Does N-hydroxyglycine inhibit plant and fungal laccases?

Article abstract of DOI:10.1016/S0031-9422(99)00302-7

The effect of N-hydroxyglycine on the oxidation of substrates, syringaldazine, tolidine, 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) and 2,6-dimethoxyphenol, by fungal and plant laccases was examined. At μM concentrations, N-hydroxyglycine decolorized solutions of substrates oxidized enzymatically by laccase or chemically by sodium periodate. This discoloration, or bleaching, could be mistaken for inhibition of laccase activity if N-hydroxyglycine was added to assays for laccase that monitored colored products, N-hydroxyglycine also affected oxygen consumption assays when some of these substrates were oxidized enzymatically or chemically. Spectral scans of the products formed during enzymatic or chemical oxidation of the substrates indicated that addition of N-hydroxyglycine caused a general decrease in absorption. Except for 2,6-dimethoxyphenol, no formation of new absorption peaks was noted. These results suggest that N- hydroxyglycine may not be a 'classical' enzyme inhibitor of laccase, but that this compound interferes with both spectrophotometric and oxygen uptake enzyme assays for laccase.

Full text of DOI:10.1016/S0031-9422(99)00302-7

Phytochemistry 52 (1999) 775±783
Does N-hydroxyglycine inhibit plant and fungal laccases?
Jingming Zhang, Richard Kjonaas, William H. Flurkey*
Department of Chemistry, Indiana State University, Terre Haute, IN, 47809, USA
Received 23 February 1999; received in revised form 3 May 1999
Abstract
The e€ect of N-hydroxyglycine on the oxidation of substrates, syringaldazine, tolidine, 2,2'-azino-bis-(3-ethylbenzothiazoline-
6-sulfonic acid) and 2,6-dimethoxyphenol, by fungal and plant laccases was examined. At mM concentrations, N-hydroxyglycine
decolorized solutions of substrates oxidized enzymatically by laccase or chemically by sodium periodate. This discoloration, or
bleaching, could be mistaken for inhibition of laccase activity if N-hydroxyglycine was added to assays for laccase that
monitored colored products. N-hydroxyglycine also a€ected oxygen consumption assays when some of these substrates were
oxidized enzymatically or chemically. Spectral scans of the products formed during enzymatic or chemical oxidation of the
substrates indicated that addition of N-hydroxyglycine caused a general decrease in absorption. Except for 2,6-dimethoxyphenol,
no formation of new absorption peaks was noted. These results suggest that N-hydroxyglycine may not be a ``classical'' enzyme
inhibitor of laccase, but that this compound interferes with both spectrophotometric and oxygen uptake enzyme assays for
laccase. # 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Plant; Fungal; Laccase; N-hydroxyglycine; Inhibition
1. Introduction
ponent of plant cell walls. In the presence of a
``mediator'', fungal laccases have also been shown to
oxidize nonphenolic compounds as well as to partici-
pate in deligni®cation (Call & Mucke, 1997). Other
roles assigned to fungal laccases include involvement
in fruit body development, spore/conidia pigmentation,
Laccase (EC 1.10.3.2; benzendiol:oxygen oxido-
reductase) is an extracellular Cu-containing enzyme
found in fungi, plants, and some bacteria (Mayer &
Harel, 1979; Mayer, 1987; O'Malley, Whetten, Bao,
Chen & Sedero€, 1993; Ferrar, Barberel, Ginger &
Walker, 1995; Yaropolov, Skorobogat'ko, Vartanov &
phytopathogenicity, and detoxi®cation (Mayer
&
Harel, 1979; Mayer, 1987; Ferrar et al., 1995;
Thurston, 1994; Call & Mucke, 1997). In contrast,
plant laccases are believed to be involved in ligni®ca-
tion rather than lignin degradation (O'Malley et al.,
1993; Dean & Eriksson, 1994).
Even though a wide variety of compounds can be
used to monitor laccase activity, there are few speci®c
or selective inhibitors of the enzyme. Because laccase
contains Cu ions that act to accept and transfer elec-
trons in the reduction of molecular oxygen, it is sus-
ceptible to agents that chelate or reduce copper. Some
of these agents include CN , sodium azide, diethyl-
dithiocarbamate, EDTA, and thioglycolic acid.
