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: anappi@svm.vetmed.wisc.edu (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. 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