Oxidation of xenobiotics by plant microsomes, a reconstituted cytochrome P450 system and peroxidase: A comparative study

Article abstract of DOI:10.1016/S0031-9422(00)00123-0

The microsomal fraction from tulip bulbs (Tulipa fosteriana, L.) contains cytochrome P450 (CYP³, EC 1.14.14.1) and peroxidase (EC 1.11.1.7.) enzymes catalyzing the NADPH - and hydrogen peroxide - dependent oxidation of the xenobiotic substrates, N-nitrosodimethylamine (NDMA), N- nitrosomethylaniline (NMA), aminopyrine and 1-phenylazo 2-hydroxynaphthalene (Sudan I), respectively. Oxidation of these model xenobiotics has also been assessed in a reconstituted electron-transport chain with a partially purified CYP fraction, phospholipid and isolated tulip NADPH:CYP reductase (EC 1.6.2.4.). Peroxidase isolated from tulip bulbs (isoenzyme C) oxidizes these xenobiotics, too. Values of kinetic parameters (K(m), V(max)), requirements for cofactors (NADPH, hydrogen peroxide), the effect of inhibitors and identification of products formed from the xenobiotics by the microsomal fraction, partially purified CYP and peroxidase C were determined. These data were used to estimate the participation of the CYP preparation and peroxidase C in oxidation of two out of the four studied xenobiotics (NMA, Sudan I) in tulip microsomes. Using such detailed study, we found that the CYP-dependent enzyme system is responsible for the oxidation of these xenobiotics in the microsomal fraction of tulip bulbs. The results demonstrate the progress in resolving the role of plant CYP and peroxidase enzymes in oxidation of xenobiotics. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0031-9422(00)00123-0

Phytochemistry 54 (2000) 353±362
www.elsevier.com/locate/phytochem
Oxidation of xenobiotics by plant microsomes, a reconstituted
cytochrome P450 system and peroxidase: a comparative study
M. Stiborovaa,*, H.H. Schmeiserb, E. Freib
aDepartment of Biochemistry, Faculty of Sciences, Charles University, Albertov 2030, 128 40 Prague 2, Czech Republic
bDepartment of Molecular Toxicology, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
Received 29 October 1999; received in revised form 29 March 2000
Abstract
The microsomal fraction from tulip bulbs (Tulipa fosteriana, L.) contains cytochrome P450 (CYP3, EC 1.14.14.1) and
peroxidase (EC 1.11.1.7.) enzymes catalyzing the NADPH Ð and hydrogen peroxide Ð dependent oxidation of the xenobiotic
substrates, N-nitrosodimethylamine (NDMA), N-nitrosomethylaniline (NMA), aminopyrine and 1-phenylazo 2-
hydroxynaphthalene (Sudan I), respectively. Oxidation of these model xenobiotics has also been assessed in a reconstituted
electron-transport chain with a partially puri®ed CYP fraction, phospholipid and isolated tulip NADPH:CYP reductase (EC
1.6.2.4.). Peroxidase isolated from tulip bulbs (isoenzyme C) oxidizes these xenobiotics, too. Values of kinetic parameters (Km,
Vmax), requirements for cofactors (NADPH, hydrogen peroxide), the e€ect of inhibitors and identi®cation of products formed
from the xenobiotics by the microsomal fraction, partially puri®ed CYP and peroxidase C were determined. These data were
used to estimate the participation of the CYP preparation and peroxidase C in oxidation of two out of the four studied
xenobiotics (NMA, Sudan I) in tulip microsomes. Using such detailed study, we found that the CYP-dependent enzyme system
is responsible for the oxidation of these xenobiotics in the microsomal fraction of tulip bulbs. The results demonstrate the
progress in resolving the role of plant CYP and peroxidase enzymes in oxidation of xenobiotics. 7 2000 Elsevier Science Ltd.
All rights reserved.
