Báo cáo khoa học: Production and utilization of hydrogen peroxide associated with melanogenesis and tyrosinase-mediated oxidations of DOPA and dopamine docx

Production and utilization of hydrogen peroxide
associated with melanogenesis and tyrosinase-mediated
oxidations of DOPA and dopamine
Maristella Mastore
1
, Lara Kohler
2
and Anthony J. Nappi
2
1 Universita
`
degli Studi dell’Insubria, Dipartimento di Biologia Strutturale e Funzionale, Laboratorio di Immunologia Comparata, Varese, Italy
2 Animal Heath and Biomedical Sciences, University of Wisconsin-Madison, Madison, WI, USA
Human melanins are heteropolymers synthesized by
such diverse cells as those comprising portions of the
skin, hair, inner ear, brain and retinal epithelium.
These multifunctional pigments are derived from a
complex series of enzymatic and nonenzymatic reac-
tions initiated by the hydroxylation of l-phenylalanine
to l-tyrosine. This reaction is mediated by the
enzyme phenylalanine hydroxylase (EC 1.14.16.1), an
iron-containing protein that requires the presence of
the cofactor (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin.
A critical two-step reaction sequence follows involving
the hydroxylation of tyrosine to DOPA (monopheno-
lase activity), and the ensuing oxidation of the o-diphe-
nol (diphenolase activity) to o-quinone (dopaquinone).
Subsequent oxidative polymerizations of indolequinones
yield brown to black eumelanins, whereas similar reac-
tions involving cysteine and glutathione conjugates
of dopaquinone form reddish-brown pheomelanins

(Fig. 1). Neuromelanin, which is also a brown-black
pigment, apparently is restricted to the substantia nigra
pars compacta and certain other regions of the mamma-
lian brain. The pigment is derived in large part from the
oxidation of dopamine (i.e. the decarboxylated deriv-
ative of DOPA) with a variety of nucleophiles, including
thiols derived from glutathione [1–3]. Some of the
numerous factors influencing pigment biogenesis in
mammalian systems include substrate availability, the
presence and concentrations of O
2
, metal ions, thiol
Keywords
hydrogen peroxide; melanogenesis; reactive
intermediates of oxygen; tyrosinase
Correspondence
A. J. Nappi, Animal Heath and Biomedical
Sciences, University of Wisconsin-Madison,
Madison, WI 53706, USA
Fax: +1 608 2627420
Tel: +1 608 2622618
E-mail:
(Received 10 December 2004, revised
3 February 2005, accepted 11 March 2005)
doi:10.1111/j.1742-4658.2005.04661.x
The synthesis and involvement of H
2
O
2
during the early stages of melano-

genesis involving the oxidations of DOPA and dopamine (diphenolase
activity) were established by two sensitive and specific electrochemical
detection systems. Catalase-treated reaction mixtures showed diminished
rates of H
2
O
2
production during the autoxidation and tyrosinase-mediated
oxidation of both diphenols. Inhibition studies with the radical scavenger
resveratrol revealed the involvement in these reactions of additional react-
ive intermediate of oxygen (ROI), one of which appears to be superoxide
anion. There was no evidence to suggest that H
2
O
2
or any other ROI was
produced during the tyrosinase-mediated conversion of tyrosine to DOPA
(monophenolase activity). Establishing by electrochemical methods the
endogenous production H
2
O
2
in real time confirms recent reports, based in
large part on the use of exogenous H
2
O
2
, that tyrosinase can manifest both
catalase and peroxidase activities. The detection of ROI in tyrosinase-medi-
ated in vitro reactions provides evidence for sequential univalent reductions

