Primary product of the horseradish peroxidase-catalyzed oxidation of pentachlorophenol

Article abstract of DOI:10.1021/es981126n

Peroxidases are a class of enzymes that catalyze the oxidation of various phenolic substrates by hydrogen peroxide. They are common enzymes in soil and are also available commercially, so that they have been proposed as agents of phenolic pollutant transformation both in the environment and in engineered systems. Previous research on the peroxidase-catalyzed oxidation of pentachlorophenol (PCP) has suggested that tetrachloro-p-benzoquinone (chloranil) is the principal product and that a considerable fraction of the PCP added to reaction mixtures appears to be resistant to oxidation. In experiments employing alternative methods of product separation and analysis, we found that both of these observation s are artifacts of extraction and analytical methods used in previous studies. The major product of the horseradish peroxidase-catalyzed oxidation of pentachlorophenol from pH 4-7 was 2,3,4,5,6-pentachloro-4-pentachlorophenoxy-2,5-cyclohexadienone (PPCHD), which is formed by the coupling of two pentachlorophenoxyl radicals. The yield of chloranil and other soluble products was negligible. PPCHD is insoluble and unreactive in aqueous media but is reactive when dissolved in various organic solvents. Substantia amounts of chloranil were formed when PPCHD was dissolved in benzene, ethyl acetate, or methanol; less was formed in hexane and acetonitrile; and negligible amounts were formed in dimethylformamide. High-pressure liquid chromatography (HPLC) analysis of PPCHD indicated that it is capable of undergoing dissociation and reduction to pentachloro phenol under typical reversed-phase HPLC conditions. Chlorinated oligomeric products are formed when PPCHD is stored in acetonitrile, either alone or with added pentachlorophenol. Our results demonstrate that the removal of PCP in peroxidase-catalyzed reactions can be much higher than indicated in previous work, as long as the initial product is separated by filtration or other physical means. The major product of the horseradish peroxidase-catalyzed oxidation of pentachlorophenol from pH 4-7 was 2,3,4,5,6-pentachloro-4-pentachlorophenoxy-2,5-cyclohexadienone (PPCHD), which is formed by the coupling of two pentachlorophenoxyl radicals. PPCHD is insoluble and unreactive in aqueous media but is reactive when dissolved in various organic solvents. High pressure liquid chromatography (HPLC) analysis of PPCHD indicated that it is capable of undergoing dissociation and reduction to pentachlorophenol under typical reversed-phase HPLC conditions. The removal of pentachlorophenol in peroxidase-catalyzed reactions can be much higher as long as the initial product is separated by filtration or other physical means.

Full text of DOI:10.1021/es981126n

Environ. Sci. Technol. 1999, 33, 1408-1412
PCP is known to be biodegradable by several bacterial
Primary Product of the Horseradish
Peroxidase-Catalyzed Oxidation of
Pentachlorophenol
and fungal species (2, 4-6). Full-scale bioremediation of PCP-
contaminated soil has been achieved (4), but PCP can be
difficult to biodegrade in contaminated soil and groundwater.
PCP-degrading microbes are not always present at contami-
nated sites, requiring inoculation with axenic cultures in such
situations (2, 4). High concentrations of PCP can be inhibitory
to the degrading organisms (2, 7), and supplemental carbon
sources are often required to sustain biodegradation over
extended periods of time (2, 8).
The oxidation of phenolic compounds, including PCP, is
readily catalyzed by a number of extracellular fungal and
plant oxidative enzymes. Enzyme-catalyzed oxidation there-
fore has been proposed as an alternative method of removing
PCP and other phenols from contaminated aqueous mixtures
C H I K O M A K A Z U N G A ,
M I C H A E L D . A I T K E N , * A N D A V R A M G O L D
Department of Environmental Sciences and Engineering,
CB 7400, School of Public Health, University of North
Carolina, Chapel Hill, North Carolina 27599-7400
Peroxidases are a class of enzymes that catalyze the
oxidation of various phenolic substrates by hydrogen peroxide.
