Production and chemiluminescent free radical reactions of glyoxal in
lipid peroxidation of linoleic acid by the ligninolytic enzyme,
manganese peroxidase
Takashi Watanabe
1
, Nobuaki Shirai
2
, Hitomi Okada
1
, Yoichi Honda
1
and Masaaki Kuwahara
1
1
Laboratory of Biomass Conversion, Wood Research Institute, Kyoto University, Gokasho, Uji, Japan;
2
Industrial Research Center of Shiga
Prefecture, Ritto, Kamitoyama, Japan
Glyoxal is a key compound involved in glyoxal oxidase
(GLOX)-dependent production of glyoxylate, oxalate and
H
2
O
2
by lignin-degrading basidiomycetes. In this paper, we
report that glyoxal was produced from a metabolite of
ligninolytic fungi, linoleic acid, by manganese peroxidase
(MnP)-dependent lipid peroxidation. In the absence of the
parent substrate of linoleic acid, the dialdehyde was
oxidized by MnP and Mn(III) chelate to start free radical
reactions with emission of chemiluminescence at 700–

2
O
2
production [1–6]. As GLOX is activated by
peroxidases, the peroxidase-dependent lignin-degradation
can be controlled by the combination of GLOX and its
substrate, glyoxal [2,7]. Thus, the importance of glyoxal
oxidation in wood decay has been recognized. However,
little is known about the biosynthetic route for the extra-
cellular production of glyoxal by wood rot fungi. In this
paper, we first report that a ligninolytic enzyme, MnP, is able
to catalyze formation of glyoxal from a metabolite of wood
rot fungi, linoleic acid [8], by lipid peroxidation. The
glyoxal produced by MnP can be converted to glyoxylate
and oxalate by GLOX [6] and these carboxylic acids are
further oxidized by MnP or LiP/VA to yield O
2
†
–
and
CO
2
†
–
, which in turn reduce free radicals and transition
metals like Fe(III) [9–12]. Thus, the present result
highlights the new roles of MnP-dependent lipid peroxi-
dation in free radical chemistry of wood rot fungi.
In lipid peroxidation of USFAs, it has been reported that
Mn(II) reacts with a chain-carrying radical, peroxyl radical

, formate anion
radical; MnP, manganese peroxidase; LiP, lignin peroxidase;
HRP, horseradish peroxidase; 13(S)-HPODE,
13(S)-hydroperoxy-9Z,11E-octadecadienoic acid; SFA, saturated fatty
acid; USFA, unsaturated fatty acid; 2,6-DMP, 2,6-dimethoxyphenol;
ESR, electron spin resonance; MDA, malondialdehyde; MSTFA,
N-methyl-N-trimethylsilyltrifluroacetamide; DFB, decafluorobenzene;
GLOX, glyoxal oxidase, TBARS, thiobarbituric acid reactive
substances; PFBHA, O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine
hydrochloride; PFBO, pentafluorobenzyl oxime; CH
3
CN, acetonitrile;
MeOH, methyl alcohol; EtOH, ethyl alcohol; DM, n-dodecyl
b-maltoside; DHMA, dihydroxymaleic acid; EI/GC/MS, electron
ionization-gas chromatography-mass spectrometer; PAH, polycyclic
aromatic hydrocarbon.
Eur. J. Biochem. 268, 6114–6122 (2001) q FEBS 2001
without the aid of the other oxidizable substrates. We now
report the formation and chemiluminescent chain reactions
of glyoxal in MnP-dependent lipid peroxidation of linoleic
acid.
MATERIALS AND METHODS
General methods
Manganese (II) sulfate and 1,2,3-trimethoxybenzene,
decafluorobenzene, 1-dodecanal, 1-decanal, 2,4-nonadienal,
1-hexanal, 1-nonanal, 1-pentanal, 1-octanal, 1-butanal,
glyoxlic acid, glycol aldehyde, glyoxal were obtained
from Wako Pure Chemical Industries (Tokyo, Japan).
trans,trans-2,4-Decadienal, cis-4-decenal was obtained from
Aldrich Chemical Company (Milwaukee, USA). trans-2-