of O
2
, most likely occurring at the enzyme active site copper. Collectively,
these observations focus attention on the possible involvement of peroxi-
dase-H
2
O
2
systems and related ROI-mediated reactions in promoting
melanocytotoxic and melanoprotective processes.
Abbreviation
ROI, reactive intermediate of oxygen.
FEBS Journal 272 (2005) 2407–2415 ª 2005 FEBS 2407
compounds, and reducing agents, the activities of mela-
nogenic enzymes and competitive oxidases, and the
availability of enzyme cofactors.
The two-step reaction sequence that converts tyro-
sine to dopaquinone is regulated by tyrosinase (EC
1.14.18.1), a ubiquitous copper-containing enzyme that
requires both O
2
and a source of reducing equivalents
(Fig. 2). The iron-containing tyrosine 3-hydroxylase
(E.C. 1.14.16.2), which is localized primarily in ner-
vous tissue, also hydroxylates tyrosine to DOPA
utilizing tetrahydrobiopterin as a cofactor, but the
enzyme does not ordinarily oxidize the o-diphenol to
o-quinone. Apparently, when sufficient amounts of
thiols are available, tyrosine 3-hydroxylase can oxidize
DOPA [4]. Peroxidase (EC 1.11.1.7), which also is an

iron-containing enzyme, can readily perform the two-
step reaction sequence, provided hydrogen peroxide
(H
2
O
2
) is present (Fig. 2). Compared to tyrosinase,
disproportionately less effort has been given to under-
standing the role of peroxidase in the early stages of
melanogenesis, despite reports of the involvement of
peroxidase–H
2
O
2
systems in later stages during the
oxidation of indolequinone precursors of eumelanin
and benzothiazinylalanine precursors of pheomelanin
[5–10]. Of considerable interest are recent studies that
have kinetically characterized both catalase (EC
1.11.1.6) (i.e. conversion of H
2
O
2
to ½O
2
and H
2
O)
and peroxygenase (H
2

O
2
-dependent oxygenation of
substrates) activities of tyrosinase [11], suggesting the
latter enzyme also can utilize H
2
O
2
, if available, to
metabolize substrates.
The role of H
2
O
2
in melanogenesis has not been
clearly defined, with some reports indicating it func-
tions to enhance pigment formation by regulating
Fig. 1. Overview of the principal melanotic
pathways and some of the proposed sites
of activity of DOPA decarboxylase (DDC),
tyrosinase (TYR), tyrosine-3-hydroxylase
(TAH), peroxidase (PER), and phenylalanine
hydroxylase (PAH). BH
4
, tetrahydrobiopterin;
GSH, reduced glutathione.
Fig. 2. Comparison of the modes of action
of tyrosinase and peroxidase in converting
tyrosine to dopaquinone. In the in vitro
tyrosinase-mediated assays conducted,

endogenous H
2
O
2
was detected, but only
when the enzyme was engaged in dipheno-
lase activity. RH
2
, compounds contributing
reducing equivalents.
Hydrogen peroxide in tyrosinase-mediated reactions M. Mastore et al.
2408 FEBS Journal 272 (2005) 2407–2415 ª 2005 FEBS
levels of tyrosinase [12], others suggesting the molecule
serves as a potent inhibitor of tyrosinase [13]. The
problem in attempting to identify and clarify peroxida-
tive activity during melanogenesis is that past assess-
ments frequently have been based in large part
on reaction rates following exposure of cells to either
exogenous H
2
O
2
[8,11], or to various reactive inter-
mediates of oxygen followed by inhibition assays.
Observations of enhanced enzyme-mediated oxida-
tions following exposure of cells to endogenous H
2
O
2
alone are insufficient to document normal peroxidative

activity during melanogenesis. Also, quantitative deter-
minations of enzyme-mediated reactions based on
spectrometric methods may be inaccurate because of
pigment-bleaching and related modifications resulting
from exogenous H
2
O
2
.
In this investigation, specific and sensitive electro-
chemical methods were employed in conjunction with
enzyme inhibition studies to ascertain H
2
O
2
production
in vitro during the tyrosinase-mediated conversion of
tyrosine to dopaquinone. Comparative quantitative
data showed that H
2
O
2
was generated only during the
oxidation of DOPA to dopaquinone, but not during
the hydroxylation of tyrosine to DOPA. Tyrosinase-
mediated diphenolase activity was enhanced by the
endogenously generated H
2
O
2