They are common enzymes in soil and are also available
commercially, so that they have been proposed as agents of
phenolic pollutant transformation both in the environment
and in engineered systems. Previous research on the
peroxidase-catalyzed oxidation of pentachlorophenol (PCP)
has suggested that tetrachloro-p-benzoquinone (chloranil)
is the principal product and that a considerable fraction
of the PCP added to reaction mixtures appears to be resistant
to oxidation. In experiments employing alternative methods
of product separation and analysis, we found that both
of these observations are artifacts of extraction and analytical
methods used in previous studies. The major product of
the horseradish peroxidase-catalyzed oxidation of
(
9). The oxidation of PCP has been reported to be catalyzed
by lactoperoxidase (10), horseradish peroxidase (HRP) (11-
3), lignin peroxidase (LIP) (13-18), Coprinus cinereus
1
peroxidase (CIP) (19), and a laccase from Trametes (Coriolus)-
versicolor (20-22). These enzymes generally catalyze one-
electron oxidations, which for phenolic substrates lead to
phenoxyl radical intermediates that can undergo various
postenzymatic reactions, including coupling reactions to
form dimeric products (23, 24). Researchers have proposed
that peroxidases might serve to detoxify PCP-contaminated
media by catalyzing the conversion of PCP to less chlorinated
products (14, 15, 22) or by facilitating the polymerization of
PCP with natural organic matter precursors via the pen-
tachlorophenoxyl radical intermediate (18, 25-27).
In several laboratory studies on the enzyme-catalyzed
oxidation of PCP in aqueous media, the predominant reaction
product was reported to be tetrachloro-p-benzoquinone
(chloranil) (11, 14, 15, 17, 20, 22), and a substantial fraction
of the added PCP was recovered after the reaction (11, 14,
pentachlorophenol from pH 4-7 was 2,3,4,5,6-pentachloro-
4-pentachlorophenoxy-2,5-cyclohexadienone (PPCHD),
which is formed by the coupling of two pentachlorophenoxyl
radicals. The yield of chloranil and other soluble products
was negligible. PPCHD is insoluble and unreactive in
aqueous media but is reactive when dissolved in various
organic solvents. Substantial amounts of chloranil were formed
when PPCHD was dissolved in benzene, ethyl acetate,
or methanol; less was formed in hexane and acetonitrile;
and negligible amounts were formed in dimethylformamide.
High-pressure liquid chromatography (HPLC) analysis of
PPCHD indicated that it is capable of undergoing dissociation
and reduction to pentachlorophenol under typical reversed-
phase HPLC conditions. Chlorinated oligomeric products
are formed when PPCHD is stored in acetonitrile, either
alone or with added pentachlorophenol. Our results
demonstrate that the removal of PCP in peroxidase-
catalyzed reactions can be much higher than indicated in
previous work, as long as the initial product is separated
by filtration or other physical means.
1
6, 19, 20, 24). Other observations, however, conflict with
these findings. For example, the HRP-catalyzed oxidation of
PCP at pH 4 results in a decrease in sample toxicity that is
far greater than predicted on the basis of recovered PCP (13,
1
9). In two other studies, significant concentrations of
chloranil and residual PCP were measured by HPLC analysis
of reaction mixture extracts, but the UV absorbance spectrum
of the crude reaction mixture itself either failed to indicate
the presence of these compounds (11) or indicated the
presence of additional compounds (14). For reactions
catalyzed by HRP over the pH range 4-7, we have found that
direct filtration of the reaction mixture consistently yields a
precipitate and that chloranil is not observed as a significant
product. We report the characterization of the precipitate
isolated from the HRP-catalyzed oxidation of PCP and explain
why chloranil and PCP were the species determined following
enzymatic oxidation in previous studies.
Methods
Enzym e and Chem icals. Purified HRP (type VIII acidic
isoenzyme) was purchased from Sigma (St. Louis, MO). The
enzyme was dissolved in 1 mL of reagent water and stored
at -15 °C when not in use. Catalytic activity was assayed just
prior to PCP oxidation by oxidation of 2,2′-azinodi(3-
ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS)
Introduction
Pentachlorophenol (PCP) has been used widely as a biocide
in many agricultural and industrial applications (1, 2). It is
a ubiquitous pollutant that has been placed on the U.S.
Environmental Protection Agency priority pollutant list, and
the use of PCP for wood preservation and other purposes
has been restricted or banned in several countries (3).