Keywords: Tulipa fosteriana; Cytochrome P450; Peroxidase; Xenobiotics
1. Introduction
as the terminal oxidase. However, it is not exactly
known which plant enzymes are the most important
for the oxidation of xenobiotics in plants. Several
types of oxidative enzymes such as CYPs, peroxidases
(EC 1.11.1.7.) and lipoxygenases (EC 1.13.11.12.) have
been suggested to be implicated in the oxidation of
xenobiotics in plants (Sandermann, 1988, 1992; Fonne-
P®ster et al., 1988; Higashi, 1988; Durst and Benve-
niste, 1993; P¯ugmacher and Sandermann, 1998).
An economically signi®cant function of plant CYPs,
and one which is only partially understood, is the role
of CYPs in the detoxication of herbicides (Moreland et
al., 1993a, 1993b; Bolwell et al., 1994; Frear, 1995;
Thies et al., 1996). This function is one which provides
the ®rst committed step in the detoxi®cation process
responsible for herbicide selectivity in a variety of
plant-herbicide combinations. Other plant CYP
The oxidation of xenobiotics in animals is thought
to be catalyzed mainly by mixed function oxidases,
usually with a cytochrome P450 (CYP, EC 1.14.14.1.)
Abbreviations: BDI, benzenediazonium ion; CYP, cytochrome
P450; HPLC, high-performance liquid chromatography; Km, Michae-
lis constant; NDMA, N-nitrosodimethylamine; NMA, N-nitroso-N-
methylaniline; 4'-OH-Sudan I, 1-(4-hydroxyphenylazo)-2-hydroxy-
naphthalene; 6-OH-Sudan I, 1-phenylazo-2,6-dihydroxynaphthalene;
4',6-di(OH)-Sudan I, 1-(4-hydroxyphenylazo)-2,6-dihydroxynaphtha-
lene; Rf, relative mobility; TLC, thin layer chromatography; Vmax
maximal velocity.
,
* Corresponding author. Tel.: +420-2-219-52-333; fax: +420-2-
219-52-331.
E-mail address: stiborov@prfdec.natur.cuni.cz (M. Stiborova).
0031-9422/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(00)00123-0

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M. Stiborova et al. / Phytochemistry 54 (2000) 353±362
354
enzymes serve critical functions in numerous biosyn-
thetic pathways, especially those involved in the pro-
duction of a diverse array of secondary metabolites
such as terpenoids, alkaloids, phenylpropanoids, phy-
tosterols, pigments, phytoalexins, giberellins, etc.
(Durst and Benveniste, 1993; Durst and O'Keefe,
1995; Hallahan and West, 1995; Schuler 1996; Schalk
et al., 1997; Chapple, 1998; Tijet et al., 1998) as well
as in several reactions related to metabolism of xeno-
biotics (Riviere and Cabanne, 1987; Higashi, 1988;
Sandermann, 1988, 1992; Frear et al., 1991; Durst and
Benveniste, 1993; Frear et al., 1993; Thies et al., 1996;
P¯ugmacher and Sandermann, 1998; Robineau et al.
1998).
The metabolism of xenobiotics in many plant species
(Dohn and Krieger, 1981; Sandermann, 1987, 1988,
1992) or plant microsomal enzymes has been pre-
viously shown (Fonne-P®ster et al., 1988; Sandermann,
1992, 1994; Moreland et al., 1993a, 1993b; Durst and
O'Keefe, 1995; Schalk et al., 1997; Batard et al., 1998).
Microsomal localization of the activity, O2 and
NADPH requirements, inhibition by CO and its rever-
sal by light provided conclusive evidence for the invol-
vement of CYPs in the oxidative metabolism.