, and by at least one
other reactive intermediate of oxygen (ROI). The
results of this investigation support studies implicating
the involvement of these potentially cytotoxic ROI in
melanogenesis [8,14,15].
Results
Initial experiments were performed with HPLC-ED to
determine if, and to what extent, H
2
O
2
was generated
during the autoxidations and tyrosinase-mediated oxi-
dations of tyrosine, DOPA, and dopamine. This sensi-
tive and specific method was effective in detecting
changes in the levels of monophenol and diphenol sub-
strates in concentrations ranging from 0.1 nm (not pre-
sented) to 0.5 nm (Fig. 3), and provided comparative
quantitative data with which to assess the effect of cata-
lase on substrate oxidation (Fig. 4). Although cata-
lase was not shown to have an inhibitory effect on the
tyrosinase-mediated oxidation of tyrosine, the oxida-
tions of both diphenol substrates were significantly
reduced by catalase. With reaction mixtures containing
catalase, the percentage of DOPA (initial concentra-
tion 0.1 mm) oxidized in 5 min incubations averaged
61.3%, compared to 38% substrate oxidation in reac-
tion mixtures lacking catalase (Fig. 4). In these experi-
ments, the rates of reaction averaged 48.4 pmÆmin
)1

with catalase, and 81.2 pmÆmin
)1
without catalase.
Similar results were obtained with the tyrosinase-medi-
ated oxidation of dopamine (initial concentration
0.01 mm), with approximately 3.5 times less substrate
oxidized with catalase than without catalase. These
catalase-inhibited oxidations of DOPA and dopamine
strongly implicate the involvement of H
2
O
2
in the
diphenolase activity of tyrosinase. Insufficient amounts
of substrate were autoxidized in 5 min assays to com-
pare, by HPLC-ED, the inhibitory effects of catalase.
To verify the involvement of H
2
O
2
in the dipheno-
lase activity of tyrosinase, reaction mixtures identical
to those used for the above HPLC-ED analyses were
monitored with the APOLLO Free Radical Detector
(APOLLO 4000) equipped with an H
2
O
2
sensor. At
a pulse voltage of +400 mV, H

2
O
2
production was
observed during the autoxidation and enzyme-medi-
ated oxidation of DOPA (Fig. 5) and dopamine (not
presented), but not in reaction mixtures containing
catalase. After 5 min incubation, 5 lL samples were
removed and analyzed by HPLC-ED to determine
rates of reaction. The rate of DOPA autoxidation
was 0.8 pmÆmin
)1
. In reaction mixtures containing
DOPA and tyrosinase, the rate of substrate oxidation
Fig. 3. Representative chromatograms of the autoxidation and
tyrosinase-mediated oxidations of DOPA, with and without cata-
lase. Peak profiles represent levels of DOPA in 5 lL samples of
separate reaction mixtures after 5 min incubation. Initial level of
DOPA (2.5 nm) prior to incubation is indicated (Æ). Reaction mixtures
contained 0.5 mm DOPA, and 10 lg each of tyrosinase (3870
UÆmg
)1
) and catalase (15 700 UÆmg
)1
), in a total volume of 100 lL
NaCl ⁄ P
i
(10 mm; pH 7.4). Chromatographic conditions were
+675 mV, 200 nA full scale, and a flow rate of 0.8 mLÆmin
)1

.
M. Mastore et al. Hydrogen peroxide in tyrosinase-mediated reactions
FEBS Journal 272 (2005) 2407–2415 ª 2005 FEBS 2409
averaged 77 pmÆmin
)1
, approximately 1.5 times faster
than in incubations with catalase. The rate of DOPA
oxidation averaged 53 pmÆmin
)1
in reaction mixtures
containing 0.1 lgÆmL
)1
catalase, and 48.9 pmÆmin
)1
in reaction mixtures containing 0.5 lg ÆmL
)1
catalase
(Fig. 5C). Similar electrochemical response profiles
were observed showing catalase inhibition of H
2
O
2
generation during tyrosinase-mediated oxidation of
dopamine (not presented), and during autoxidation
when varying amounts of the substrate were added to
a solution containing only buffer (pH 7.4) (Fig. 6). A
concentration-dependent electrochemical response was
generated with 50–500 nm dopamine.
Because endogenous H
2

