Protoporphyrinogen oxidase: High affinity tetrahydrophthalimide radioligand for the inhibitor/herbicide-binding site in mouse liver mitochondria

Article abstract of DOI:10.1021/tx960074h

Protoporphyrinogen oxidase (protox), the last common enzyme in heme and chlorophyll biosynthesis, is the target of several classes of herbicides acting as inhibitors in both plants and mammals. N-(4-Chloro-2-fluoro-5- (propargyloxy)phenyl)-3,4,5,6-tetrahydrophthalimide (a potent protox inhibitor referred to as THP) was synthesized as a candidate radioligand ([³H]-THP) by selective catalytic reduction of 3,6-dihydrophthalic anhydride (DHPA) with tritium gas followed by condensation in 45% yield with 4-chloro- 2-fluoro-5-(propargyloxy)aniline. Insertion of tritium at the 3 and 6 carbons of DHPA as well as the expected 4 and 5 carbons resulted in high specific activity [³H]THP (92 Ci/mmol). This radioligand undergoes rapid, specific, saturable, and reversible binding to the inhibitor/herbicide binding site of the protox component of cholate-solubilized mouse liver mitochondria with an apparent K(d) of 0.41 nM and B(max) of 0.40 pmol/mg of protein. In the standard assay, mouse preparation (150 μg of protein) and [³H]THP (0.5 nM) are incubated in 500 μL of phosphate buffer at pH 7.2 for 15 min at 25 °C followed by addition of ammonium sulfate and filtration with glass fiber filters. The potencies of five nitrodiphenyl ethers and two other herbicides as inhibitors of [³H]THP binding correlate well with those for inhibition of protox activity (r² = 0.97, n = 7), thus validating the binding assay as relevant to enzyme inhibition. It is also suitable to determine in vivo block as illustrated by an ~50% decrease in [³H]THP binding in liver mitochondria from mice treated ip with oxyfluorfen at 4 mg/kg. This is the first report of a binding assay for protox in mammals. The high affinity and specific activity of [³H]THP facilitate quantitation of protox and therefore research on a sensitive inhibition site for porphyrin biosynthesis.

Full text of DOI:10.1021/tx960074h

Chem. Res. Toxicol. 1996, 9, 1135-1139
1135
P r otop or p h yr in ogen Oxid a se: High Affin ity
Tetr a h yd r op h th a lim id e Ra d ioliga n d for th e In h ibitor /
Her bicid e-Bin d in g Site in Mou se Liver Mitoch on d r ia
Norman B. Birchfield and J ohn E. Casida*
Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science,
Policy and Management, University of California, Berkeley, California 94720-3112
Received April 26, 1996X
Protoporphyrinogen oxidase (protox), the last common enzyme in heme and chlorophyll
biosynthesis, is the target of several classes of herbicides acting as inhibitors in both plants
and mammals. N-(4-Chloro-2-fluoro-5-(propargyloxy)phenyl)-3,4,5,6-tetrahydrophthalimide (a
potent protox inhibitor referred to as THP) was synthesized as a candidate radioligand ([3H]-
THP) by selective catalytic reduction of 3,6-dihydrophthalic anhydride (DHPA) with tritium
gas followed by condensation in 45% yield with 4-chloro-2-fluoro-5-(propargyloxy)aniline.
Insertion of tritium at the 3 and 6 carbons of DHPA as well as the expected 4 and 5 carbons
resulted in high specific activity [3H]THP (92 Ci/mmol). This radioligand undergoes rapid,
specific, saturable, and reversible binding to the inhibitor/herbicide binding site of the protox
component of cholate-solubilized mouse liver mitochondria with an apparent Kd of 0.41 nM
and Bmax of 0.40 pmol/mg of protein. In the standard assay, mouse preparation (150 µg of
protein) and [3H]THP (0.5 nM) are incubated in 500 µL of phosphate buffer at pH 7.2 for 15
min at 25 °C followed by addition of ammonium sulfate and filtration with glass fiber filters.
The potencies of five nitrodiphenyl ethers and two other herbicides as inhibitors of [3H]THP
binding correlate well with those for inhibition of protox activity (r2 ) 0.97, n ) 7), thus
validating the binding assay as relevant to enzyme inhibition. It is also suitable to determine
in vivo block as illustrated by an ∼50% decrease in [3H]THP binding in liver mitochondria
from mice treated ip with oxyfluorfen at 4 mg/kg. This is the first report of a binding assay
for protox in mammals. The high affinity and specific activity of [3H]THP facilitate quantitation
of protox and therefore research on a sensitive inhibition site for porphyrin biosynthesis.
opmental effects including embryotoxicity, growth retar-
dation, and teratogenicity (skeletal and cardiovascular)
(8-10), and in one study fetal proto was greatly elevated
(11).