Desferal, a transition metal ion chelator, has been
reported to inhibit laccase, but the I50 for desferal was
approximately 30 mM (De Pinto & Ros Barcelo,
Varfolomeyev, 1994; Thurston, 1994; Dean
&
Eriksson, 1994; Call & Mucke, 1997). In the presence
of a suitable electron donor, the enzyme catalyzes the
four-electron reduction of molecular oxygen to water.
Mono/di/polyphenols, aminophenols, aromatic amines,
methoxyphenols, ascorbic acid, and even some inor-
ganic ions can act as arti®cial electron donors and
become oxidized in the process of reducing molecular
oxygen. Most, if not all, white rot fungi excrete lac-
cases that can degrade lignin, a polyphenolic com-
* Corresponding author. Tel.: +1-812-237-2245; fax: +1-812-237-
2232.
E-mail address: ch¯urke@scifac.indstate.edu (W.H. Flurkey)
0031-9422/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(99)00302-7

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J. Zhang et al. / Phytochemistry 52 (1999) 775±783
Table 1
E€ect of HG on the oxidation of syringalazine and tolidine by plant
and fungal laccases
1996). CTAB, a quaternary ammonium cationic deter-
gent, has also been shown to inhibit laccases from a
variety of sources (Ferrar et al., 1995; Walker &
McCallion, 1980). However, the Ki values for CTAB
were in a range of approximately 1±20 mM. These
large I50 and Ki values seem to indicate a lack of speci-
®city compared to other enzyme inhibitors. In con-
trast, some halides, especially F , are potent inhibitors
of fungal laccases (Xu, 1996).
Enzyme source
Enzyme activity (A/min/ml)
Syringaldazine o-tolidine
HG
0.96
0.22
+HG
HG
0.03
+HG
0
P. oryzae (ICN)
P. oryzae (Sigma)
C. hirsutus
0.29
0.06
0.07
0
0.33
8.7
0
Recently, N-hydroxyglycine (HG), a natural product
isolated from Penicillim citrinum, was reported to be a
speci®c inhibitor of Coriolus versicolor laccase (Murao
et al., 1992). Murao et al. (1992) indicated that N-
hydroxyglycine did not inhibit tyrosinase, ascorbate
oxidase, bilirubin oxidase or plant phenol oxidase, but
that it was a potent (I50 of 0.1 mmol) and selective in-
hibitor for C. versicolor laccase. Since that initial
report, other investigators have used N-hydroxyglycine
to inhibit laccase. For example, HG inhibited laccase
from P. oryzae and Azospirillum lipoferum using o-
and p-aminophenol and syringaldazine as substrates
(Faure, Bouillant & Bally, 1995; Jacoud, Faure,
E€osse, Wadoux & Bouillant, 1995). I50 values ranged
from 50 to 800 mM for HG inhibition of laccase in
these reports. Flurkey, Ratcli€, Lopez, Kuglin and
Dawley (1995) also reported the use of HG to selec-
tively discriminate between laccase and tyrosinase ac-
tivities and to inhibit laccases in Agaricus bisporus,
Crimini, Enoki, Oyster, and Shiitake mushrooms.
All of the reports describing the use of HG as an in-
hibitor of laccase used colorimetric or spectrophoto-
metric assay methods to monitor laccase activity. In
the analysis of tyrosinase, another Cu containing
oxido-reductase, Kahn and co-workers have shown
that some types of compounds can interfere with color
formation in spectrophotometric tyrosinase assays, but
have little or no e€ect on oxygen consumption assays
(Kahn, Schved & Lindner, 1993; Kahn, Lindner &
Zakin, 1995). Interference was thought to occur
through reactions with product or product intermedi-
ates. In the course of synthesizing HG and derivatives
of HG as potential inhibitors of laccase, we noticed
some unexpected behavior of HG in solution and
when added to enzyme assays to inhibit laccase ac-
tivity. In this report, we show that HG has little, if
any, inhibitory e€ect on laccase. Instead this com-
pound interferes with formation of colored product in
spectrophotometric assays and can also interfere in
some oxygen consumption assays for laccase.
22.0
0.1
0.13
0
A. bisporus
R. vernicifera
6.67
0.21
0.25
0.04
0
(HG) by the method of Goto, Kawakita, Okutani and
Miki (1986), but in our hands the ®nal step led only to
a mixture of intractable products. We then attempted
to synthesize HG using the method of Jahngen and
Rossomando (1982) which involves the sodium cyano-
borohydride reduction of glyoxalic acid oxime. The
product obtained by this method was contaminated
with a large amount of an unidenti®ed impurity. A
minor modi®cation in the original procedure circum-
vented the problem; we used only half the amount of
solvent so that much of the product crystallized out of
solution during the reaction.