O
2
generation during the oxi-
dations of DOPA and dopamine very likely resulted
from the univalent reduction of O
2
, we were interested
to learn if other intermediates of oxygen also were gen-
erated during the oxidations of these two diphenols. In
subsequent experiments the radical scavenger resvera-
trol was used in reaction mixtures to ascertain the
involvement of additional ROI during the autoxidation
of diphenols. In these experiments, varying amounts of
dopamine were introduced into reaction mixtures and
then monitored by the free radical detector. With resve-
ratrol, there was a significant decrease in the amount of
H
2
O
2
produced (Fig. 6). With 50 nm dopamine, 1.1 lm
of H
2
O
2
was produced in reaction mixtures containing
resveratrol, compared to 3.5 lm of H
2
O
2

in mixtures
lacking resveratrol (Fig. 7). With 100 nm dopamine,
2.5 lm H
2
O
2
was produced in reaction mixtures con-
taining resveratrol, compared to 8.7 lm H
2
O
2
in mix-
tures lacking resveratrol. Thus, both H
2
O
2
and at least
one additional ROI were generated during the tyrosin-
ase-mediated oxidations of DOPA and dopamine. The
identity of the ROI could not be determined by inhi-
bition studies using resveratrol, which reportedly
effectively scavenges other partially reduced oxygen
intermediates, including superoxide anion (ÆO
2
–
) and
the hydroxyl radical (ÆOH) [16–18], species that precede
and follow, respectively, H
2
O

2
production by sequen-
tial univalent reduction reactions of O
2
(Eqns 1–6).
The addition of superoxide dismutase (SOD; (EC
1.15.1.1), which converts ÆO
2
–
to H
2
O
2
(Eqn 7), into
reaction mixtures containing tyrosinase and either
DOPA (Fig. 8) or dopamine (not shown) produced a
slight but statistically significant (P<0.05) increase in
the tyrosinase-mediated oxidations of the two diphen-
ols. In reaction mixtures incorporating both tyrosinase
(0.05 lgÆlL
)1
) and SOD (0.4 lgÆlL
)1
), the rate of
tyrosinase-mediated oxidation of DOPA averaged
215 pmÆmin
)1
,15±2pmÆmin
)1
higher than in control

incubations lacking SOD. With the concentration of
tyrosinase increased to 0.1 lgÆlL
)1
, the rate of DOPA
oxidation averaged 368 pmÆmin
)1
,22±4pmÆmin
)1
higher than in incubations lacking SOD (Fig. 8). No
DOPA oxidation was recorded in control mixtures
containing SOD, but lacking tyrosinase.
O
2
þ e
À
!ÁO
2
À
ð1Þ
ÁO
À
2
þ e
À
! HO
2
ð2Þ
HO
2
þ e

À
þ H
þ
! H
2
O
2
ð3Þ
H
2
O
2
þ e
À
!ÁOH þ HO
À
ð4Þ
HO
À
þ e
À
! H
2
O ð5Þ
Fe
2þ
þ H
2
O
2

! Fe
3þ
þÁOH þ HO
À
ðFenton reactionÞð6Þ
O
À
2
þ O
À
2
þ 2Hþ!
SOD
H
2
O
2
þ O
2
ð7Þ
Discussion
Melanogenesis entails the conversion of the amino
acid tyrosine, through a series of intermediates, to
yield dopaquinone derivatives that eventually polymer-
Fig. 4. Effects of catalase on the tyrosinase-
mediated oxidations of tyrosine, DOPA and
dopamine during 5 min incubations. Except
for the differences specified in the
concentrations of each substrate tested
(0.01–0.1 m

M), reaction mixture compo-
nents were identical to those given in
Fig. 3, as were the chromatographic condi-
tions established for the assays. Data pre-
sented represent means and ranges for at
least three replicate experiments.
Hydrogen peroxide in tyrosinase-mediated reactions M. Mastore et al.
2410 FEBS Journal 272 (2005) 2407–2415 ª 2005 FEBS
ize to form pigment. The identity and mode of action
of the enzymes involved in the different steps of mel-
anogenesis have long been intensely investigated, in
large measure to elucidate the etiology of certain pig-
mentation disorders, and to better understand the fac-
tors underlying melanoprotective and melanocytotoxic
processes [10,19]. It is now generally acknowledged
that the key regulatory enzyme of melanogenesis in
melanocytes and melanoma cells is tyrosinase (Chun
et al. 2001), which normally utilizes O
2
to catalyze the
initial two-step conversion of tyrosine to dopaquinone
[19]. A peroxidase–H
2
O
2
system appears to be involved
during the terminal stages of melanogenesis, acting
solely or collaboratively with tyrosinase in the oxida-
tive polymerizations of pigment precursors [5,14]. Sur-
prisingly, very few reports have considered a more