Although losses of PCP can occur in the environment (3), it
is persistent at highly contaminated sites (4).
(
28). The assay reaction mixture contained 67 mM phosphate
buffer (pH 6.0), 1.7 mM ABTS, 0.83 mM hydrogen peroxide
), and enzyme. One unit (U) of catalytic activity is
2 2
(H O
defined as the amount of enzyme required to form 1 µmol
of product/ min.
PCP and ABTS were purchased from Aldrich (Milwaukee,
*
Corresponding author phone: (919)966-1481; fax: (919)966-7911;
e-mail: mike_aitken@unc.edu.
WI). 14C-Labeled PCP (specific activity 7.9 mCi/ mmol; purity
1
4 0 8
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 33, NO. 9, 1999
10.1021/es981126n CCC: $18.00

1999 Am erican Chem ical Society
Published on Web 03/19/1999

>
98%), deuterated chloroform, trifluoroacetic acid, trifluo-
roacetic anhydride, and fuming nitric acid were purchased
from Sigma. All solvents were HPLC or spectrophotometric
grade, and all other chemicals were ACS reagent grade or
equivalent.
2
,3,4,5,6-Pentachloro-4-pentachlorophenoxy-2,5-cyclo-
hexadienone (PPCHD) was synthesized by oxidizing 10.6 g
of PCP with 4.2 mL of fuming nitric acid as a suspension in
3
0 mL of trifluoroacetic acid and 45 mL of trifluoroacetic
anhydride, as described by Reed (29).
FIGURE 1. Structure of 2,3,4,5,6-pentachloro-4-pentachlorophenoxy-
Enzym atic Oxidation of PCP. PCP oxidation was carried
out at pH 4, 5, 6, and 7. Since the solubility of PCP increases
with pH, we used higher initial PCP concentrations with
increasing pH to facilitate product recovery. Reactions at pH
13
2
,5-cyclohexadienone. The assigned C NMR shifts (in ppm) are C1
168.2; C2 (C6) ) 132.4; C3 (C5) ) 132.3; C4 ) 95.1; C7 ) 147.1; C8
C12) ) 132.3; C9 (C11) ) 145.2; C10 ) 128.9.
)
(
4
2 2
were conducted with 10 µM PCP, 6 µM H O , and 0.2 unit/
EPR spectra were recorded on a JES-RE1X spectrometer
JEOL, USA Inc.) Typical acquisition parameters were mi-
crowave power of 1 mW, a sweep width of 50-100 G, a field
modulation of 0.32-2 G, and a scan time of 2-4 min.
mL of enzyme in a 10 mM sodium citrate/ citric acid buffer.
Reactions at pH 5 were conducted with 75 µM PCP, 45 µM
(
H
2
O
2
, and 0.2 unit/ mL of enzyme in a 10 mM sodium citrate/
citric acid buffer. Reactions at pH 6 and 7 were conducted
with 200 µM PCP, 120 µM H , and 0.2 unit/ mL of enzyme
in a 10 mM potassium phosphate buffer. Controls were
equivalent to sample vials except no H was added. For
2
O
2
Results
2
O
2
NMR and EPR Spectra of PPCHD. The oxidation of pen-
tachlorophenol by fuming nitric acid yielded an orange-
yellow solid (mp 175-177 °C) whose color and melting point
corresponded to that of PPCHD (Figure 1), as described by
Reed (29). A well-resolved 1 C NMR spectrum of PPCHD was
recorded in chloroform-d using a standard 13C acquisition
time (15 s). In contrast to other perchlorinated aromatic
compounds, the addition of a spin relaxation reagent was
not required. This finding is consistent with the observation
by EPR spectroscopy (vide infra) indicating dissociation of
PPCHD in chloroform to give a low concentration of
pentachlorophenoxyl radical, which acts to enhance spin
relaxation.
1
4
experiments with C-labeled PCP, 20 000 disintegrations per
min (dpm) were added to reaction vials and controls.