In tr od u ction
Protoporphyrinogen oxidase (EC 1.3.3.4) (protox),1 the
last common enzyme in the heme and chlorophyll bio-
synthetic pathways, catalyzes the dehydrogenation of
protoporphyrinogen IX (protogen) to protoporphyrin IX
(proto). Many herbicides acting via peroxidation of lipid
membranes are potent inhibitors of protox from plants
and animals (1, 2). The human hereditary disease
variegate porphyria is associated with half-normal protox
activity (3) and results in an array of dermatological and
neurological problems (4) and an elevated incidence of
liver cancer (5). Test animals survive high doses of
selected protox inhibitors, but in subacute or chronic
feeding studies, these compounds increase porphyrin
levels in feces and blood and carboxylated porphyrins in
liver of rats and mice, much the same as occurs in
variegate porphyria in humans (6, 7). Rats given single
or chronic doses of many protox inhibitors show devel-
Extensively-studied protox inhibitors include the ni-
trodiphenyl ethers (e.g., acifluorfen and oxyfluorfen) and
the even more potent tetrahydrophthalimides (12). The
optimization of these and other herbicides as protox
inhibitors has enabled the development of radioligand
probes which have been successfully applied to plant
protox (13-16), but there is no comparable report for
mammalian protox. A suitable radioligand probe for
protox in mammals must have both high affinity and high
specific activity. N-(4-Chloro-2-fluoro-5-(propargyloxy)-
phenyl)-3,4,5,6-tetrahydrophthalimide (THP) was se-
lected for study for three reasons: it has outstanding
herbicidal activity and inhibits plant protox by 50% at 7
nM (14); it is a single isomer of relatively simple
structure; tritium might be introduced into the tetra-
hydrophthalimide moiety at high specific activity. This
paper describes the radiosynthesis of [3H]THP, the
development of a binding assay, and validation of [3H]-
THP as a probe for the inhibitor/herbicide-binding site
in protox from mouse liver mitochondria.
* To whom correspondence should be addressed, at the Environ-
mental Chemistry and Toxicology Laboratory, Department of Envi-
ronmental Science, Policy and Management, 114 Wellman Hall,
University of California, Berkeley, CA 94720-3112; phone: (510) 642-
5424; fax: (510) 642-6497; e-mail address: ectl@nature.berkeley.edu.
X Abstract published in Advance ACS Abstracts, September 1, 1996.
1
Abbreviations: the aniline, 4-chloro-2-fluoro-5-(propargyloxy)-
aniline; DHPA, 3,6-dihydrophthalic anhydride; EtOAc, ethyl acetate;
I50, concentration for 50% inhibition; HOAc, glacial acetic acid; LSC,
liquid scintillation counting; MeOH, methanol; Pd/C, palladium on
activated carbon; proto, protoporphyrin IX; protogen, protoporphyrino-
gen IX; protox, protoporphyrinogen oxidase; THP and [3H]THP, N-(4-
chloro-2-fluoro-5-(propargyloxy)phenyl)-3,4,5,6-tetrahydrophthal-
imide (also referred to in the literature as S-23142) and its 3H-labeled
form as a radioligand; THPA and [3H]THPA, 3,4,5,6-tetrahydrophthalic
anhydride and its labeled form.
Exp er im en ta l P r oced u r es
Syn th esis of [3H]THP (Sch em e 1). (A) Ra d iosyn th esis
Rou te. All reactions were initially optimized with hydrogen
and deuterium under analogous conditions to those ultimately
used with tritium. The radiosynthesis involved selective cata-
S0893-228x(96)00074-4 CCC: $12.00 © 1996 American Chemical Society

1136 Chem. Res. Toxicol., Vol. 9, No. 7, 1996
Birchfield and Casida
Sch em e 1a
the solution and rinse were passed through glass wool and a
poly(tetrafluoroethylene) filter to remove Pd/C. Glacial acetic
acid (HOAc) (0.5 mL) was injected into the conical flask,
resulting in a faint blue-green color (not observed in preliminary
experiments using hydrogen or deuterium). The solution was
then evaporated to ∼0.1 mL followed by another addition of
HOAc (0.5 mL). This process was repeated one more time to
effectively remove EtOAc so the condensation step could be
performed in HOAc.