However, the metabolism of xenobiotics by puri®ed
plant CYPs reconstituted with NADPH:CYP reductase
(EC 1.6.2.4.) was studied only in a few cases. CYP iso-
lated from avocado was found to metabolize xeno-
biotics (e.g. 4-chloro-N-methylaniline) (O'Keefe and
Leto, 1989). Demethylation of this substrate was also
measured in yeast transformed with recombinant
CYP71A1 from avocado (Bozak et al., 1992), demon-
strating that CYP71A1 product oxidizes this xeno-
biotic. A recombinant plant cinnamate 4-hydroxylase
CYP73A (EC 1.14.13.11.) (from Helianthus tuberosus
tubers) produced in yeast was highly speci®c for its
natural substrate (Pierrel et al., 1994; Urban et al.,
1994; Schalk et al., 1997). However, it also demethy-
lated herniarin (Urban et al., 1994; Pierrel et al., 1994),
hydroxylated 2-naphthoic acid (Durst et al., 1996;
Schalk et al., 1997) and oxygenated ®ve xenobiotics
including the herbicide chlorotoluron (Urban et al.,
1994; Pierrel et al., 1994). An inducible CYP76B1
enzyme isolated from Helianthus tuberosus dealkylated
a model xenobiotic compound 7-ethoxycoumarin and
metabolized also a wide range of xenobiotics, including
alkoxycoumarins, alkoxyresoru®ns, and several herbi-
cides of the class of phenylureas (Robineau et al.
1998). Another CYP oxidizing xenobiotics such as
phenylurea herbicides was a gene product of heterolo-
gously expressed CYP cDNA (CYP71A10) (Siminszky
et al., 1999). We have shown that a protein fraction
highly enriched for CYPs, isolated from tulip (Tulipa
fosteriana, L.) bulbs, is active in the in vitro reductase-
mediated oxidation of N-nitrosamines and aminopyr-
ine (Hansikova et al., 1994, 1995).

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M. Stiborova et al. / Phytochemistry 54 (2000) 353±362
355
Care should be taken into account of any interpret-
ation of data from crude microsomal fractions. Plant
microsomes contain not only CYPs as do microsomes
of animals, but peroxidases are also present in rela-
tively high levels in this plant subcellular system (Chia-
pella et al., 1995). These enzymes were assumed to be
even more e€ective in metabolism of several xeno-
biotics in plants than CYPs (Sandermann, 1988; Sti-
borova and Anzenbacher, 1991; Stiborova et al.,
1988a, 1990, 1992; Plewa and Wagner, 1993; Gichner
et al., 1994, 1995; Plewa et al., 1996; Stavreva et al.,
1997).
The aim of the present work is to elucidate the par-
ticipation of CYPs and peroxidases in oxidation of
xenobiotics by plant microsomes. Here, we complete
the in vitro comparative study of oxidation of N-nitro-
sodimethylamine (NDMA), N-nitroso-N-methylaniline
(NMA), aminopyrine and 1-phenylazo-2-naphthol
(Sudan I) using enzyme systems (microsomes, CYP
and peroxidase) prepared from tulip bulbs.
as a heterocyclic ligand, binding to both the oxidized
and reduced form of CYP heme, competing with sub-
strate and oxygen, inhibited more eciently the oxi-
dation of NDMA, NMA and Sudan I than the
oxidation of aminopyrine (Table 1). Unfortunately, in-
hibitors of peroxidase, which are often used in the in
vitro experiments (Gichner et al., 1994, 1995; Chiapella
et al., 1995), could not be utilized in the study. We
found that most of such well known peroxidase inhibi-
tors (i.e. CN-, diethyldithiocarbamate, pyrogallol) also
inhibit CYP (results not shown).
Oxidation of the compounds was measured in the
reaction mixture, which contained microsomal
enzymes, NADPH as cofactor and various concen-
trations of the xenobiotic compounds. The reactions
followed the Michaelis-Menten kinetics. Among the
studied xenobiotics, NMA is the best substrate for
plant microsomal enzymes. Under the conditions used,
the highest value of the maximal velocity (Vmax) and
the lowest value of the apparent Michaelis constant
(Km) for NMA were observed (Table 2). The values of
kinetic parameters for the reactions catalyzed by the
microsomal system, in which hydrogen peroxide was
used, were not determined because of the low eciency
of microsomes to oxidize the studied xenobiotics under
the presence of this cofactor (see Table 1).