)
2
SO
4
and then purified by gel filtration on
Superdex 75 PG (1.6 Â 60 cm, Amersham Pharmacia
Biotech, Sweden) using 20 m
M sodium succinate buffer
containing 0.1
M NaCl as an eluent. Fractions showing MnP
activities were collected, and desalted with Centriprep
YM-30 (cut off, 30,000, Millipore, USA). MnP was further
purified by preparative IEF as described previously [15]
[pI 3.40, Reinheitzahl (RZ, A at l
max
/A 280) value: 3.0,
1.0 U ¼ 8.75 Â 10
211
mol]. Low molecular mass com-
pounds were removed by successive washings with Milli-
Q
TM
water with a Centricut N-10 ultrafiltration concentrator
(cut off, 10 000, Kurabo, Japan) before use. For the time
course experiments of aldehyde production, the enzyme
purified on Superdex 75 PG was desalted with distilled
water in Centricut N-10 and used without further
purification (15 U
:
mL

H
2
O
2
. Lipoxygenase activity was measured by O
2
uptake in
a reaction system containing 1. 5 m
M linoleic acid, 1 mM
n-dodecyl b-maltoside (DM) and 20 mM Tris/HCl buffer
(pH. 9. 0). One unit of lipoxygenase activity is defined as
the amount of enzyme that absorbs 1 mmol of O
2
in 1 min.
GLOX activity was measured by O
2
uptake in a
reaction system containing 3 m
M glyoxal in 20 mM sodium
tartrate (pH 3. 0), acetate (pH 4. 5) or phosphate (pH 6. 0)
buffers.
Electron ionization/gas chromatography/mass
spectrometetry (EI/GC/MS) analysis of oxidation
products by MnP
Linoleic acid and aldehydes were reacted with 250 mU of
the purified MnP, 0.5 m
M of Mn(II) and 50 mM of H
2
O
2

, and maintained at 250 8C for 20 min. The
time course of glyoxal production by MnP was analyzed as
described above after the reaction with and without linoleic
acid in formate and tartrate buffers. EI/GC/MS analyses of
authentic aldehydes and ketones were carried out using a
0.6-m
M methanol solution after derivatization with PFBHA
under the conditions described above. Tetramethylsilation
by N-methyl-N-trimethylsilyltrifluroacetamide (MSTFA)
was carried out as described previously [18].
Chemiluminescence measurements
Chemiluminescence was measured by an ultra-high sensi-
tive photon counter (ARGUS-50/VIM, Hamamatsu Photo-
nics, Hamamatsu, Japan) equipped with a charge-coupled
device (CCD) camera connected with an image intensifier
and ARGUS-50 image processor. The wavelength range of
the detector was 350–650 nm, 512 Â 483 pixels, and the
noise count was 0.15 c.p.s. The reactions were carried out in
a cuvette for a 96-well microplate reader. The conditions for
each experiment are described in the figure legends.
Inactivation of MnP was carried out by heating the MnP
in a boiling water bath for 10 min
The chemiluminescence spectra were measured by a
simultaneous multiwavelength analyzer CLA-SP2 (Tohoku
Electronic Industries Co. Ltd, Sendai, Japan) with an
q FEBS 2001 Production of glyoxal in lipid peroxidation by MnP (Eur. J. Biochem. 268) 6115
incident slit width of 1.0 mm. The wavelength range of the
spectrometer was 370–820 nm. Experimental conditions
are described in the legend of each figure.
RESULTS

MnP/Mn(II)/lipid system proceeds by complex radical
reactions involving the formation of unusual carbonyl
species. Tetramethylsilation with MSTFA did not change
the mass chromatogram at m/z 181 that originates from C–O
bond cleavage products of pentafluorobenzyl oxime [26]
(data not shown).
The reactions of MnP in four different buffers clearly
demonstrate that the formation of glyoxal was significantly
stimulated by the presence of tartrate. Therefore, the
reaction was carried out with and without linoleic acid in
sodium formate and tartrate buffers (Figs 1 and 3). GC/MS
analysis demonstrated that glyoxal was explosively
produced after 6 h in tartrate buffer containing linoleic
Fig. 1. Mass chromatograms of PFBO derivatives of products of lipid peroxidation by C. subvermipora MnP and soybean lipoxygenase at
m/z 181. (A) Products of the oxidation of linoleic acid by MnP in sodium acetate buffer for 19 h. The reaction system (500 mL) contained 3 m
M
linoleic acid, 500 mM MnSO
4
,50mM H
2
O
2
, 0.02% of Tween 20, 250 mU of purified MnP and 10 mM sodium acetate buffer (pH 4.5). (B) As (A) but
10 m
M sodium formate buffer (pH 4.5) was used instead of acetate buffer. (C), As (A) but 10 mM sodium lactate buffer (pH 4.5) was used instead of
acetate buffer. (D) As (A) but 10 m
M sodium tartrate buffer (pH 4.5) was used instead of acetate buffer. (E) As (B) but the reaction was carried out
without addition of linoleic acid. (F) As (D) but the reaction was carried out without addition of linoleic acid. (G) Products of the oxidation of linoleic
acid with soybean lipoxygenase. Linoleic acid (3 m
M) was reacted with soybean lipoxygenase (10 U) in 40 mM Tris/HCl buffer (pH 9.0) containing