(D) Syn th esis a n d P u r ifica tion of [3H]THP . Attempts to
condense the aniline with [1H]THPA were unsuccessful in
EtOAc and mixtures of EtOAc and HOAc but successful in
HOAc only. Accordingly, the aniline (9.3 mg, 0.0467 mmol) in
0.2 mL of HOAc was injected into the [3H]THPA solution (0.6
mL of HOAc) followed by two 0.1-mL rinses of HOAc. The
solution was refluxed for 2 h at 110 °C (bath temperature)] (17,
18) in a nitrogen atmosphere, and then the solvent was stripped
and the solid material lyophilized overnight. After adding
freshly-distilled THF, the [3H]THP was purified on normal-
phase HPLC with a Merck LiChrospher column (10 cm, Si-60,
5 µm) using hexane-THF (88:12) as the mobile phase at 1.5
mL/min. UV absorbance was monitored at 285 nm and radio-
activity with a â-ram detector. The total yield was 6.9 mg
(0.0207 mmol, 45% based on the aniline), and the specific
activity was 92 Ci/mmol determined by HPLC UV response and
liquid scintillation counting (LSC).
a
(a) NH4OH, MeOH; (b) Pd/C, H2 or 2H2, or 3H2, EtOAc; (c)
HOAc, 110 °C.
Ta ble 1. 1H NMR Sh ifts for En vir on m en ta lly-Sen sitive
P r oton sa
compd
H-3
H-4
DHPA
THPA
THP
2.23
1.58
1.89
1.88
5.05
0.85
1.06
1.04
[3H]THP
a
C6D6 with 5-mm probe. Bruker AM-400 spectrometer except
for [3H]THP with IBM AF-300 spectrometer. All signals are
multiplets.
Mitoch on d r ia l P r ep a r a tion s. Mouse liver mitochondria
(19) were solubilized by stirring with sodium cholate (1% w/v)
for 1 h at 0 °C and centrifuging for 1 h at 100000g. The clear
yellow-brown supernatant was removed (avoiding fatty depos-
its), rapidly frozen in liquid nitrogen, and stored at -80 °C. No
loss in enzyme or binding activity was observed on frozen
storage for up to 30 days at -80 °C. There was a rapid increase
in binding activity for the first 90 min after thawing without
further increase at 120 min (the recommended time for use in
assays).
lytic reduction of the 4-ene of 3,6-dihydrophthalic anhydride
(DHPA) with tritium gas followed by condensation of labeled
3,4,5,6-tetrahydrophthalic anhydride ([3H]THPA) with 4-chloro-
2-fluoro-5-(propargyloxy)aniline (the aniline). The radiosyn-
thesis involved only two chemical steps and one chromato-
graphic purification.
(B) In ter m ed ia tes. Sources for the chemicals were as
follows: DHPA from Frinton Laboratories (Vineland, NJ ); THPA
from Aldrich Chemical Co. (Milwaukee, WI); THP (98% purity)
from Sumitomo Chemical Co. (Osaka, J apan). The aniline,
previously reported from reduction of the corresponding nitro
compound (12, 17), was prepared here by dissolving THP (350
mg) in a minimal amount of methylene chloride (CH2Cl2) and
then diluting with methanol (MeOH) (30 mL), followed by slow
addition of ammonium hydroxide (15 mL) until the solution
became cloudy, standing for 30 min at room temperature, and
evaporation with gentle heat under vacuum. Isolation involved
dissolving the residue in MeOH and introduction into a silica
gel column from which the aniline was eluted with CH2Cl2.