2. Results and discussion
2.1. Oxidation of NDMA, NMA, aminopyrine and
Sudan I by tulip microsomes
Microsomes isolated from tulip bulbs (20.921.8 mg
protein/g fresh weight of tulip bulbs) contained CYP
(45.023.2 pmol/mg protein), cytochrome P420 (0.052
0.01 pmol/mg protein), cytochrome b5 (0.10 2
0.01nmol/mg protein) and exhibited the activity of
NADPH:CYP reductase with respect to cytochrome c
as a substrate (2.71 2 1.91 nmol/min per mg protein)
and the activity of peroxidase with guaiacol and benzi-
dine as substrates (13.2 2 1.4 nmol/min per mg and
14.1 2 1.4 nmol/min per mg protein, respectively).
Hence, we have con®rmed that tulip microsomes con-
tain not only CYPs, but also the peroxidase activity.
Four structurally diverse chemicals (NDMA, NMA,
aminopyrine, and Sudan I) are oxidized by this micro-
somal fraction. Their oxidation by microsomes had an
absolute requirement for NADPH. The reaction was
negligible when the NADPH regenerating system was
omitted (Table 1). NADH is less ecient cofactor than
NADPH. Hydrogen peroxide (the peroxidase cofactor)
is even less ecient cofactor for oxidation of NDMA,
NMA and Sudan I than NADH. Only demethylation
of aminopyrine, in the presence of hydrogen peroxide,
was greater than that in the presence of NADH
(Table 1). The oxidation of NDMA, NMA and Sudan
I was signi®cantly inhibited by CO and this inhibition
was reversible by light (Table 1). The inhibition of the
aminopyrine demethylation was less e€ective and it
was only partially reversible by light. Likewise, metyra-
pone (2-methyl-1,2-di-pyridyl-1-propanone), which acts
2.2. Oxidation of NDMA, NMA, aminopyrine and
Sudan I by a CYP reconstitution system and peroxidase
All studied xenobiotics are also oxidized by partially
puri®ed CYP (a protein fraction highly enriched for
CYPs) reconstituted with NADPH:CYP reductase and
by peroxidase (one of the most abundant isoenzyme of
tulip bulbs ± isoenzyme C) (Tables 1 and 2). The reac-
tions catalyzed by the CYP reconstituted system were
not detectable when the NADPH-regenerating system
or the reductase was omitted. Likewise, the presence of
hydrogen peroxide was absolutely required for the ac-
tivity of isolated peroxidase (Table 1). A molar ratio
of 0.65 of CYP to the reductase, which was shown to
be the most appropriate for the catalysis of the metab-
olism of xenobiotics (Hansikova et al., 1995), was used
to determine the kinetic parameters of the oxidation of
all studied xenobiotics. The reactions followed Michae-
lis-Menten kinetics. The apparent Km values and maxi-
mal velocities (Vmax) for the oxidation of xenobiotics
by this system are shown in Table 2. Similarly to the
crude microsomal fraction, NMA was also the best
substrate for the reconstituted system containing par-
tially puri®ed CYP.
The reaction of NDMA, aminopyrine and Sudan I
oxidation catalyzed by isolated tulip peroxidase fol-
lowed the Michaelis-Menten kinetics, too. Among the
studied xenobiotics, aminopyrine is the best substrate
for tulip peroxidase (Table 2). We had described pre-

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M. Stiborova et al. / Phytochemistry 54 (2000) 353±362
356
viously that non-Michaelian saturation curves were
observed when the initial velocity of NMA oxidation
catalyzed by isoenzymes of tulip peroxidase was
plotted as a function of NMA concentrations (Stibor-
ova and Hansikova, 1997). Therefore, we cannot
characterize eciency of NMA oxidation by plant per-
oxidases using the values of Vmax and apparent Km.
The values of apparent Km obtained for NDMA,
NMA and Sudan I as the substrates of CYP reconsti-
tuted system in vitro are similar to those obtained for
these substrates in microsomes (Table 2). A di€erent
Km value was, however, determined for aminopyrine.
Other CYP enzymes di€erent from the isolated enzyme
fraction or other enzymes, which oxidize aminopyrine,
but have lower anities for aminopyrine, are present
in the tulip microsomes.
On the basis of found e€ects of inhibitors, the
requirement for cofactors and values of kinetic par-
ameters, we can speculate that CYP isolated from tulip
bulbs should be responsible for the NDMA, NMA
and Sudan I oxidation in microsomes of tulip bulbs.