2 2
Reactions were initiated by adding H O , and the vials
3
were shaken for 2 h on a rotary shaker at 250 rpm. The reaction
mixtures were then vacuum filtered through a 0.2-µm
polycarbonate filter (Poretics Corp.; Livermore, CA) or a 0.02-
µm alumina membrane filter (Anodisc, Whatman Scientific;
Maidstone, England). The precipitate collected on the filter
was dissolved in 2 mL of acetonitrile for immediate analysis,
and the unamended filtrate was analyzed separately. For the
experiment in which a mass balance of radiolabel was
evaluated, the filter and the filtrate were added to 9 mL of
scintillation fluid (Scintisafe, Fisher Scientific; Pittsburgh,
PA). Radioactivity (measured in dpm) was determined for 4
min on a scintillation counter (model 1900 TR, Packard
Instruments; Meriden, CT).
In accord with the σ-plane including C1, C4, C7, and C10,
eight resonances were apparent as two sets of four, with an
∼2:1 intensity ratio. The more intense set of resonances can
be attributed to the carbons related by reflection in the
σ-plane, and the less intense set can be atributed to the unique
carbons. Assignment of the latter set is readily made by
comparison with chemical shifts reported for chloranil and
perchlorinated phenyl ethers: C1, 168.2; C7, 147.1; C10, 128.9;
and C4, 95.1 ppm (30-33). The carbonyl carbon C1 and the
Optical Spectra and HPLC Analysis. Ultraviolet/ visible
(
UV/ VIS) spectra were recorded on a U-3300 spectropho-
tometer (Hitachi Instruments, Danbury, CT) between 195
and 500 nm with a slit width of 0.5 nm. HPLC analysis was
performed with a Waters (Milford, MA) system equipped
with a model 616 pump, a 600S controller, a 717 plus
autosampler, and a 996 photodiode array detector that
allowed the spectra to be scanned between 200 and 500 nm.
A C
Inc., Bellefonte, PA) was used for product separation. The
mobile phase was 55% acetonitrile: 45% H O containing 0.1%
trifluoroacetic acid for 5 min, increasing linearly to 100%
acetonitrile over 10 min, and holding for 10 min before
returning to initial conditions.
13C Nuclear Magnetic Resonance Spectroscopy (NMR).
PPCHD (50 mg) was dissolved in 1 mL of deuterated
3
sp -hybridized C4 are expected to appear at the lowest and
highest fields, respectively. The relative shifts of C7 and C10
are established on the basis of an upfield position predicted
for C10 para to the ether oxygen. The resonances of the
symmetry-related carbons are assigned according to the
following rationale. On the basis of relative intensities,
resonances at 132.4 and 132.3 (partially resolved from a
slightly less intense signal) have similar relaxation times and
are assigned to vinylic carbons C2 (C6) and C3 (C5),
respectively, a slight downfield shift assumed for the carbons
adjacent to the carbonyl group. Since phenyl carbons ortho
to the ether oxygen are expected to exhibit an upfield shift
relative to the meta positions, the remaining signals at 132.3
and 145.2 ppm are assigned to C8 (C12) and C9 (C11),
respectively.
8
column (25 cm × 4.6 mm, 5 µm particle size; Supelco,
2
3
chloroform (CDCl ) and stored in an ice bath until analysis
1
3
(
∼1 h). The C NMR spectrum was recorded on a Bruker
AM-500 spectrometer at 125 MHz, with shifts reported in
ppm relative to the carbon resonance of tetramethylsilane
(
TMS). The centerline of the CDCl
3
triplet (77 ppm) served
PPCHD has been reported to be EPR active at ambient
temperatures in carbon tetrachloride and benzene through
dissociation of the dimer into pentachlorophenoxyl radicals
(31). Chloroform solutions of PPCHD were also EPR active,
giving a signal 37 G in width with no hyperfine structure,
similar to the signals obtained in carbon tetrachloride and
benzene. The g-value of 2.0076 in chloroform confirms that
the signal originates from a pentachlorophenoxyl radical in
this solvent as well. Dissociation to the radical species in
as a standard.
Mass Spectrom etry. Mass spectrometric analysis was
performed on a VG 70 250SEQ mass spectrometer. Electron
impact (EI) spectra were obtained using a direct insertion
probe at 60 and 15 eV. Synthetic PPCHD was added to the
probe as a solid, while the enzymatic product was added to
the probe tube dissolved in carbon tetrachloride, which was
then allowed to evaporate.