Solvent evaporation gave the aniline as an oil which solidified
after several days. Silica gel TLC yielded a single red spot
following ninhydrin treatment of Rf 0.53 in hexane/ethyl acetate
Bin d in g Assa ys. Binding assays were performed in 100 mM
sodium phosphate buffer (pH 7.2) at 25 °C by sequential
additions of 5 µL aliquots of Me2SO containing [3H]THP (0.5
nM final concentration in standard assays), competing inhibitor,
and acifluorfen (none for total binding or 500 µM final concen-
tration to measure nonspecific binding at each ligand or
inhibitor concentration). Solubilized mitochondrial preparation
(150 µg of protein) was added last in a 10 µL aliquot; then the
mixture was vortexed and incubated 15 min at 25 °C. Analysis
involved addition of 60% saturated ammonium sulfate adjusted
to pH 7.2 (500 µL), followed by vortexing the mixture and
allowing it to sit for 5 min before filtering on a Brandel Model
M-24R cell harvester (Gaithersburg, MD) through glass fiber
filters (Whatman GF/C). The filters were then rinsed with three
aliquots (2 mL) of cold 10 mM phosphate buffer (pH 7.2), placed
in scintillation vials with 1 mL of Optiphase HiSafe 2 scintil-
lation cocktail (Wallac OY, Turku, Finland), and soaked for at
least 2 h before LSC. Specific binding was defined as the
difference between total and nonspecific binding in the presence
of acifluorfen. Inhibitors were assayed between 0.1 and 105 nM
at 10-fold dilutions with duplicate samples in each series and
two sets of experiments. The concentrations for 50% inhibition
(I50 values) were based on the 1-mL volume after adding
ammonium sulfate solution, recognizing thereby the continuing
association and dissociation during this assay step. Inhibition
curves were analyzed using an iterative nonlinear least-squares
regression on Sigma Plot software (J andel Scientific Software,
San Rafael, CA).
1
(EtOAc) 7:3 and 0.63 in CH2Cl2. The H NMR spectrum of the
aniline [i.e., (CDCl3) δ (ppm): 2.54 (1H, t, J ) 2 Hz), 4.69 (2H,
d, J ) 2 Hz), 6.55 (1H, d, J ) 8 Hz), 7.03 (1H, d, J ) 10 Hz)]
was similar to the reported spectra for the aniline and THP (17).
GC/MS using chemical ionization yielded the expected molecular
mass.
(C) Syn th esis of [3H]THP A. Reduction of DHPA with
hydrogen or deuterium gave THPA or [2H]THPA in 100% yield
in 15 min with product characterization by comparison to
authentic THPA by 1H NMR (Table 1) and IR spectroscopy (data
not given). In the radiosynthesis, the reduction time was
extended to 45 min (for potential isotope effects), and [3H]THPA
was used directly for the next reaction. More specifically, DHPA
(19.9 mg, 0.133 mmol) was dissolved in EtOAc (1 mL) with 10%
palladium on activated carbon (Pd/C) (21.5 mg) and stirred
under tritium gas (610 mmHg) for 45 min at room temperature.
After tritium and some solvent were removed under vacuum,
the residual solvent was frozen with liquid nitrogen and the
remaining gaseous tritium removed under high vacuum. The
flask was repressurized with nitrogen; the solution was thawed,
then refrozen and placed under high vacuum again. This
process was repeated one more time. EtOAc was then added
to the solution which was transferred by needle to a 5-mL
conical flask followed by a rinse of EtOAc. In the transfer step
En zym e Assa ys. The assay measures the formation of proto
in incubations containing mitochondrial preparation and pro-
togen, with and without inhibitor. Protogen was prepared by
reducing proto by a described procedure (19, 20) with the
following exceptions: proto (Sigma Chemical Co., Milwaukee,
WI) was reduced with coarse 5% sodium mercury amalgam
(ground in hot glassware under nitrogen for consistent quality
even with high humidity); all solutions were previously degassed
with nitrogen; the reduced solution was passed through a

Binding Site of Protoporphyrinogen Oxidase
Chem. Res. Toxicol., Vol. 9, No. 7, 1996 1137
F igu r e 2. Binding parameters for [3H]THP in solubilized
mouse liver mitochondria. Solubilized preparation (180 µg of
protein) was incubated with 0.025-3.0 nM [3H]THP in phos-
phate buffer (0.5 mL) at pH 7.2 for 15 min at 25 °C followed by
addition of 60% ammonium sulfate solution (0.5 mL) and
filtration. Specific binding is defined as the difference between
total binding and nonspecific binding (with 500 µM acifluorfen)
at each ligand concentration. The inset plot shows linearity for
the 0.025-0.18 nM range in which nonspecific binding is
minimal. The Scatchard plot at the right is based on two
experiments on different days and includes the data (circles)
from the plot of specific binding on the left.
F igu r e 1. 3H NMR spectrum of [3H]THP showing proton-
decoupled chemical shifts and integrals. IBM AF-300 spectrom-
eter at 320 MHz using C6D6 and 5-mm probe. No other signals
are observed.