2.2. E€ect of HG on laccase activity
Several compounds were examined for potential use
as substrates in the inhibition studies using HG. Some
of these substrates included guaiacol, p-phenylenedia-
mine, catechol, pyrogallol, dopa, syringaldazine
(SYR), o-tolidine (TOL), 2,2'-azino-bis-(3-ethylben-
zothiazoline-6-sulfonic acid (ABTS), and 2,6-dimethox-
yphenol (DMP). SYR, TOL, ABTS, and DMP were
chosen as substrates in our studies because: (1) they
are frequently used as substrates for laccase; (2) they
can be monitored at di€erent wavelengths because of
characteristic product absorption spectra; (3) the oxi-
dized substrates yield di€erent absorption spectra dis-
tinct from the starting material; and (4) each oxidized
product has a di€erent color. When SYR and TOL
were used as substrates, they were oxidized to di€erent
extents by three types of fungal laccases and one plant
laccase (Table 1). C. hirsutus laccase showed greatest
activity with SYR while low activities with SYR were
observed using the other laccases. A. bisporus and C.
hirsutus laccase showed high activity with TOL as the
substrate while P. oryzae and Rhus vernicifera laccases
showed much lower activities with TOL. C. hirsutus
and A. bisporus laccase were used in subsequent studies
because of their greater activity with SYR,TOL and
also ABTS and DMP (data not shown). When the lac-
cases were assayed with SYR or TOL in the presence
of HG, an apparent decrease in activity was observed
2. Results and discussion
2.1. Synthesis of N-hydroxyglycine
Initially, we attempted to prepare N-hydroxyglycine

J. Zhang et al. / Phytochemistry 52 (1999) 775±783
777
Fig. 1. E€ect of HG on the enzymatic oxidation of SYR by C. hirsutus laccase. C. hirsutus, 20 ml, laccase (1 mg solid/ml; 1 mg protein/ml) was
incubated with 0.2 ml of 0.5 mM SYR (0.1 mmole) and 1.8 ml of acetate bu€er (pH 5.0). Various amounts (0.05 mmole/10 ml) of 5 mM HG dis-
solved in acetate bu€er were added at zero time. Rates of the individual reactions at 525 nm were determined in A/min between 0 and 0.2 A and
0.6 to 0.9 A.
compared to when HG was absent. This decrease was
noted if changes in absorbance were measured during
the ®rst few min of the reaction, assuming these ®rst
few min represent the initial rate of the reaction.
an enzyme reaction containing C. hirsutus laccase and
SYR, and the reaction monitored for a longer period
of time. As seen in Fig. 2(A), excess HG added at time
zero inhibited the reaction for approximately 12 min,
at which time a pink color began to form. We let this
reaction proceed until an absorbance of approximately
0.7 at 525 nm was reached and then more HG was
added. An immediate decrease in absorbance, ``bleach-
ing of product'', took place and the absorbance
dropped to a baseline level. After some time (10±
12 min), the color began forming again but did not
reach as high an absorbance as earlier. Addition of
more HG caused bleaching and an immediate decrease
in absorbance.
This process is better illustrated in Fig. 2(B) in
which smaller amounts or HG were added to a C. hir-
sutus laccase catalyzed reaction in progress. We
allowed the reaction to proceed until an absorbance of
approximately 0.55 was reached. At this time small
amounts of HG were added. Addition of HG caused
an immediate decrease in absorbance; this decrease
reached a minimum and color formation began again.
It appeared that HG was consumed in some process
and the rate of product formation proceeded as before.
This cycle could be repeated several times. Doubling
the amount of HG added, decreased the absorbance
by a factor of approximately 2. Upon depletion, pro-
duct formation resumed and could be ``bleached'' in a
repetitive fashion several more times. Eventually, with
enough added HG, the amount of product formed
began to decline, perhaps due to eventual substrate de-
2.3. E€ect of HG on the enzymatic oxidation of SYR
If one assumes that this apparent inhibitory e€ect
by HG is real, then I50 values for HG can be deter-
mined for di€erent substrates and di€erent laccases.