Electron Param agnetic Resonance (EPR) Spectroscopy.
PPCHD (50 mg) was dissolved in 1 mL of deuterated
3
chloroform (CDCl ) immediately prior to analysis. X-Band
1
3
chloroform is consistent with indications from C NMR
spectrometry of enhanced relaxation of the 13C nuclear spins.
A capillary sample of PPCHD in acetonitrile exhibited a similar
VOL. 33, NO. 9, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
1 4 0 9

TABLE 1. Distribution of Radiolabel after HRP-Catalyzed
14
a
Oxidation of C-Labeled PCP at Different pH Values
pH
recovery of
DPM (%)
4
5
6
7
filtrate
sam ple
control
precipitate
sam ple
control
total
7.9
79.2
4.5
85.8
6.0
94.4
22.2
93.2
87.4
9.1
91.7
7.4
87.1
4.3
71.0
3.7
sam ple
control
95.3
88.3
96.2
93.2
93.1
98.7
93.2
96.9
a
All sam ples and controls were filtered through a 0.02-µm filter.
FIGURE 2. UV/VIS spectra of synthetic PPCHD and the precipitate
obtained from the HRP-catalyzed oxidation of PCP at pH 5 as
compared to the spectrum for PCP. Samples were dissolved in
acetonitrile and analyzed immediately. Spectra were normalized
with respect to the maximum absorbance of each species.
FIGURE 3. HPLC chromatograms (225 nm detection) of (a) an
acetonitrile solution of the precipitate from the HRP-catalyzed
oxidation of PCP at pH 6, dissolved and analyzed immediately after
collection; (b) a solution of PPCHD in acetonitrile analyzed
immediately; and (c) a solution of PPCHD in acetonitrile after
incubation for 2 weeks. PCP standards eluted at 8 min and chloranil
standards eluted at 5.8 min under the same HPLC conditions (not
shown).
EPR spectrum, supporting the occurrence of monomer-
dimer equilibration in polar media as well.
Enzym atic Oxidation of PCP. The HRP-catalyzed oxida-
tion of PCP over the pH range 4-7 led to the formation of
a yellow precipitate. The precipitate could be collected on
a 0.2-µm filter except for the material obtained at pH 7, which
required a 0.02-µm filter. Mass balance data for the HRP-
standard. This result may be explained by the dissociation
of PPCHD and subsequent reduction, perhaps by trace metals
in the column packing or other components of the HPLC
system, since it has been shown that PPCHD is readily
reduced to PCP by metals such as zinc in an alcohol or acid
medium (29, 31). The presence of trace reducing-contami-
nants associated with the column packing is supported by
the observation that, after conditioning the column by
repeated injections of PPCHD, it was possible to obtain
consistent chromatograms having a predominant peak at a
longer elution time (18 min) than that of PCP (8 min) (Figure
3, panels a and b). The precipitated enzymatic reaction
product and a PPCHD standard produced identical chro-
matograms on the conditioned column, and the peak at 18
min had a UV spectrum identical to that of the PPCHD
standard. No peak corresponding to chloranil was detected
in either the standard or the enzymatic oxidation product.
The enzymatic reaction product and a PPCHD standard
gave identical mass spectra (not shown), with a base cluster
centered at m/ z 266 corresponding to PCP. Weak ion clusters
1
4
catalyzed oxidation of C-labeled PCP over this pH range
revealed that most of the radioactivity added was recovered
in the precipitate (Table 1). In contrast, most of the radiolabel
in the control samples was recovered in the filtrate.
The UV/ VIS spectrum of the precipitate from the enzy-
matic reaction (at all pH values) matched the spectrum of
the synthetic PPCHD standard (Figure 2). The spectra,
recorded in acetonitrile, were characterized by an absorbance
maximum at 218 nm with a shoulder at 259 nm. This spectrum
is distinct from that of chloranil in acetonitrile, which exhibits
an absorbance maximum at 288 nm. The ratio of molar
extinction coefficients ꢀ288/ ꢀ218 for the precipitate (0.05) and
chloranil (4.2) provides a strong indication that little or no
chloranil is present in the HRP-derived product. The UV
spectrum of PCP is characterized by the absence of significant
absorbance at 259 nm.
