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Oxidation of melatonin and tryptophan by an hrp cycle involving compound iii

Biochemical and Biophysical Research Communications 287, 130 –134 (2001)
doi:10.1006/bbrc.2001.5557, available online at http://www.idealibrary.com on Oxidation of Melatonin and Tryptophanby an HRP Cycle Involving Compound III Valdecir F. Ximenes,* Luiz H. Catalani,† and Ana Campa*,1*Faculdade de Cieˆncias Farmaceˆuticas and Instituto de Quı´mica, Universidade de Sa˜o Paulo,CEP 05508-900, Sa˜o Paulo, Brazil Received July 16, 2001 pound I, compound II and native enzyme (3). Peroxi- We recently described that horseradish peroxidase
dases also exhibit oxidase activity in the presence of (HRP) and myeloperoxidase (MPO) catalyze the oxi-
NADH, in a enzyme cycle producing compound III and dation of melatonin, forming the respective indole
ferrous peroxidase (4, 5). In the latter case, hydroxy- lated products are formed, probably at expenses of kynuramine (AFMK) (Biochem. Biophys. Res. Commun.
hydroxyl radical produced from superoxide anion and 279, 657– 662, 2001). Although the classic peroxidatic
enzyme cycle is expected to participate in the oxida-
The increased interest in the peroxidase-catalyzed tion of melatonin, the requirement of a low HRP:H O
oxidation of biological indoles is noteworthy in the re- ratio suggested that other enzyme paths might also be
cent literature (1, 2, 7–9). The reactions of tryptophan operative. Here we followed the formation of AFMK
or melatonin with compound I and II of MPO were under two experimental conditions: predominance of
recently reported. Tryptophan reacts rapidly with com- HRP compounds I and II or presence of compound III.
pound I (7) and melatonin reacts efficiently with both Although the consumption of substrate is comparable
compound I and compound II (8). Although these data under both conditions, AFMK is formed in significant
support a common peroxidatic cycle in the oxidation of amounts only when compound III predominates dur-
these compounds, our initial observation that the oxi- ing the reaction. Using tryptophan as substrate, N-
dation of indolic compounds by HRP and MPO requires formyl-kynurenine is formed in the presence of com-
high concentrations of H O is an indication that other pound III. Both, melatonin and tryptophan efficiently
enzyme paths might also be operative.
prevents the formation of p-670, the inactive form of
HRP. Since superoxide dismutase (SOD) inhibits the

Here, we report the HRP-catalyzed production production of AFMK, we proposed that compound
of indole ring-opening products of melatonin and III acts as a source of O• or participates directly in
the reaction, as in the case of enzyme indoleamine
(AFMK) and N-formyl-kynurenine (NFK), respectively, under two different reaction conditions: where there is 2001 Academic Press
Key Words: HRP; horseradish peroxidase; compound
a predominance of HRP compounds I and II of the III; indolic compounds; indoleamine 2,3-dioxygenases,
peroxidatic cycle or where compound III is present.
kynurenine, melatonin; oxidation; p-670; peroxidase;
superoxide anion; tryptophan.

MATERIALS AND METHODS Catalase (EC 1.11.1.6; from bovine liver), superoxide dismutase (SOD; EC 1.15.1.1; from bovine erythrocytes), horseradish peroxi- We recently described that, when high concentra- dase (HRP; EC 1.11.1.7; type VI), melatonin, L-tryptophan, DL- tions of H O are used, HRP and MPO catalyse the kynurenine, mannitol and NADH were from Sigma. Hydrogen per- oxidation of indole compounds in a reaction that con- oxide (60%, from Interox) was diluted to the appropriate stockconcentration and spectrophotometrically measured (10). The con- sumes oxygen, triggers chemiluminescence and forms centration of HRP was determined by absorption at 403 nm using a indole ring opening products (1, 2).
molar absorptivity of 1.02.105 M⫺1 cm⫺1 (11).