central role for a peroxidase–H
2
O
2
mechanism in initi-
ating melanogenesis [9].
In this investigation, extremely sensitive and specific
electrochemical methods detected and quantitatively
measured, in real time, the production of H
2
O
2
dur-
ing tyrosinase-mediate oxidations of the DOPA and
dopamine. Comparative analyses of reaction rates
Fig. 5. Profiles of electrochemical responses generated by the
endogenous production H
2
O
2
during the autoxidation (A) and
tyrosinase-mediated oxidations (B,C) of
L-DOPA. For analyses by
APOLLO 4000 Detector, reaction mixtures initially contained 0.1 m
M
L
-DOPA in a total volume of 2 mL NaCl ⁄ P
i
(10 mM pH 7.4).
Enzyme(s) was(were) introduced 2–3 min after equilibration of the

H
2
O
2
sensor, and separate reaction rates were determined with
HPLC-ED by analyzing 5 lL of each reaction mixture at 5 min postin-
cubation. Catalase was included in C (0.5 lgÆlL
)1
). Chromatographic
conditions for determining reaction rates were +675 mV, 200 nA full
scale, and a flow rate of 0.8 mLÆmin
)1
. Pulse voltage was maintained
at +400 mV.
Fig. 6. Electrochemical responses resulting from H
2
O
2
production
during the autoxidation of dopamine following the addition of vary-
ing amounts of the diphenol into solutions of NaCl ⁄ P
i
(pH 7.4) that
lacked catalase (A and B) and those with catalase (C). Arrows indi-
cate times when dopamine was incorporated in the reaction mix-
tures. Pulse voltage was maintained at +400 mV.
M. Mastore et al. Hydrogen peroxide in tyrosinase-mediated reactions
FEBS Journal 272 (2005) 2407–2415 ª 2005 FEBS 2411
showed the tyrosinase-mediated oxidations to be sig-
nificantly diminished in the presence of catalase, indi-

cating that the H
2
O
2
generated during these reactions
was utilized as a cofactor in generating the corres-
ponding o-quinones. Additionally, inhibition studies
with resveratrol showed that at least one ROI also
was generated during the tyrosinase-mediated oxida-
tion of DOPA and dopamine. Experiments showing
slightly enhanced tyrosinase-mediated oxidations in
the presence of SOD implicate ÆO
2
–
in the process,
with SOD converting the radical to H
2
O
2
. Collec-
tively, the results of this investigation support in part
the recent observations made by Yamazaki and co-
workers [11], who reported that in the presence of
exogenous H
2
O
2
tyrosinase exhibited both catalase
and peroxidase activities, and the studies by Wood
et al. [20] showing the enzyme to be activated by low

concentrations of H
2
O
2
.
Unquestionably, the generation of H
2
O
2
and other
ROI during tyrosinase-mediated melanogenesis repre-
sent a potentially dangerous situation, but one that is
apparently successfully circumvented by the enzyme
employing these molecules to metabolizing substrates.
A likely scenario for the production of these mole-
cules involves the partial reduction of O
2
caused by
sequential univalent transfers (Eqns 1–5). The latter
reactions are readily initiated by catalytic metals (e.g.
Cu
+
and Fe
2+
), which normally are sequestered or
otherwise rendered unavailable for such reactivity in
biological systems. Metalloenzymes, such as tyrosinase
and peroxidase, represent important sources for these
metal catalysts. Substrate binding by these enzymes
can expose active site copper and iron, respectively,

and initiate localized univalent reductions of O
2
that
sequentially produce superoxide anion (ÆO
2
–
), H
2
O
2
,
and ÆOH, en route to forming H
2
O. With the in vitro
system used in this study, the copper-containing tyro-
sinase was the only known source of metal. Presuma-
bly, normal enzyme activity either prevents catalytic
engagement of the active site copper with H
2
O
2
, or the
enzyme capitalizes on this reactivity to metabolize sub-
strates. However, it would be detrimental for a single
enzyme to engage in the simultaneous reduction of O
2
and Cu
2+
or Fe
3+