and the Fenton reaction (Fig. 4). Lipoxygenase is an enzyme
that abstracts hydrogen from the bis-allylic position of
unsaturated fatty acids containing cis,cis-1,4-pentadienyl
moiety. In the reaction with linoleic acid, the fatty acid is
oxidized to yield a pentyl radical [27] and 12-oxododecyl-
cis-9-enoic acid [28] via b-scission of hydroperoxide
intermediates, leading to production of 1-hexanal [29] and
1-pentanal as shown in Fig. 1G. When linoleic acid was
oxidized by soybean lipoxygenase, emission of chemilumi-
nescence was close to the background level, both in the
presence and absence of Fe(II). In the Fenton system,
chemiluminescence was also below the background level,
except for a weak emission of light from linoleic acid after 2
days (Fig. 4).
In contrast to these oxidation systems, reactions of glyoxal
with MnP in tartrate buffer emitted strong chemilumines-
cence. As shown in Figs 5 and 6, intensive light emission
was observed immediately after the reaction started. The
photon emission reached a maximum (35 000 counts
:
h
21
)
within 30 min, and then decreased, but chemiluminescence
of < 9000 counts
:
h
21
was observed even after 1 h. In
lactate, formate and acetate buffers, the photon emission

M linoleic acid, 500 mM
MnSO
4
,50mM H
2
O
2
, 0.02% of Tween 20, MnP (250 mU) and 10 mM
sodium acetate buffer (pH 4.5). (B) Authentic standard of glyoxal.
* 1/10 of the original signal intensity.
Fig. 3. Time course of glyoxal formation by MnP. (A) Glyoxal formed by the reaction of linoleic acid with MnP in sodium tartrate buffer. The
reaction system (500 mL) contained 3 m
M linoleic acid, 500 mM MnSO
4
,50mM H
2
O
2
, 0.02% of Tween 20, MnP (250 mU) and 10 mM sodium
tartrate buffer (pH 4.5). (B) As (A) but linoleic acid was omitted. (C) As (A) but 10 m
M sodium formate buffer (pH 4.5) was used instead of sodium
tartrate bufer. (D) As (C) but linoleic acid was omitted.
q FEBS 2001 Production of glyoxal in lipid peroxidation by MnP (Eur. J. Biochem. 268) 6117
Fig. 4. Time course of light emission during
oxidation of linoleic acid by soybean
lipoxygenase (I) and the Fenton reaction (II).
(I): (A) The reaction system (200 mL) contained
4m
M linoleic acid, 10 U of lipoxygenase, 0.05%
of Tween 20, 10 m

250 mU of MnP, 500 m
M MnSO
4
, 0.2 mM H
2
O
2
,
0.05% of Tween 20 and 10 m
M sodium tartrate
buffer (pH 4.5). (B) As (A) but glyoxal was added
instead of linoleic acid. (C) As (A) but
trans-2-nonenal was added instead of linoleic acid.
(D) As (A) but 1-dodecanal was added instead of
linoleic acid. (E) As (A) but 1-hexanal was added
instead of linoleic acid. (F) As (A) but
2,4-nonadienal was added instead of linoleic acid.
(G) As (A) but MDA was added instead of linoleic
acid. (H) As (A) but linoleic acid was omitted.
Inset shows the time course of the reactions (A–H)
during 2.5 h.
Fig. 6. Chemiluminescence emitted by the
oxidation of glyoxal with MnP. (I): (A) The
reaction system (200 mL) contained 4 m
M glyoxal,
250 mU of MnP, 500 m
M MnSO
4
, 0.2 mM H
2