Peroxidase uses H O to form the active compound I UV-Vis spectra were recorded on a Shimadzu Multispec-1501 spec- that catalyses the dehydrogenation of several sub- trophotometer. The reaction mixtures were analyzed by high perfor-mance liquid chromatography using a SHIMADZU LC-10A system strates in a peroxidatic cycle, classically involving com- coupled to SPD-10A UV-Vis and RF535 fluorescence detectors. Theanalyses were carried out on a Luna C-18 reversed phase column 1 To whom correspondence should be addressed. Fax: (55-11) 3813- (25 ⫻ 4.6 mm, 5 ␮m) in isocratic mode using 1 mmol/L KH PO , 2197. E-mail: anacampa@usp.br.
pH4.0/acetonitrile 3:1 as mobile phase at a constant flow rate of 1 0006-291X/01 $35.00 Copyright 2001 by Academic PressAll rights of reproduction in any form reserved.
Vol. 287, No. 1, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Absorption spectral changes of HRP during melatonin oxidation under the standard reaction condition a. Compound II HPLC profile of the melatonin/HRP/H2O2 standard reac- (Soret band at 420 nm and absorbances at 527 and 554 nm) predom- tion obtained after 30 min. From the top to the bottom the profiles inates during the first 20 min, at which time it changes to the native are: melatonin (MLT), condition a and b. For each profile, the upper form. Scans were recorded every 3 min.
and lower lines correspond to absorbance and fluorescence detection,respectively.
mL/min. The mass spectra were obtained employing a Hewlett– 20 min, the spectrum returns to that of the native Packard 5988 quadrupole mass spectrometer attached to a 5890 gas chromatograph using a HP1 (12 m ⫻ 0.25 mm ⫻ 0.25 ␮m) column.
The formation of AFMK during the reaction was followed in a SPEX- Under condition b, the HRP:H O ratio is 1:2000 FLUOROLOG 1681 fluorometer with a cooled photomultiplier.
and, as shown in Fig. 2, the characteristic compound Unless otherwise stated, the standard reaction mixture was: III spectrum predominates over the entire 30 min of [HRP] ⫽ 1 ␮mol/L, [H2O2] ⫽ 10 ␮mol/L (condition a) or 2 mmol/L reaction (Soret band at 418 nm and absorbances at 544 (condition b), melatonin and tryptophan ⫽ 50␮mol/L in 0.05 mol/Lphosphate buffer pH 7.4, at 37°C and final volumes of 3 mL. Typi- and 577 nm). During the reaction an increased absor- cally, the reaction was initiated by addition of hydrogen peroxide.
bance is clearly observed in the 340 nm region. Thiscorresponds to the absorption of the indole ring- opening product from melatonin, AFMK. After 30 min,the HPLC profiles of the reactions performed under The formation of indole ring-opening products and condition a and b (Fig. 3) show similar consumption of peroxidase spectral features were followed under two melatonin. However, a prominent signal corresponding conditions: at low concentration of H O , where com- to formation of AFMK (␭ ⫽ 340 nm; ␭ ⫽ 460 nm; pounds I and II prevail (condition a), and at high H O MS (m/z): 264(7), 176(69), 160(100), 150(24), 117(13)) concentration, where there is a predominance of com- was observed only in condition b.
pound III (condition b).
The differences in AFMK production under condi- Under condition a, the HRP:H O ratio is 1:10 and tions a and b can be clearly seen in Fig. 4. The con- the rate liming step is the conversion of compound II to sumption of melatonin (initial 50 ␮M) in three experi- the native form; if melatonin is present, the spectrum ments was found to be 30 ⫾ 3 and 32 ⫾ 7 ␮M under is dominated by compound II (Soret band at 420 nm conditions a and b, respectively, while the formation of and absorbances at 527 and 554 nm) (Fig. 1). After AFMK under condition a is only 13% of that observedfor condition b.