Millipore Millex-HV (0.45 µm) filter; the pH was adjusted to
∼10 with degassed 20% phosphoric acid. Whereas proto in
solution is burgundy red, the protogen solution is clear and
stable for several weeks when stored at -10 °C under paraffin
oil. Fluorescence measurements showed little or no proto but
upon oxidation (enzymatic or at low pH) the protogen solution
produced the fluorescence and absorbance spectra of proto. The
incubation pH of 8.2 was selected to minimize non-protox
oxidation of protogen. For fluoresence assay (19, 21), the
incubations were made in 3.0 mL of 100 mM Tris buffer (pH
8.2) with 1 mM EDTA, 0.2% Tween 20, and 10 mM GSH. To
this solution were added Me2SO (10 µL) alone or containing
inhibitor, mitochondrial preparation (675 µg of protein, 45 µL),
and finally the above protogen solution (75 µL, 5 µM final assay
concentration based on the amount of proto subjected to
reduction). Proto was measured fluorometrically by exciting at
405 nm and monitoring at 635 nm for 15 min with a Perkin
Elmer LS50B Luminescence spectrometer equipped with a
rotating, four-cuvette-holding turret for multiple samples. En-
zyme activity was defined as the difference between the rate of
proto production in the presence and absence of 3 µM oxyfluo-
rfen (which completely inhibits protox activity leaving only
background oxidation which was 10-25% of the protox-
catalyzed rate). Inhibition curves for I50 values were analyzed
as above.
Develop m en t of [3H]THP Bin d in g Assa y. Intact
mitochondria are not suitable giving apparent nonspecific
binding of 90-100% (or even higher with a nonpolar
competing inhibitor that may induce ligand precipitation)
presumably due to partitioning of the apolar ligand into
lipid membranes. This problem was solved by solubiliz-
ing the protox activity and [3H]THP binding site prior to
the assays and by adding the water-soluble acifluorfen
to determine nonspecific binding. Sodium cholate was
used in solubilization for several reasons: it extracts
nearly 100% of the protox activity from mitochondria; it
does not appear to lower enzyme stability as described
for other detergents (22); it does not wash the solubilized
enzyme through the GF/C filter to the extent observed
with other solubilizing agents. Poly(ethylenimine)-
treated filters are not suitable to harvest protox solubi-
lized with cholate or several other ionic or nonionic
detergents. Dilution of the cholate-treated preparation
below the critical micelle concentration allows some
specific binding to be collected on untreated filters, and
this is improved by adding ammonium sulfate (23) to
precipitate the solubilized enzyme reaching a maximum
for specific binding with ammonium sulfate at around
30% saturation before harvesting. Specific binding is
identical before and after ammonium sulfate addition,
suggesting that it does not hinder binding or denature a
significant amount of the enzyme. The binding assay,
relative to the enzyme assay, is less sensitive to change
in pH and does not require reducing or chelating agents.
Bin d in g P a r a m eter s for [3H]THP (F igu r e 2). The
specific binding is saturable with Kd 0.41 nM and Bmax
0.40 pmol/mg of protein. Attempts to show inhibition of
[3H]THP binding by protogen at levels in the range of
its Km (5.6 µM) (24) were not successful, resulting instead
in unacceptable levels of nonspecific binding. Under the
standard assay conditions, specific binding is character-
ized as follows (data not shown): completely abolished
by heating the preparation at 90 °C for 10 min; linear
with protein level in the range of 10-200 µg; equilibrium
achieved in ∼10 min at 25 °C (leading to 15 min as the
standard incubation time); dissociation rapid and binding
completely reversible using either unlabeled acifluorfen
or oxyfluorfen for displacement.
In Vivo Exp er im en ts. Oxyfluorfen was administered ip at
doses of 2-64 mg/kg to male albino Swiss-Webster mice using
Me2SO (25 µL/mouse) as the carrier vehicle. The mice were
sacrificed after 19 h for preparation of liver mitochondria and
assay of [3H]THP binding as above.