The HPLC analysis of PPCHD proved to be difficult,
initially yielding only a single peak that coeluted with a PCP
•+
were centered at m/ z 495 and 456, consistent with PPCHD -
1
4 1 0
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 33, NO. 9, 1999

metric conversion of PCP to 2,3,4,5,6-pentachloro-4-pen-
tachlorophenoxy-2,5-cyclohexadienone (PPCHD): (i) the
majority of the radioactivity in reactions carried out with
TABLE 2. Solvent Effects on the Formation of Chloranil from
a
the Decomposition of 200 µM PPCHD
1
4
chloranil yield (µM)b
C-labeled PCP is associated with the precipitate (Table 1);
ii) analysis of the precipitate by UV/ VIS spectroscopy (Figure
), HPLC (Figure 3), and mass spectrometry indicated that
solvent
(
2
dim ethylform am ide
hexane
0.4 ( 0.4
12.8 ( 3.5
17.3 ( 4.8
64.9 ( 2.5
85.1 ( 3.9
87.1 ( 0.9
the precipitate is virtually identical to the synthesized PPCHD;
and (iii) we have seen no evidence for any other significant
product of the reaction. PPCHD is formed by the coupling
of two pentachlorophenoxyl radicals (11, 29), the expected
products of one-electron oxidation reactions catalyzed by
HRP and other peroxidases. The formation of pentachlo-
rophenoxyl radicals from the HRP-catalyzed oxidation of PCP
has been observed (11), but the coupling product has not
been identified previously as the major product of this
reaction.
acetonitrile
c
benzene
m ethanol
ethyl acetate
c
a
Sam ples were incubated for 24 h in 50 m L of solvent at 33 °C and
then analyzed by HPLC. b Mean and standard deviation of three
replicates. Sam ples were blown to dryness, resuspended in an
c
equivalent am ount of acetonitrile, and then analyzed by HPLC.
In earlier studies on the peroxidase- or laccase-catalyzed
oxidation of PCP, chloranil and PCP were reported to be the
predominant compounds remaining after the reactions were
terminated (11, 14, 15, 17, 20, 21, 25). The apparent residual
PCP was assumed to be resistant to oxidation based on its
persistence after the addition of fresh enzyme (17, 19).
Chloranil has been proposed to form by an enzyme-mediated
one-electron oxidation of the pentachlorophenoxyl radical
to a carbocation, followed by the addition of water (11, 14).
However, in most of the previous studies on the enzymatic
oxidation of PCP, the products were extracted from the
reaction mixture with an organic solvent followed by
chromatographic separation of the extract. In this study, we
examined the behavior of PPCHD and demonstrated that it
is unstable under these analytical conditions. PPCHD
decomposes to chloranil and PCP on standing in several
organic solvents, with a significant yield of chloranil within
a 24-h period (Table 2). Particularly noteworthy is the
formation of chloranil in ethyl acetate, a solvent commonly
used for extraction, and in methanol, commonly used as a
mobile phase in HPLC analysis. PPCHD is also easily reduced
to PCP by sodium iodide and zinc in alcohol or acidic media
FIGURE 4. HPLC radiochromatogram of an acetonitrile solution
14
initially containing 63 µM PPCHD and 12.5 µM C-labeled PCP
after incubation for 3 days. Chromatogram represents 56 samples
collected at 32-s intervals. The radioactivity injected for both the
sample and the control was 20 000 dpm.
(
29, 34) and is reduced by sodium borohydride and sodium
iodide to chlorinated phenoxyphenols (31, 32).
PPCHD is relatively insoluble in water and does not react
nor decompose in water at temperatures up to 100 °C (29).
Neither the precipitate from the enzymatic reaction nor the
synthesized product decomposed in aqueous suspension at
pH 4 over incubation periods as long as 12 days (not shown).
We took advantage of these characteristics to isolate PPCHD
directly from the enzymatic reaction mixture by filtration.
Under acidic conditions, the collection by filtration is efficient,
while at neutral pH the product is generated in fine
suspension and must be collected on filters of pore size e0.02
µm.