, because this activity also can gen-
erate cytotoxic ÆOH by the Fenton reaction (Eqn 6),
with the enzyme inactivated, if not destroyed, along
with any bound ligand. Thus, it would be imperative
for metalloenzymes engaging O
2
in their metabolism of
A
B
Fig. 7. Effects of the radical scavenger resveratrol on the H
2
O
2
generation during autoxidation of 50 and 100 nM dopamine in
NaCl ⁄ P
i
. Diminished H
2
O
2
levels in presence of resveratrol impli-
cates involvement of one or more additional ROI in the autoxidation
of diphenols. Chromatographic conditions were +675 mV, 200 nA
full scale, and a flow rate of 0.8 mLÆmin
)1
.
Fig. 8. Representative chromatographs showing the effects of
SOD on tyrosinase-mediated oxidation of DOPA. Peak profiles rep-
resent levels of DOPA in 5 lL samples of separate reaction
mixtures after 1 min incubations. Reaction mixtures contained

0.1 mm DOPA, 40 lg of SOD (30 000 UÆmg
)1
), and either 5 or
10 lg tyrosinase (3870 unitsÆmg
)1
), in a total volume of 100 lL
NaCl ⁄ P
i
(10 mm; pH 7.4). Chromatographic conditions were
+675 mV, 500 nA full scale, and a flow rate of 0.8 mLÆmin
)1
.
Hydrogen peroxide in tyrosinase-mediated reactions M. Mastore et al.
2412 FEBS Journal 272 (2005) 2407–2415 ª 2005 FEBS
substrates to employ a spatial or temporal separation
between univalent reductions of O
2
and the redox cyc-
ling of metal ions at the active site. Aberrant redox
activity involving catalytic metals at the enzyme active
site activity may cause or contribute to certain
pathologies associated with melanogenesis. The results
of this investigation provide a focus for future studies
to clarify reports that correlate elevated levels of tyro-
sinase in melanoma cell lines with cytotoxicity [5], as
well as numerous reports attributing cytoprotective
roles to melanin and the enzymes involved in pigment
biosynthesis.
Experimental procedures
Chemicals

All reagents used in this study were obtained from Sigma
Chemical Company (St. Louis, MO, USA). Stock solutions
of all components were prepared daily in ultrapure reagent-
grade water obtained with a Milli-Q system (Millipore,
Bedford, MA, USA), filtrated on Acrodisc LC13 PVDF
0.2 lm and immediately used or kept at 4 °C for a maxi-
mum period of 3 h and then discarded.
Reaction mixtures and enzyme assays
Substrate concentrations used to measure rates of autoxi-
dations and enzyme-mediated oxidations ranged from
0.1 mm to 1 mm in a total volume of 100 lL of phos-
phate-buffered saline (NaCl ⁄ P
i
) pH 7.4. Unless specified
otherwise, enzyme-mediated reaction mixtures contained
10 lg tyrosinase (EC 1.14.18.1; 3870 UÆmg
)1
), either with
or without equal amounts of catalase (EC 1.11.1.6;
15 7000 UÆmg
)1
) or superoxide dismutase (EC 1.15.1.1;
30 000 U). Quantitative determinations of the monopheno-
lase activity of tyrosinase were made by measuring the
exact amount of DOPA formed during each incubation.
This was made possible by the addition of ascorbic
acid (0.1 mm) to the reaction mixture, which prevented
any subsequent enzyme-mediated oxidation of DOPA to
dopaquinone. Quantitative determinations of the dipheno-
lase activity of tyrosinase were made by measuring the