As illustrated in Fig. 1, C. hirsutus laccase was assayed
in the presence of a constant amount of SYR and var-
ied amounts of added HG. We observed that a distinct
lag period occurred in all assays. This lag period, the
time in which it took before color began to develop,
seemed to increase with increasing [HG]. The initial
rate measurements (0±0.2 A) decreased as the [HG]
was increased in the assays. If one used these initial
rate measurements to determine I50 values, an I50 of
50 mM for SYR and 65 mM for TOL was obtained
(data not shown). These I50 values are similar to those
reported elsewhere (Murao et al., 1992; Faure et al.,
1995). However, if the reaction was allowed to proceed
for a longer period of time, color formation began
again and the rate of color formation approached the
rate in the absence of added HG (rates measured at
0.4±0.8 A/time). This suggested that small amounts of
HG may not be inhibiting the enzyme but changing
the product into a non-colored material.
To determine if HG had an e€ect on the product
formation, successive amounts of HG were added to

778
J. Zhang et al. / Phytochemistry 52 (1999) 775±783
cally generated colored oxidation products, a similar
phenomenon should be observed with chemical oxi-
dation of laccase substrates. Four laccase substrates,
SYR, TOL, ABTS, and DMP, were oxidized in the
presence of sodium periodate. In the absence of HG,
each compound was oxidized to a colored product and
the rate of color formation was monitored with time.
In the presence of large amounts of added HG at time
zero, the rate of product formation decreased substan-
tially. Adding HG to the reaction in progress
decreased the rate of product as indicated by a
decrease in absorbance. Eventual loss of color or
decreased color was reached using all four laccase sub-
strates. These results indicated an enzymatic reaction is
not needed for HG to have a ``bleaching'' e€ect on
oxidized laccase substrates.
2.5. E€ect of HG on oxygen uptake during enzymatic
oxidation of laccase substrates
Laccase activity can also be monitored by following
oxygen uptake during the enzymatic reaction. HG can
apparently a€ect spectrophotometric assays for laccase
by reducing the amount of colored product, but can it
a€ect oxygen uptake assays? To examine this possi-
bility, enzymatic oxidation of SYR, TOL, ABTS, and
DMP was followed using a Clark oxygen electrode
after successive additions of HG (Fig. 3). Addition of
small amounts of HG had little e€ect on decreasing
oxygen uptake in assays using SYR, TOL, DMP and
ABTS as substrates with A. bisporus laccase. In fact,
HG caused an apparent increase in oxygen consump-
tion using SYR, DMP, and ABTS as substrates when
HG was added in amounts exceeding the amount of
substrate. If HG were behaving as a laccase inhibitor,
one would expect a signi®cant decrease, not an
increase, in oxygen consumption. However, we
observed little to no decrease in oxygen consumption
under these conditions. Because the oxygen electrode
reaction chambers were transparent, we observed that
addition of HG caused an immediate and/or fast
bleaching of any colored product formed without a
corresponding decrease in oxygen consumption.
Therefore, product or color disappeared with HG ad-
dition but oxygen consumption stayed the same or
actually increased. We also observed that excessive
amounts of added HG during later times of the reac-
tions caused a large increase in oxygen consumption
(data not shown).
Fig. 2. E€ect of HG on spectrophotometric enzyme assays monitor-
ing the oxidation of SYR by C. hirsutus laccase. (A) 25 ml of C. hir-
sutus laccase (1 mg solid/ml; 1 mg protein/ml) was incubated with
0.1 ml of 0.5 mM SYR (0.05 mmole), 1.8 ml of acetate bu€er and
0.1 ml of 5 mM HG (0.5 mmole) dissolved in acetate bu€er. After
the absorbance increased and began to taper o€, a second aliquot of
5 mM HG was added. This process was repeated a third time. (B)
10 ml of C. hirsutus laccase was added to 1.8 ml acetate bu€er and
0.1 ml of 0.5 mM SYR (0.05 mmole) and the absorbance monitored
with time. The absorbance was allowed to increase to approximately
0.55 A, at which time 10 ml of 5 mM HG (0.05 mmole/10 ml) was
added. The process was repeated several times with 10 and 20 ml ad-
ditions of HG.
pletion or decreased enzyme activity.
A similar
phenomenon was also observed using SYR, TOL,
ABTS, DMP, and quaiacol and A. bisporus laccase
with additions of HG. N-hydroxylglycine added to a
reaction in progress with these substrates bleached pro-
duct/color formation almost immediately (data not
shown).