2
O
2
was
omitted. (D) As (A) but glyoxal was omitted. (E)
As (A) but inactivated MnP was used instead of
native MnP.
6118 T. Watanabe et al. (Eur. J. Biochem. 268) q FEBS 2001
observed with MDA, trans-2-nonenal and linoleic acid, but
the intenisity was less than 1/10 of that observed in the
oxidation of glyoxal. Light emissions from 1-dedecanal,
1-hexanal, 2,4-nonadienal were almost at the same level as
that observed without addition of linoleic acid/aldehyde. No
photon emission was observed in the reactions of these
oxidizable substrates with Mn(II)–tartrate complex (data
not shown). These results demonstrate that MnP catalyzes
oxidation of Mn(II) to Mn(III), which in turn reacts with
glyoxal to generate electronically excited species. Co-oxida-
tion of tartrate is essential to carry the chemiluminescent
chain reactions over several days.
Figure 8 shows emission spectra obtained by glyoxal
oxidation with (A) Mn(III)–lactate complex, and (B) MnP/
Mn(II)/H
2
O
2
in tartrate buffer. The spectra showed a broad
single peak with emission maxima at 700 and 710 nm,
respectively. Figure 2C is the chemiluminescence spectrum
of singlet oxygen formed by the reaction of HClO

MnP reached a maximum around day 4 and then gradually
decreased, coincident with the peroxidation of the fatty
acids. Thus, accumulated data supports the involvement of
MnP-dependent lipid peroxidation in wood decay by white
rot fungi.
With regard to the radicals produced in the lipid
peroxidation by MnP, we reported that MnP oxidized
linoleic acid to generate acyl radicals in acetate and tartrate
buffers in the presence of Mn(II) [15]. The formation of acyl
radicals strongly suggests that hydrogen abstraction from
aldehydes is involved in the chain propagation cycle of the
MnP-dependent lipid peroxidation. Therefore, aldehydes
formed by the MnP/linoleic acid/Mn(II)/H
2
O
2
reactions
were analyzed by EI/GC/MS after derivatization to PFBO
(Figs 1–3). The GC/MS analysis demonstrated that
oxidation of linoleic acid with MnP produced glyoxal,
1-hexanal and 1-pentanal. Time course experiments of the
MnP reactions showed that glyoxal was not formed on
initiation of the lipid peroxidation but after 6 h (Fig. 3). In
contrast to the oxidation of linoleic acid, the reaction of
MnP with 13(S)-HPODE selectively produced 1-hexanal,
indicating that the glyoxal formation is not catalyzed by the
direct breakdown of lipid hydroperoxide with MnP and
Mn(III) chelates.
In wood decay, a supply of extracellular hydrogen
peroxide is essential to initiate peroxidase-dependent free

As (A) but 2,4-nonadienal was added instead of
linoleic acid. (G) As (A) but MDA was added
instead of linoleic acid. (H) As (A) but linoleic acid
was omitted.Time course of the photon emission
from glyoxal is shown separately from that of the
other oxidizable compounds due to the difference
of emission intensity.
q FEBS 2001 Production of glyoxal in lipid peroxidation by MnP (Eur. J. Biochem. 268) 6119
reported that lignin peroxidase deomposed a b-O-4 lignin
model compound with production of glycol aldehyde, a
substrate of GLOX [6]. The glycol aldehyde formed by this
process was converted to oxalate with the production of 2.8
equivalent of H
2
O
2
. Therefore, they proposed a pathway
producing oxalate from the b-O-4 lignin model compound
via glyoxal and glyoxylate. However, there has been no
direct evidence for the glyoxal formation from lignin by the
LiP/GLOX system. The finding of glyoxal formation by
MnP-dependent lipid peroxidation indicates that MnP and
Mn(III) chelate part in the formation of glyoxal, leading to
the enzymatic production of glyoxylate, oxalate and H
2
O
2
by GLOX. Mn(III) stabilized by the former two carboxylic
acids can diffuse into the wood cell wall region. At the same
time, glyoxylate and oxalate are oxidized by Mn(III) to