In order to con®rm this speculation, special emphasis
was laid on the comparison of metabolites formed by
these three used plant enzyme systems. NMA and
Sudan I were used for such a detailed comparative
study.
cofactor (Table 3). Methylaminophenol was not
detected in the peroxidase system.
Major metabolites produced from Sudan I by tulip
microsomes with the NADPH-regenerating system [4'-
OH-Sudan I,6-OH-Sudan I and the colourless product,
which was identi®ed previously as compound derived
from the benzenediazonium ion (BDI) (Stiborova et
al., 1988b; Stiborova et al., 1995)] are the same as the
major products formed from this compound by par-
tially puri®ed CYP (Table 3). A minor product is 4',6-
di(OH)-Sudan I (Table 3, Fig. 2). The formation of
Sudan I oxidative products by microsomes were negli-
gible when hydrogen peroxide was used as cofactor
(Table 3). Tulip peroxidase oxidized Sudan I to form
only a small amount of 6-OH-Sudan I, BDI and two
other products with Rf 0.35 and 0.7 (Table 3, Fig. 2).
These two products decomposed during mass spec-
troscopy. The fragmentation peaks at m/z 77 and 105
in the mass spectra of both products indicate the pre-
sence of phenyl and phenyldiazonium ion fragments,
respectively,whereas the most prominent peak at m/z
247 in the mass spectrum of the products with Rf 0.7
apparently belongs to the Sudan I ion. We compared
the fragmentation patterns of the two products with
those of Sudan I. The most prominent peaks in the
mass spectrum of Sudan I arise from the phenyl ion
(m/z 77), the phenyldiazonium ion (m/z 105), and the
Sudan I ion (m/z 247). Therefore, we suggest that the
two products may be dimers and/or oligomers of
Sudan I, being formed from Sudan I radicals. Their
complete structure elucidation by NMR spectroscopy
was precluded by their instability. These metabolites
are formed neither by microsomes nor by CYP. Tulip
peroxidase is less ecient with respect to oxidation of
Sudan I than CYP (Table 3).
2.3. Metabolites generated from NMA and Sudan I by
tulip microsomes, a CYP reconstitution system and
peroxidase
Formaldehyde, methylaniline, aniline, p-methylami-
nophenol and p-aminophenol were found to be metab-
olites formed from NMA by the tulip CYP
reconstituted with NADPH:CYP reductase, and also
by the NADPH-dependent reactions catalyzed by the
crude microsomal system (Table 3, Fig. 1). Phenol was
not detected in these systems under the experimental
conditions used (Table 3 and Hansikova et al., 1995).
This metabolite was produced by peroxidase and, in a
very low amount, also by microsomes with H2O2 as
The identi®cation of metabolites formed from NMA
and Sudan I con®rmed our suggestion (see above) that
the CYP-dependent enzyme system is mainly respon-
sible for the oxidation of both xenobiotics in the
microsomal fraction of tulip bulbs.
The biological signi®cance of both CYP- and peroxi-
Table 2
Kinetics of metabolism of di€erent xenobiotic substrate by microsomes, a reconstituted CYP system and peroxidase from tulip bulbsa
Substrate
Crude microsomal system
CYP reconstituted system
Peroxidase
b
b
c
KmmM
Vmax
Km, mM
Vmax
Km, mM
Vmax
NDMA
NMA
151232
4523
33002280
682263
1.2320.210
1.2520.230
0.7120.072
0.5320.062
142227
4421
117229
635260
2.5120.230
2.4220.030
0.1120.016
0.5320.030
420260
n.d.d
0.15020.003
n.d.d
Aminopyrine
Sudan I
8029
893253
1.95020.090
0.32020.030
a Means and standard deviations from 3 parallel experiments are presented.
b nmoles HCHO or Sudan I/min per nmol CYP.
c nmoles HCHO or Sudan I/min per nmol peroxidase.
d n.d.: not detectable.

