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

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

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

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

, 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

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

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

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

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

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

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-

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,

, 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

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

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

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-

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