
were externally calibrated using Protein Calibration Stand-
ard II (Bruker Daltonics).
For MS of trypsin-digested samples, inactivated APX in
50 mm sodium phosphate, pH 7.0, was precipitated with
acetone and dissolved in 4 m urea and 50 mm ammonium
bicarbonate, pH 8.0. Sequencing-grade trypsin (Promega,
Madison, WI, USA) was added to the solution at a molar
ratio of 1 : 50 and incubated at 37 °C for 6 h. The resulting
reaction mixture was separated on a C18 reversed-phase
column (4.6 · 150 mm; TSK gel ODS-100S; Toso, Tokyo,
Japan). Peptide and heme were separated by delivery of
0.1% trifluoroacetic acid for 10 min, followed by a linear
gradient of 0–10% acetonitrile over 15 min, a linear gradi-
ent of 10–40% acetonitrile over 30 min, and a linear gradi-
ent of 40–100% acetonitrile over 5 min. The peptide
fraction showing both heme and peptide absorbance was

isolated and concentrated by evaporation. The sample was
loaded on to a ZipTip C18 microcolumn (Millipore) and
eluted with 60% acetonitrile and 0.1% formic acid for ana-
lysis using a Q-TOF Ultima mass spectrometer (Waters
Co., Milford, MA, USA). MS and MS ⁄ MS data were
acquired and processed automatically using MassLynx 4.0
software (Waters Co.).
Acknowledgements
We thank Ms. Yuki Shinzaki for technical assistance.
This study was supported in part by the Research
Association for Biotechnology, which is subsidized by
the Ministry of Economy, Trade and Industry of
Japan.
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O
2
-mediated inactivation of stromal APX S. Kitajima et al.
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