2.6. E€ect of HG on oxygen uptake during chemical
oxidation of laccase substrates
2.4. E€ect of HG on the chemical oxidation of various
laccase substrates
If HG causes an apparent increase in O2 consump-
tion in enzymatic assays then a similar increase might
be observed in chemical oxidation of laccase sub-
If HG is interfering with the formation of enzymati-

J. Zhang et al. / Phytochemistry 52 (1999) 775±783
779
Fig. 3. E€ect of HG on enzymatic oxygen uptake assays for laccase with di€erent substrates. (A) 0.1 ml of SYR (0.1 mmole), 3.9 ml of acetate
bu€er, and 30 ml of A. bisporus laccase (2 mg protein/ml) were mixed in the electrode cell. Indicated amounts of 5 mM HG (0.5 mmole/0.1 ml)
were added at the designated times. (B) 25 ml of 40 mM tolidine (1 mmole), 3.85 ml of acetate bu€er, and 30 ml of A.bisporus laccase were mixed
in the electrode cell. At the times indicated, speci®ed amounts of 10 mM HG (1 mmole/01. ml) were added. (C) 3.6 ml of 0.5 mM ABTS (1.8
mmole) in acetate bu€er and 30 ml of A. bisporus laccase were mixed in the electrode cell. At indicated times, 0.1 ml of 10 mM HG (1 mmole) was
added. (D) 0.2 ml of 5 mM DMP (1 mmole) in acetate bu€er, 3.6 ml of acetate bu€er, and 10 ml of A. bisporus laccase were mixed in the elec-
trode cell. At selected intervals, 0.1 ml of mM HG (0.5 mmole) was added. Oxygen uptake was followed with time using a Clark oxygen elec-
trode.
strates, SYR, TOL, and ABTS, by sodium periodate.
Oxygen consumption was monitored in bu€ered sub-
strates containing HG that were oxidized with sodium
periodate. In general, addition of HG showed a slight
increase or no change in oxygen consumption. When
excess periodate was added, there was immediate for-
mation of a colored product. If more HG was added,
the intensity of the color decreased. In all later ad-
ditions, little to no change in O2 consumption was
noted and no decrease in oxygen consumption was
observed. Similarly, addition of HG to bu€ered DMP
caused a small increase in oxygen uptake but no
change in color. In contrast to the above, periodate
addition to the DMP reaction caused an immediate
coloration that was not blocked by HG.
2.7. Incubation experiments with laccase and HG
If the literature values of the I50 for HG are in the
micromolar range, one might expect the enzyme to

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J. Zhang et al. / Phytochemistry 52 (1999) 775±783
have a low Ki for HG and thus signi®cant inhibition
should occur if the enzyme was incubated with large
concentrations of HG. Even on dilution, the enzyme
might be expected to be signi®cantly inhibited. To test
this, A. bisporus laccase (0.2 mM ®nal concentration)
was incubated with 1 mM HG (5 mM after dilution)
for 30 min. Control experiments contained enzyme
added to bu€er. At the end of 30 min, tolidine and
bu€er were added, and the enzyme assayed spectro-
photometrically. Enzyme activity decreased slightly
from 8.5 units/ml to 8.0 units/ml when incubated in
the absence of HG compared to the presence of HG.
Although this does not prove lack of inhibition by
HG, it does suggest that incubation with 1 mM sol-
ution should have resulted in a greater diminution of
enzyme activity.
2.8. E€ect of HG on the absorption spectra during
enzymatic oxidation of laccase substrates
The e€ect of HG on the spectral products generated
by enzymatic oxidation of SYR, TOL, DMP, and
ABTS was also examined using scanning absorption
spectrophotometry. Oxidation of SYR by C. hirsutus
laccase showed an increase in absorbance at 525 nm
with time as expected. Addition of HG after the reac-
tion was in progress for 5 min caused the disappear-
ance of this absorption with time (Fig. 4(A)), while
addition of HG at time zero prevented the appearance
of this absorption peak for at least 16 min (Fig. 4(B)).
Similar results were observed for the e€ect of HG on
the enzymatic oxidation of TOL (Fig. 4(C) and (D)).