Resu lts
Syn th esis of [3H]THP . [3H]THP was synthesized in
45% yield at 92 Ci/mmol. The product was characterized
by comparison to authentic unlabeled THP (17) and had
identical properties on HPLC (Rt 8.8 min), cochromato-
graphed on two-dimensional TLC (Rf ) 0.40 in hexane/
EtOAc 7:3 and 0.43 in CH2Cl2), and also gave similar
inhibition in the low nanomolar range in the protox
activity assay. The radiochemical purity was >99% by
two-dimensional TLC as above and autoradiography. The
1H NMR spectrum of [3H]THP was identical to that of
the unlabeled standard except the tetrahydrophthalimide
proton signals were reduced, verifying both the structure
3
and H labeling positions. 1H NMR of [3H]THP (benzene)
δ (ppm): 1.04 (4H, m), 1.88 (4H, m), 1.96 (1H, s), 4.04
(2H, s), 6.83 (1H, d, J ) 9 Hz), 6.93 (1H, d, J ) 6 Hz);
splitting of the propargyl protons was not observed due
to the low sample concentration. 3H NMR showed the
insertion of approximately one tritium atom at carbons
3 and 6 for two at carbons 4 and 5 (Figure 1).
Cor r ela tion betw een I50 Va lu es for [3H]THP Bin d -
in g a n d P r otox Activity (F igu r e 3). The inhibitory

1138 Chem. Res. Toxicol., Vol. 9, No. 7, 1996
Birchfield and Casida
expected additions at carbons 4 and 5, making this
procedure useful for synthesis of ligands with high
specific activity.
The favorable correlation with several types of inhibi-
tors between the I50s for [3H]THP binding and protox
activity has two implications. First, the [3H]THP binding
site is intimately associated with enzymatic activity and
may be the substrate or catalytic site based on competi-
tion studies for both plant and animal protox by enzy-
matic (rather than binding) procedures (1); unfortunately,
high nonspecific binding of [3H]THP at micromolar or
higher protogen concentrations precluded a direct test
of this hypothesis. Second, a large variety of inhibitors
bind at the same or a closely-coupled site in plants
(15, 24), and this may also apply to mammals.
The characteristics of mammalian protox reported here
appear to differ somewhat from those of plant protox
based on literature cited above. Solubilized mouse pro-
tox, in contrast to solubilized maize enzyme (14), is not
trapped on poly(ethylenimine)-treated filters, suggesting
that mouse protox is less acidic than the maize enzyme.
The Bmax of 0.40 pmol/mg of protein for the [3H]THP site
in mouse mitochondrial protox in this study is 10- to 73-
fold lower than the value of 4 pmol/mg of protein for
analogs of THP in maize etioplasts (14, 15) and 16-29
pmol/mg of protein for [3H]acifluorfen in corn and pea
preparations (13, 24). This difference may be due in part
to comparing heme biosynthesis as a basal rate in liver
to a rapidly growing stage in plants. However, the protox
concentration in mouse liver mitochondria is considerably
higher based on relative enzyme activity determined
during purification to homogeneity (25, 26) than on the
Bmax in binding assays reported here, indicating the
possibility of incomplete recoveries by the present pro-
cedure involving addition of ammonium sulfate and
filtration.
F igu r e 3. Correlation for peroxidizing herbicides between
potency for inhibiting [3H]THP binding and protox activity in
solubilized mouse liver mitochondria. The inhibitors are com-
mercial or candidate herbicides available from previous studies
in this laboratory as follows: 1 oxyfluorfen, 2 acifluorfen methyl,
3 oxadiazon, 4 phenopylate, 5 nitrofen, 6 acifluorfen, and 7 (a
nonherbicidal analog) 4′-fluoro-4-nitrodiphenyl ether. The mean
I
50 values plotted from both the enzyme and binding assays are
from two experiments involving duplicate samples with average
deviations of 21% and 32% of the mean, respectively. The protein
level, buffer, and incubation time differ in the two assays as
indicated in the text.
[3H]THP binding is a sensitive and convenient method
for measuring inhibition of mammalian protox in vivo
as illustrated in a dose-response experiment with oxy-
fluorfen. The inhibition measured suggests that oxy-
fluorfen or an active metabolite is present at the protox
binding site or in the liver 19 h after treatment. It is
clear that the binding assay has the requisite sensitivity
to facilitate studies on the toxicology of other protox
inhibitors as well.
This is the first report of a binding assay for protox in
mammals. [3H]THP appears to be highly potent and
specific in quantitating this enzyme and should be useful
in research on the mechanisms of heme biosynthesis and
the toxicology of peroxidizing herbicides in plants and
mammals.