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M. Stiborova et al. / Phytochemistry 54 (2000) 353±362
357
dase-mediated oxidation of NMA and Sudan I metab-
olism in intact plants in vivo is still unknown and can-
not be derived from the results as shown here. The
determination of metabolites of both studied xeno-
biotics formed in tulip plants in vivo would resolve
this question. Such study is planned for the future
work. From this point of view, metabolites which are
formed as unique products of oxidation by CYP or
peroxidase (e.g. phenol and methylaminophenol
formed from NMA by peroxidase and CYP, respect-
ively, C-hydroxy derivatives and oligomeric products
exclusively formed from Sudan I by CYP and peroxi-
dase, respectively) will serve as marker metabolites for
a CYP- or peroxidase-mediated metabolism of both
compounds in the intact tulip plant.
OH-Sudan I) and 1-(4-hydroxyphenylazo)-2,6-dihy-
droxynaphthalene [4',6-di(OH)-Sudan I] were syn-
thesized (Stiborova et al., 1988b) and puri®ed by
column chromatography on basic alumina and by thin
layer chromatography (TLC) on silica gel (Woelm).
[14C]-Labelled Sudan I (20 MBq/mmol) was syn-
thesized as shown earlier (Stiborova et al., 1988b) and
stored in methanol at 178C.
3.2. Preparation of microsomes
Tulip bulbs (Tulipa fosteriana L., var. Red Emperor)
were extracted without any inducing pretreatment.
Microsomes were prepared as described (Hansikova et
al., 1994, 1995) and resuspended in 0.1 M sodium
phosphate (pH 7.4), containing 30% glycerol (v/v) and
stored at 808C.
3. Experimental
3.3. Puri®cation of CYP
3.1. Chemicals
The CYP enzyme used for the reconstitution exper-
iments was isolated by the procedure we described pre-
viously (Hansikova et al., 1994, 1995). The speci®c
content of CYP in the ®nal preparation (a protein
fraction highly enriched for CYPs, see Hansikova et
al., 1995) was 5.2 nmol/mg protein. The CYP prep-
aration was stored in 10 mM potassium phosphate
(pH 7.4) at 808C.
Chemicals were obtained from the following sources:
cytochrome c, NADP+, NADPH and dilauroylpho-
sphatidylcholine from Fluka (Switzerland), Sudan I
from British Drug Houses (UK), NADH from Boeh-
ringer (Germany), dithiothreitol from Koch-Light
(UK), Emulgen 911 from Atlas Co. (Japan), benzene-
diazonium hexa¯uorophospate from Aldrich (USA)
and all other chemicals were reagent grade or better.
NMA and NDMA were synthesized as described
previously (Stiborova et al., 1992). The derivatives
1-(4-hydroxyphenylazo)-2-hydroxynaphthalene (4'-OH-
Sudan I),1-phenylazo-2,6-dihydroxynaphthalene (6-
3.4. Puri®cation of NADPH CYP reductase
A homogeneoues preparation of NADPH:CYP re-
ductase was isolated from solubilized tulip bulb micro-
Table 3
In vitro metabolism of NMA and Sudan I by microsomes, a CYP reconstituted system and peroxidase from tulip bulbsa
Metabolite
Yields (%)c
Crude microsomal system
with NADPH
CYP reconstituted system
Peroxidase
with H2O2
N-Methylaniline
Phenol
Aniline
3.5420.40
n.d.
n.d.b
3.6020.39
n.d.
0.3220.02
5.20d20.43
0.8220.09
n.d.
0.1020.03
0.0220.01
n.d.
3.8420.40
5.0620.51
7.5420.80
3.1020.30
0.8020.08
6.6720.63
1.6920.16
n.d.
4.0020.41
5.2020.49
8.2520.80
2.5220.27
0.7520.08
6.9920.71
1.9520.18
n.d.
p-Methylaminophenol
p-Aminophenol
BDIe
n.d.
1.4020.11
0.5120.05
n.d.
0.0820.01
n.d.
n.d.
4',6-di(OH)-Sudan I
4'-OH-Sudan I
6-OH-Sudan I
Unknown
n.d.
n.d.
n.d.