In addition, a second absorption peak at 366 increased
Fig. 4. E€ect of HG on the absorption spectra of substrates oxidized enzymatically by laccase. HG (®nal concentration 1.25 mM) was added to
bu€er containing SYR (®nal concentration 0.17 mM) at time zero (B) or after the reaction was initiated with C. hirsutus laccase (A). HG (®nal
concentration 1.0 mM) was added to bu€er containing TOL (®nal concentration 2.0 mM) at time zero (D) or after the reaction was initiated
with C. hirsutus laccase (C). HG (®nal concentration 0.91 mM) was added to bu€er containing ABTS (®nal concentration 1.8 mM) at time zero
(F) or after the reaction was initiated with C. hirsutus laccase (E). HG (®nal concentration 1.54 mM) was added to bu€er containing DMP (®nal
concentration 1.54 mM) at time zero (H) or after the reaction was initiated with C. hirsutus laccase (G). Spectral scans were carried out at time
intervals indicated in the boxes.

J. Zhang et al. / Phytochemistry 52 (1999) 775±783
781
and decreased with HG addition after the reaction was
initiated (Fig. 4(C)). Again, similar results were
observed for the e€ects of HG on the products from
enzymatic oxidation of ABTS (Fig. 4(E) and (F)).
However, if the reaction was allowed to continue for a
long period of time, approximately 40 min, the absor-
bance and absorption peak at 420 nm began to
increase again (Fig. 4(E)). Oxidation of DMP by lac-
case caused an increase in absorbance at 470 nm (Fig.
4(G)). In contrast, HG addition appeared to cause the
appearance of a new absorption peak at 390 nm that
was generated irrespective whether HG was added at
time zero or after the reaction was initiated (Fig.
4(H)). This suggests that HG may be reacting with the
product of DMP oxidation to generate a di€erent
compound.
products derived from chemical oxidation of SYR,
TOL, ABTS, and DMP was also examined. HG ad-
dition at time zero prevented an increase in absorbance
at the wavelengths usually used to monitor SYR
(525 nm), TOL (630 nm), ABTS (420 nm), and DMP
(470 nm) oxidation. N-hydroxylglycine addition after
the chemical oxidation was initiated caused the absor-
bance to decrease at these wavelengths, in e€ect caus-
ing a ``bleaching'' of color. Similarly, HG decreased
the absorbance/absorption peak at 470 nm during oxi-
dation of DMP if added at some later time. Much like
enzymatic oxidation of DMP, addition of HG at time
zero almost prevented absorbance increases at 470 nm
but showed an increase in the absorption peak at
390 nm.
2.10. Does HG inhibit laccase activity?
2.9. E€ect of HG on the absorption spectra during
chemical oxidation of laccase substrates
All spectral changes suggest that HG is reacting
with products from the enzymatic or chemical oxi-
dation of SYR, TOL, ABTS, and DMP. Thus, it
The e€ect of HG on the absorption spectra of the
Fig. 4 (continued)

782
J. Zhang et al. / Phytochemistry 52 (1999) 775±783
1
appears that HG causes a decrease in the absorption
at those wavelengths used to monitor laccase activity,
resulting in the ``apparent'' but unreal inhibition of en-
zymatic activity. Although de®nite evidence showing
that HG inhibits laccase is not available, there appears
to be ample evidence that HG can interfere with the
product or intermediates in the reactions catalyzed by
laccase. In this sense, HG does not behave as a classi-
cally de®ned enzyme inhibitor. Until de®nitive exper-
iments to determine if HG can inhibit laccase, such as
equilibrium dialysis or radio-isotope tagged HG bind-
ing studies, can be carried out, the current study
suggests that HG does not inhibit laccase by acting as
a classical enzyme inhibitor.
(KBr) 3105, 1601, 1536, 1483, 1399, 1310 cm ;
1H-
NMR d D2O) 3.87 (0CH20); 13C-NMR d (D2O)
56.0, 173.0; MS (FAB) m/z (M-1) 1)90. H and 13C-
1
NMR spectra were recorded at 250 MHZ. Chemical
shifts were reported in ppm down®eld from sodium tri-
methylsilylpropanoate. High resolution fast atom bom-
bardment mass spectra analysis was carried out by the
Nebraska Center for Mass Spectrometry. To con®rm
the identity of HG, X-ray crystallography was carried
out by John Hu€man at the Indiana University
Department of Chemistry Molecular Structure Center
(data not shown).
The crude reaction mixture from which the N-hydro-
xyglycine had been ®ltered was evaporated to dryness
in vacuo at 40 8C to give a mixture of N-hydroxygly-
cine and a substantial amount of a least one unidenti-
®ed compound.