•
+
Cl and PPCHD -2Cl, respectively. The base peak most likely
arises from the protonation of the dissociation productsthe
pentachlorophenoxyl radicalsin the ion source. Given the
lability of PPCHD, the absence of the molecular ion is not
surprising. No chloranil molecular ion (cluster with base peak
at m/ z 246) was observed in either spectrum.
Chloranil was reported to be a product of the peroxidase-
catalyzed oxidation of PCP when organic solvents were used
to extract products from the reaction mixtures or as cosolvents
in enzymatic reactions (11, 14, 15, 17, 20, 22). The recovery
of chloranil from a 200 µM solution of PPCHD in various
solvents varied from negligible in the case of dimethylfor-
mamide to 87 µM in the case of ethyl acetate (Table 2).
Variable amounts of PCP were also found, but it is unclear
how much of the PCP may have originated from dissociation
and reduction of PPCHD on the HPLC column.
Incubation of PPCHD in acetonitrile for 2 weeks resulted
in the formation of several other compounds, as indicated
by HPLC analysis (Figure 3c). The reactivity of PPCHD in
acetonitrile was evaluated further by incubating PPCHD with
C-labeled PCP in acetonitrile for 3 days. Some of the
radiolabel initially associated with C-labeled PCP was
distributed among higher molecular weight compounds
The reaction of PPCHD with spin relaxation enhancers
1
3
normally added to perchlorinated hydrocarbons for C NMR
spectrometry has prevented acquisition of NMR data (31).
However, it proved possible to record the 13C NMR spectrum
of PPCHD in the absence of added relaxation enhancers.
The explanation for this unexpected behavior lies in the
dissociation of the dimer to a low concentration of pen-
tachlorophenoxyl radical, which provided the necessary spin
relaxation pathway. Such equilibration has been reported
for oligomeric ethers of other perchlorinated compounds
(35) and has been observed for PPCHD in carbon tetrachloride
and benzene (31). We have demonstrated that dissociation
also occurs in chloroform and acetonitrile. Consistent with
reversible homolytic dissociation, on standing in acetonitrile,
PPCHD forms other oligomeric products (Figure 3c) and
incorporates 14C-labeled PCP into oligomers (Figure 4).
It is clear that the removal of PCP by peroxidase-catalyzed
oxidation can be much more extensive than has been
indicated in other work and that potentially toxic products
1
4
1
4
(
Figure 4), suggesting that PPCHD reacts with PCP in
acetonitrile to form oligomers.
Discussion
We infer from the following evidence that horseradish
peroxidase catalyzes the extensive and essentially stoichio-
VOL. 33, NO. 9, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
1 4 1 1

such as chloranil are not likely to be found in the aqueous
phase from such reactions. The present study also illustrates
that sample preparation and analytical methods must be
carefully evaluated in subsequent work on the enzyme-
catalyzed oxidation of PCP and in other systems involving
the one-electron oxidation of PCP.
(15) Mileski, G. J.; Bumpus, J. A.; Jurek, M. A.; Aust, S. D. Appl. Environ.
Microbiol. 1988, 54, 2885.
(
16) Aitken, M. D.; Venkatadri, R.; Irvine, R. L. Water Res. 1989, 23,
43.
4
(
(
17) Chung, N.; Aust, S. D. Arch. Biochem. Biophys. 1995, 322, 143.
18) R u¨ ttimann-Johnson, C.; Lamar, R. T. Appl. Environ. Microbiol.
1
996, 62, 3890.
(
(
(
(
19) Sarr, D. H. MSEE Technical Report. University of North
Carolina: Chapel Hill, NC, 1993.
20) Konishi, K.; Inoue, Y. Mokuzai Gakkaishi 1972, 18, 463 (in
Japanese).
Acknowledgments
We thank David Sarr for conducting preliminary experiments
that led to the present study, Asoka Ranasinghe for assistance
with mass spectrometry analyses, Ramiah Sangaiah for
21) Roy-Arcand, L.; Archibald, F. S. Enzyme Microb. Technol. 1991,
1
3, 194.
1
3
assistance with C NMR analyses, and Chris Ryan and Emily
Warmoth for assistance with EPR analyses. The National
Institute of Environmental Health Sciences supported this
work under its Superfund Basic Research Program (Grant
P42ES05948). The U.S. Environmental Protection Agency
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