depletion of diphenol substrates (DOPA and dopamine)
in reaction mixtures lacking a reductant. Following incu-
bation at 22 ° C, 5 lL aliquots were removed from each
reaction mixture and analyzed by high performance
liquid chromatography with electrochemical detection
(HPLC-ED). Control experiments were conducted by
excluding substrate, enzyme, or H
2
O
2,
as was appropriate
for different experiments. Tyrosinase activity was
expressed as pmÆmin
)1
of product formed (monopheno-
lase activity) or substrate oxidized (diphenolase activity)
oxidized.
HPLC-ED analyses
The HPLC system consisted of a Gilson (Madison, WI,
USA) 119 UV ⁄ visible spectrophotometer and a Bioanalyti-
cal Systems (West Lafayette, IN, USA) LC-4B ampero-
metric detector with a glassy carbon working electrode
and an Ag ⁄ AgCl reference electrode. The working elec-
trode was maintained at an oxidative potential
(+675 mV). Rates of autoxidation and enzyme-mediated
oxidations were determinate by calculating amounts of
products synthesized (monophenolase activity) or sub-
strates depletion (diphenolase activity). Instrument sensitiv-
ity established for each assay is specified with the data
presented. The solvent system used to quantitatively deter-

mine rates of both autoxidations and enzyme-mediated
reactions was comprised of 50 mm citrate buffer (pH 2.9)
containing 0.4 mm Na
2
EDTA, 0.2 m m sodium octyl sul-
fate, and 5% (v ⁄ v) acetonitrile. The pH was adjusted to
3.0 with 1 m NaOH prior to the addition of acetonitrile.
All separations were made with Alltech Spherisorb
ODS 2.5 lm reverse phase column using a flow rate of
0.8 mLÆmin
)1
.
Quantitative determinations of H
2
O
2
production
The APOLLO 4000 Free-Radical Analyzer (World Preci-
sion Instruments, Inc., Sarasota, FL, USA) was used to
monitor in real-time the production of H
2
O
2
during the
autoxidations and tyrosinase-mediated oxidations of
DOPA and dopamine. A pulse voltage (+400 mV) main-
tained on a sensitive and selective H
2
O
2

sensor (ISO-
HOP2) ensured that the electrochemical responses (redox
current) generated at the working electrode were
derived only from the oxidation of any H
2
O
2
formed,
and that these responses were proportional to the con-
centration of the reactive molecule. Quantitative deter-
minations were made following the establishment of
calibration curves for the H
2
O
2
electrode prior to and
following all tests. The latter was obtained by plotting
changes in current (pA) against changes in H
2
O
2
concen-
tration. Test conditions, such as temperature and pH, were
identical to those under which the instrument was
calibrated.
To assess H
2
O
2
production, the electrode was

allowed to equilibrate for 1–3 min in reaction mix-
tures (2 mL 10 mm NaCl ⁄ P
i
, pH 7.4) that were stirred
continuously by a magnetic agitator. Some reaction mix-
tures contained substrate (tyrosine, DOPA or dopam-
ine) prior to enzyme treatment, whereas in other mixtures
substrate was introduced at specific intervals follow-
ing equilibration. At specific times after incubation,
5 lL samples were removed and processed by
HPLC-ED as described above to determine rates of
oxidation.
M. Mastore et al. Hydrogen peroxide in tyrosinase-mediated reactions
FEBS Journal 272 (2005) 2407–2415 ª 2005 FEBS 2413
Electrochemical analyses of ROI production
Resveratrol, a non flavonoid polyphenolic radical scavenger
[23–26] was used to determine to what extent additional
ROI were produced in conjunction with the H
2
O
2
gener-
ated during the autoxidation of diphenols. For these studies
50 and 100 nm of dopamine were introduced into reaction
mixtures that either contained resveratrol (500 nm), or
lacked the scavenger. Comparative levels of H
2
O
2
produc-

tion in presence and absence of the radical scavenger were
measured by the APOLLO 4000 Free-Radical Analyzer as
described above.
Statistical analysis
Differences between mean values were evaluated using
the Student’s paired t-test and considered significant when
P < 0.05. All experiments were replicated at least three
times.
Acknowledgements
We acknowledge with appreciation the financial
support received for these investigations from the
National Science Foundation (IBN 0342304) and the
National Institutes of Health (GM 059774).
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