0.7320.08
1.2420.13
1.4220.14
Unknown
n.d.
n.d.
n.d.
a Values represent the means and standard deviations of three experiments. Experimental conditions are described in Section 3.
b n.d.: not detectable.
c The values of 100% were 300 nmoles NMA/min and 5 nmoles Sudan I/min.
d Corrected for 20% recovery of phenol from peroxidase.
e Other products formed from BDI could also be present (Stiborova et al., 1988a, 1988b).

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M. Stiborova et al. / Phytochemistry 54 (2000) 353±362
358
somes by chromatography on DEAE-Sepharose CL
6B and anity chromatography on a 2',5'-ADP
Sepharose 4B column (Hansikova et al., 1994). The
®nal preparation of NADPH:CYP reductase had a
speci®c activity of 77.5 nmol/min per mg protein. The
reductase was stored in 10 mM potassium phosphate
(pH 7.4) containing 0.02% Emulgen 911 after addition
of 2 mM ¯avinmononucleotide at 808C.
DEAE-cellulose DE 52 (Shannon et al., 1996; Stibor-
ova and Hansikova, 1997). Fraction C (one of the
most abundant peroxidase isoenzyme of tulip bulbs),
which was used for detailed kinetic measurements with
xenobiotics, was then puri®ed by repeated chromatog-
raphy on CM-Sephadex until elution patterns indi-
cated that tulip peroxidase C was free from other
peroxidase fractions or other protein contaminants.
3.5. Isolation of peroxidases
3.6. Analytical methods
A slight modi®cation of the procedure for isolation
of peroxidases from horseradish roots described by
Shannon et al. (1966) was used. Chromatography on
CM-Sephadex C-25 was used for isolation of cationic
peroxidases B, C, D and E. Three anionic peroxidases
(A1, A2, A3) were isolated by chromatography on
Quantitative determination of CYP, cytochrome
P420 and cytochrome b5 was carried out according to
Omura and Sato (1964). The concentration of
NADPH:CYP reductase was estimated as described
(Vermilion and Coon, 1978). The activity of NADPH:
CYP reductase was measured according to Sottocasa
Fig. 1. Proposed metabolic scheme of NMA in the tulip peroxidase system (A,B) and the tulip CYP reconstituted system (B,C). (1) N-nitro-
somethylaniline; (2) benzenediazoniumhydroxide; (3) benzenediazonium ion; (4) phenol; (5) methylaniline; (6) aniline; (7) p-methylaminophenol;
(8) p-aminophenol. The metabolites in parentheses are unstable and therefore not detectable.

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M. Stiborova et al. / Phytochemistry 54 (2000) 353±362
359
et al. (1967) using cytochrome c as the substrate (i.e.
as NADPH:cytochrome c reductase). The peroxidase
activity was measured according to Chiapella et al.
(1995) using guaiacol and benzidine as substrates. Pro-
tein concentrations were estimated according to Brad-
ford (1976) with bovine serum albumin as a standard.
Kinetic analyses were carried out by using the non-lin-
ear square methods as described by Cleland (1983).
The composition of the assay mixtures for N-
demethylation reactions of NMA, NDMA and amino-
pyrine was described earlier (Hansikova et al., 1995).
Brie¯y, 60 pmol CYP from tulip bulbs, 90 pmol of the
tulip NADPH:CYP reductase, 50 mM potassium phos-
phate pH 7.4, 7.5 mM MgCl2, 8 mM semicarbazide,
NADPH regenerating system, 60 mg of D,L-dilauroyl-
phosphatidylcholine and 0.1±50 mM NMA, NDMA
or aminopyrine. The amount of formaldehyde formed
was measured as described by Nash (1953). The same
reaction mixtures were used for demethylation of the
compounds by peroxidase except that 200 mg tulip per-
oxidase was used instead of the CYP-dependent
enzyme system. These mixtures contained 1 mM H2O2
as a second substrate (cofactor) instead of the
NADPH regenerating system.