F igu r e 4. Effect of oxyfluorfen in vivo on [3H]THP binding
activity in solubilized mouse liver mitochondria. Analyses made
19 h after ip treatments with oxyfluorfen are related to controls
in the same experiment with carrier vehicle only. The mean
value for the controls was 15 500 dpm specific binding/mg of
protein. Each point is based on two to four mice involving
triplicate samples. Error bars represent the standard error of
the mean (n ) 6-12).
potency of the nitrodiphenyl ethers (1, 2, and 5-7) and
oxadiazon (3) and phenopylate (4) for [3H]THP binding
correlates well (r2 ) 0.97) with that for protox activity,
suggesting that the [3H]THP binding site is the same as
that causing inhibition of protox activity.
Effect of Oxyflu or fen in Vivo on [3H]THP Bin d in g
Activity (F igu r e 4). Oxyfluorfen, 19 h after ip treat-
ment, blocks [3H]THP binding in a dose-dependent man-
ner with ∼50% inhibition at 4 mg/kg.
Ack n ow led gm en t. The project described was sup-
ported by Grant PO1 ES00049 from the National Insti-
tute of Environmental Health Sciences, NIH, and the
University of California Toxic Substances Research and
Teaching Program. Tom Cromartie (Zeneca Agricultural
Products, Richmond, CA) provided useful advice for the
initial studies. We acknowledge our current or former
laboratory colleagues Loretta Cole, Phillip J efferies,
Bachir Latli, Qing-Xiao Li, Gary Quistad, Andrew Wa-
terhouse, and Edgardo Wood for useful suggestions and
Nick Norberg for help performing protox activity assays.
Hiromi Morimoto and Philip Williams of the National
Tritium Labeling Facility (Lawrence Berkeley Labora-
tory, University of California at Berkeley) performed the
Discu ssion
The radiosynthesis of [3H]THP from [3H]THPA should
be applicable to radiolabeling many 3,4,5,6-tetrahydro-
phthalimides. Insertion of tritium at the 3 and 6 carbons
of DHPA, possibly via a radical stabilized by the doubly-
allylic nature of these methylenes, increased the specific
activity of the [3H]THP by ∼30 Ci/mmol beyond the
3
radiosynthesis and H NMR analysis, respectively; this

Binding Site of Protoporphyrinogen Oxidase
Chem. Res. Toxicol., Vol. 9, No. 7, 1996 1139
that protoporphyrinogen oxidase specifically binds acifluorfen.
Eur. J . Biochem. 209, 861-868.
(14) Mito, N., Sato, R., Miyakado, M., Oshio, H., and Tanaka, S. (1991)
In vitro mode of action of N-phenylimide photobleaching herbi-
cides. Pestic. Biochem. Physiol. 40, 128-135.
(15) Nicolaus, B., J ohansen, J . N., and Bo¨ger, P. (1995) Binding
affinities of peroxidizing herbicides to protoporphyrinogen oxidase
from corn. Pestic. Biochem. Physiol. 51, 20-29.
(16) Che, F. S., Asami, T., Wang, J . M., and Yoshida, S. (1994) Binding
protein of protoporphyrinogen oxidase-inhibiting herbicide, S-23142,
in stromal extracts. Eighth IUPAC International Congress of
Pesticide Chemistry, J uly 1994, Abstract 664.
(17) Nagano, E., Hashimoto, S., Yoshida, R., Matsumoto, H., and
Kamoshita, K. 1984. Tetrahydrophthalimides, and their produc-
tion and use as herbicides. US Patent 4,484,941.
(18) Ohta, H., Suzuki, S., Watanabe, H., J ikihara, T., Matsuya, K.,
and Wakabayashi, K. (1976) Structure-activity relationship of
cyclic imide herbicides. I. N-Substituted phenyl-3,4,5,6-tetrahy-
drophthalimides and related compounds. Agric. Biol. Chem. 40,
745-751.
facility is supported by NIH Grant RR01237, National
Center for Research Resources.
Refer en ces
(1) Camadro, J .-M., Matringe, M., Scalla, R., and Labbe, P. (1991)
Kinetic studies on protoporphyrinogen oxidase inhibition by
diphenyl ether herbicides. Biochem. J . 277, 17-21.
(2) Corrigall, A. V., Hift, R. J ., Adams, P. A., and Kirsch, R. E. (1994)
Inhibition of mammalian protoporphyrinogen oxidase by aci-
fluorfen. Biochem. Mol. Biol. Int. 34, 1283-1289.
(3) Deybach, J . C., de Verneuil, H., and Nordmann, Y. (1981) The
inherited enzymatic defect in porphyria variegata. Hum. Genet.
58, 425-428.