3. Experimental
3.1. Materials and equipment
3.3. Enzyme assays
Pyricularia oryzae laccase was obtained from Sigma
(lot 87F-7704 and 119F-798, St. Louis, MO) and ICN
(lot 61938, Costa Mesa, CA). C. hirsutus laccase was
purchased from Calbiochem (lot 185891, San Diego,
CA), and Rhus vernicifera laccase was from Sigma (lot
96H06761). Extracellular culture ®ltrates from A. bis-
porus, containing laccase, were a gift from R. Kerrigan
(Sylvan Spawn, Worthington, PA). Guaiacol, o-toli-
dine, syringaldazine, 4-aminophenol, 1-hydroxybenzo-
triazole, catechol, dopa, and pyrogallol were obtained
from Sigma (St. Louis, MO). 2,6-dimethoxyphenol and
2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)
were purchased from Sigma-Fluka (Milwaukee, WI).
Sodium periodate and hydroxylamine hydrochloride
were from Fisher Scienti®c (St. Louis, MO). All other
chemicals were of reagent grade.
Oxidations of SYR, TOL, DMP, and ABTS were
monitored spectrophotometrically in 0.1 M sodium
acetate (pH 5.0) at 525, 630, 470, and 420 nm, respect-
ively as described by Flurkey et al. (1995) and Miller,
Kuglin, Gallagher and Flunkey (1997) Sanchez-Amat
and Solano (1997) and Palmieri et al. (1997). Stock
solutions of syringaldazine and o-tolidine were pre-
pared in 95% ethanol. DMP and ABTS stock sol-
utions were prepared fresh in 0.1 M acetate bu€er
pH 5.0. Changes in color/absorbance were followed
using a Hitachi 100-60 recording spectrophotometer
connected to a strip chart recorder. Initial rates were
calculated from the linear portion of the absorbance vs
time curves. Speci®c conditions for each experiment
are noted in the legends. One unit of activity was
de®ned as a change in one absorbance unit per min.
Oxidation of substrates was also monitored using oxy-
gen consumption assays with a Clark oxygen electrode
connected to a YSI model 5300 biological oxygen
meter. Assays were conducted in a circulating water
bath at 23±24 8 C. Changes in oxygen were based on
an air saturated bu€er solution as a reference at
100%.
3.2. Synthesis and characterization of N-hydroxylglycine
N-hydroxyglycine was prepared by a modi®cation of
a procedure reported by Jahngen and Rossomando
(1982). To a stirring solution of sodium cyanoborohy-
dride (0.63 g, 10 mmol) and bromocresol green (5 mg)
in 10 ml of methanol, glyoxalic acid oxime (0.89 g,
10 mmol) was added at room temperature. This sol-
ution was stirred for 3 h, and during that time 12 M
HCl was added dropwise as needed to maintain a yel-
low color; this typically took about 10 drops over the
3 h period. At about 10 min into the reaction time, a
precipitate began to form. At the end of the reaction
time, the crystalline product was recovered by vacuum
®ltration. The compound was recrystalized by dissol-
ving the product in about 4 to 5 ml of hot water
(some bubbling occurred), cooling to room, and then
cooling with an ice bath to give, after drying in vacuo,
0.3 g (33%) N-hydroxyglycine: mp 140 8C(dec); IR
3.4. Chemical oxidation assays
Oxidations of SYR, TOL, DMP, and ABTS were
carried out by additions of a stock solution of sodium
periodate (5 mM) dissolved in acetate bu€er. Changes
in color/absorbance were observed visually and were
also monitored spectrophotometrically as above.
Changes in oxygen consumption, after chemical oxi-
dation of the substrates, were monitored as above.

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Changes in the chemical or enzymatic spectra from
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monitored by scanning absorption spectrophotometry.
Spectra were recorded using a Cary 5 UV-VIS-NIR
spectrophotometer from 350 to 750 nm at a scan rate
of 1000 nm/min. Reactions contained HG added at
zero time or added at some time after the reaction
was initiated. Enzymatic reactions used C. hirsutus
laccase while chemical oxidation reactions used
sodium periodate. Speci®c conditions are noted in the
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Acknowledgements
We appreciate the services of Rhonda Pearson for
all the computer graphics generated. We would like to
thank L. Rosenhein and M. O'Sullivan for a critical
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