The composition of the assay mixture and the pro-
cedure for demethylation reactions of NMA, NDMA
and aminopyrine catalyzed by microsomes were the
same as that described for the reconstitution exper-
iments except that 0.2 nmol of CYP (measured in
microsomal preparations diluted to contain 2±3 mg/ml
protein) were used instead of the reconstituted system.
The incubation mixtures containing NMA as a sub-
strate were applied to a C18 SepPak cartridge to quan-
tify the products formed from NMA in the used plant
enzyme systems. The cartridge was washed with 0.1
mM NaOH and the products eluted with 50% metha-
Fig. 2. Proposed metabolic scheme of Sudan I in the tulip CYP reconstituted system (A,B) and tulip peroxidase (B,C). (1) Sudan I; (2) 6-OH-
Sudan I; (3) 4'-OH-Sudan I; (4) 4',6-di(OH)-Sudan I; (5) benzenediazonium ion; (6) 1,2-dihydroxynaphthalene; (7) unknown; (8) unknown. The
metabolite in parentheses was not detected.

Â
M. Stiborova et al. / Phytochemistry 54 (2000) 353±362
360
nol in water. The products were separated by high-per-
formance liquid chromatography (HPLC) on
methanol, were separated by HPLC on a Separon
SGX C18 column with a linear gradient of methanol
in water (v/v): 90% methanol 0±6 min, 90±91%
methanol 6±9 min and 91±100% methanol 9±18 min;
¯ow rate 0.5 ml/min, UV detection at 260 nm. The
retention times of 4',6-di(OH)-Sudan I, 6-OH-Sudan I,
4'-OH-Sudan I and Sudan I were 3.0, 5.7, 6.9 and 15.1
min, respectively. Recoveries of products were around
90% in the presence of enzymes without cofactors
(NADPH or hydrogen peroxide).
The 1 ml assay mixture for oxidation reactions of
Sudan I catalyzed by microsomes contained 50 mM
potassium phosphate, pH 7.4, 0.2 nmol of CYP, 0.4
units of glucose-6-phosphate dehydrogenase, 4 mM
glucose-6-phosphate, 7.5 mM MgCl2, 0.01±0.5 mM
[14C]Sudan I. The procedure was the same as that
described for the experiments with the pure enzymes
(see above).
a
Separon SGX C18 column as described (Hansikova et
al., 1995). Reaction products were identi®ed by com-
parison of their retention times with authentic stan-
dards ( p-aminophenol, p-methylaminophenol, phenol,
aniline and N-methylaniline, having the retention times
2.2, 3.5, 9.15, 10.6 and 17.1 min, respectively). The
amounts were calculated by peak areas by comparison
with external standards. Recoveries of products were
around 80% after 10 min incubation in the presence of
enzymes without cofactors (NADPH or hydrogen per-
oxide), except the recovery of phenol (it was around
60%). The recovery of phenol when benzenediazonium
hexa¯uorophosphate was added to the incubation mix-
ture containing peroxidase but without hydrogen per-
oxide was about 20% after 10 min incubation.
The assay mixture for the oxidation reactions of
Sudan I contained, in 1 ml, 60 pmol CYP from tulip
bulbs, 90 pmol NADPH:CYP reductase from the same
source, 50 mM potassium phosphate pH 7.4, 7.5 mM
MgCl2, 4 mM glucose-6-phosphate, 0.4 units of glu-
cose-6-phosphate dehydrogenase, 100 mg of D,L-dilaur-
oylphosphatidylcholine and 0.01±0.5 mM [14C]Sudan I
(or unlabelled Sudan I) dissolved in 20 ml dimethyl
sulfoxide. The reaction was started by addition of
NADPH (®nal concentration was 0.5 mM). The same
reaction mixtures were used for the oxidation of
Sudan I by peroxidases except that 200 mg tulip per-
oxidase was used instead of the CYP-dependent
enzyme system. These mixtures contained 1 mM H2O2
Carbon monoxide inhibition of oxidation of sub-
strates was performed as shown previously (Hansikova
et al., 1995).
Acknowledgements
This study was supported by grants of the Ministry
of Education of the Czech Republic (VS96141) and the
Grant Agency of the Czech Republic (203/99/1628).
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