(4) Eales, L., Day, R. S., and Blekkenhorst, G. H. (1980) The clinical
and biochemical features of variegate porphyria: an analysis of
300 cases studied at Groote Schuur Hospital, Cape Town. Int. J .
Biochem. 12, 837-853.
(5) Kauppinen, R., and Mustajoki, P. (1988) Acute hepatic porphyria
and hepatocellular carcinoma. Br. J . Cancer 57, 117-120.
(6) Krijt, J ., Pleskot, R., Sanitrak, J ., and J anousek, V. (1992)
Experimental hepatic porphyria induced by oxadiazon in male
mice and rats. Pestic. Biochem. Physiol. 42, 180-187.
(7) Krijt, J ., Vokurka, M., Sanitrak, J ., J anousek, V., van Holsteijn,
I., and Blaauboer, B. J . (1994) Effect of the protoporphyrinogen
oxidase-inhibiting herbicide fomesafen on liver uroporphyrin and
heptacarboxylic porphyrin in two mouse strains. Food Chem.
Toxicol. 32, 641-649.
(19) Brenner, D. A., and Bloomer, J . R. (1980) A fluorometric assay
for measurement of protoporphyrinogen oxidase activity in mam-
malian tissue. Clin. Chim. Acta 100, 259-266.
(20) Camadro, J .-M., Matringe, M., Scalla, R., and Labbe, P. (1993)
Fluorometric assay of protoporphyrinogen oxidase in chloroplasts
and in plant, yeast, and mammalian mitochondria. In Target
Assays for Modern Herbicides and Related Phytotoxic Compounds
(Bo¨ger, P., and Sandmann, G., Eds.) pp 29-34, Lewis, Boca Raton.
(21) J acobs, N. J ., and J acobs, J . M. (1982) Assay for enzymatic
protoporphyrinogen oxidation, a late step in heme synthesis.
Enzyme 28, 206-219.
(8) Gosselin, R. E., Smith, R. P., and Hodge, H. C. (1984) Clinical
Toxicology of Commercial Products, 5th ed., p II-343, Williams &
Wilkins, Baltimore.
(22) Camadro, J .-M., Thome, F., Brouillet, N., and Labbe, P. (1994)
Purification and properties of protoporphyrinogen oxidase from
the yeast Saccharomyces cerevisiae. J . Biol. Chem. 269, 32085-
32091.
(23) El-Refai, M. F. (1984) Assay of soluble receptors. In Membranes,
Detergents, and Receptor Solubilization (Venter, J . C., and Har-
rison, L. C, Eds.) pp 99-108, Alan R. Liss, New York.
(24) Dailey, H. A., and Karr, S. W. (1987) Purification and character-
ization of murine protoporphyrinogen oxidase. Biochemistry 26,
2697-2701.
(25) Varsano, R., Matringe, M., Magnin, N., Mornet, R., and Scalla,
R. (1990) Competitive interaction of three peroxidizing herbicides
with the binding of [3H]acifluorfen to corn etioplast membranes.
FEBS Lett. 272, 106-108.
(26) Proulx, K. L., and Dailey, H. A. (1992) Characteristics of murine
protoporphyrinogen oxidase. Protein Sci. 1, 801-809.
(9) Von Burg, R. (1994) Toxicology update (oxadiazon). J . Appl.
Toxicol. 14, 69-71.
(10) Kawamura, S., Kato, T., Matsuo, M., Sasaki, M., Katsuda, Y.,
Hoberman, A. M., and Yasuda, M. (1995) Species difference in
developmental toxicity of an N-phenylimide herbicide between
rats and rabbits and sensitive period of the toxicity to rat embryos.
Congenital Anomalies 35, 123-132.
(11) Machemer, L., Schmidt, U., and Holzum, B. (1992) Specific and
non-specific developmental effects. In Risk Assessment of Prena-
tally-Induced Adverse Health Effects (Neubert, D., Kavlock, R. J .,
Merke, H.-J ., and Klein, J ., Eds.) pp 85-100, Springer-Verlag,
Berlin.
(12) Duke, S. O., and Rebeiz, C. A., Eds. (1994) Porphyric Pesticides:
Chemistry, Toxicology, and Pharmaceutical Applications, ACS
Symposium Series 559, 317 pp, American Chemical Society,
Washington, DC.
(13) Matringe, M., Mornet, R., and Scalla, R. (1992) Characterization
of [3H]acifluorfen binding to purified pea etioplasts, and evidence
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