2
formation generates hydroxyl radicals. Thus, MnP-
dependent lipid peroxidation provides the substrate of
GLOX to produce active oxygen species in combination
with redox cycle of transition metals.
Oxidation of glyoxal by MnP
In lipid peroxidation involving aldehyde oxidation, it has
been postulated that acyl radicals are formed from aldehydes
by hydrogen abstraction with radicals (X†) [22] or transition
metals [23] according to:
X† 1 RVCHO ! XH 1 RCO† ð1Þ
M
31
ðM
21
Þ 1 RCHO ! M
21
ðM
1
Þ 1 RCO† 1 H
1
ð2Þ
With regard to the light emission from acyl radicals, four
different pathways can be discussed (Fig. 9). As shown in
Fig. 9 (pathway 2) two acyl radicals can recombine to
produce a biacyl triplet [32]. The light emission reported in
excited biacyl compounds like biacetyl (l max at 515 and
560 nm) [32] is different from the emission spectra observed
in the MnP reactions. a-Oxidation of aldehydes via a
dioxetane intermediate also produces excited triplet

and 10 m
M sodium tartrate buffer (pH 4.5). (B) The reaction system
(500 mL) contained 3 m
M glyoxal, 0.02% of Tween 20, and 2 mM
Mn(III)–lactate. A Mn(III)–lactate solution (10 mM) was prepared by
dissolving 0.1 m mol of Mn(III)–acetate in 10 mL of 0.1
M sodium
lactate buffer (pH 4.5). The reaction was initiated by adding 100 mLof
this solution. Therefore, the final concentration of lactate in the reaction
system was 20 m
M. (C) The reaction was started by adding 1 mL of
30% H
2
O
2
and 3 mL of 10% NaClO solution. Scanning time for (A) (B)
(C) were 20, 15, and 5 min, respectively.
6120 T. Watanabe et al. (Eur. J. Biochem. 268) q FEBS 2001
chemiluminescence emission from acyl radical is a forma-
tion of triplet carbonyls from a-hydroxyperoxyl radicals
(Fig. 9- [3]) that has been reported in the oxidation of
aetaldehyde with xantine oxidase [33]. However, the
emission maximum of the chemiluminescence by this
mechanism was lower than 500 nm [33] (Fig. 9). Thus, the
MnP-dependent light emission from glyoxal at 700 nm is a
new chemical event difficult to explain by the excited
species from acyl radicals reported before.
As shown in Fig. 8, the reaction of Mn(III)–lactate with
glyoxal emitted the chemiluminescence similar to that
observed in the reactions of MnP. Therefore, it can be

Mn(III)–tartrate complex [35,36]. However, no special
attention was paid to the oxidation of tartrate itself in these
studies. This may be due to the understanding that the
reactivity of tartrate is too low to be involved in the free
radical reactions by Mn(III). For instance, Perez reported
that veratryl alcohol oxidation by LiP is not affected by the
presence of tartrate in the presence or absence of Mn(II) and
Mn(III) [37]. More recently, Collins reported that the rate
of 2,2
0
-azinobiz(3-ethylbenzo-6-thiazolinesulfonic acide)
(ABTS†
1
) reduction is enhanced by the presence of
malonate, glyoxylate and oxalate but no stimulating effects
of tartrate on the ABTS†
1
reduction was observed [38]. In
contrast, the results obtained in the present study clearly
indicate that tartrate itself was oxidized by MnP to produce
glyoxal (Figs 1–3), thereby assisting chain reactions of the
aldehyde accompanied by photon emission (Fig. 7,8). In
lipid peroxidation of linoleic acid by MnP, the consecutive
formation of aldehydes and acyl radicals was observed in
acetate and formate buffers as well as in tartrate buffer.
Therefore, we propose that the enzymatic process produces
counterpart compounds like tartrate to assist the chain
propagation reactions of acyl radicals in combination with
redox cycle of Mn(II)/Mn(III).
In conclusion, the first evidence for the production of

6106–6110.
Fig. 9. Formation of electronically excited
species from acyl radicals reported before
[28,30–32]. Aldehydes are oxidized by carbon-
centered radicals and metal salts to form acyl
radicals [22,23]. Triplet carbonyls, biacyl triplet,
and singlet oxygen are formed from the acyl
radicals [28,30–32]. Their chemiluminescence
spectra are distinguishable from the electronically
excited species formed by the reactions of glyoxal
with MnP/Mn(II)/H
2
O
2
or Mn(III) chelate.
q FEBS 2001 Production of glyoxal in lipid peroxidation by MnP (Eur. J. Biochem. 268) 6121
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