The formation of p-670, an inactive form of HRP, is expected in the presence of high concentrations of H O Absorption spectral changes of HRP during melatonin oxidation under the standard reaction condition b. Compound III(Soret band at 418 nm and absorbances at 544 and 577 nm) predom- Kinetics of AFMK production under condition a versus inates over the entire 30 min of reaction. Scans were recorded every condition b measured by fluorescence at 460 nm (␭ melatonin/H2O2 control reaction is also shown.
Vol. 287, No. 1, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS HPLC profile of the melatonin/HRP/H2O2 system ob- Effect of melatonin (50 ␮mol/L and 0.5 mmol/L, B and D, tained after 30 min under condition b at different pHs: 5.0 (B); 6.0 respectively) and of tryptophan (0.5 mmol/L, C) in preventing p-670 (C); 7.4 (D); and 9.0 (E). Profile A corresponds to melatonin (50 formation during the reaction of HRP/H 2O2 (A). The reactions were run under condition b. The inset corresponds to the HRP absorptionspectra of reactions A and B taken after 15 min, showing the degra-dation of compound III and formation of p-670 (I) and conservation of but, in the latter case, there is a pronounced formation compound III (II), respectively.
To verify whether a different compound III generat- ing system was also able to catalyse the formation of and occurs via the decomposition of compound III (11).
AFMK, the reaction between NADH and HRP was Figure 5 shows the formation of p-670 when HRP is tested. At pH 5.0, which is the classical condition for mixed with H O at a 1:2000 ratio. The presence of production of compound III (12), there is no melatonin melatonin clearly causes a protection of HRP, preclud- consumption or AFMK formation (data not shown).
ing the formation of p-670670. This protection depends Since neutral to basic pH seems to be required for on the melatonin concentration.
AFMK production, the NADH/HRP system was tested The effect of SOD (166 U/mL) and the hydroxyl rad- at pH 7.4. In this condition, we found that continuous ical scavenger mannitol (100 mM) on the oxidation of bubbling of O resulted, after 8 min of reaction, in a melatonin and AFMK production were examined. The spectrum dominated by compound III absorption (Fig.
addition of SOD inhibit the production of AFMK (Fig.
8). Employing this condition, melatonin is consumed 6), without affecting melatonin consumption (data not and AFMK is formed (Fig. 8, inset). The addition of shown). Mannitol had no effect on either melatonin catalase (150 U/mL) did not affect the compound III consumption or AFMK production.
spectrum or AFMK production (data not shown).
The effect of pH on melatonin consumption and In some selected experiments, tryptophan was em- AFMK production was studied in the 5.0 to 9.0 range ployed to determine whether it could also be oxidized in using phosphate buffer. Figure 7 shows that, at acidic a similar way as melatonin. Figure 9 shows the com- pHs, the consumption of melatonin is much faster.
pound III spectrum in the presence of tryptophan at a However, no AFMK could be detected at pH 5.0 andonly a small amount was observed at pH 6.0. Theconsumption of melatonin is similar at pH 7.4 and 9.0 Generation of compound III in the NADH system. A clear spectrum of compound III (—) appears at around 8 min under thereaction conditions: [HRP], 4 ␮mol/L; [NADH], 1.5 mmol/L; [MLT], Effect of SOD (166 units/mL) and mannitol (100 mmol/L) 0.5 mmol/L in 50 mmol/L phosphate buffer, pH 7.4, at 37°C with on the kinetics of AFMK production measured by fluorescence at 460 continuous O2 bubbling. The predominance of native enzyme can be observed after 30 min of reaction ( . . ). The inset shows the HPLC 340 nm). The control is the reaction under condition b without any addition.
profile after 30 min of reaction.
Vol. 287, No. 1, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Native enzyme, compound I, compound II, compound III and ferrous enzyme are interconvertible forms, de-pending on the conditions. It is probable that, undercondition b and in the HRP/NADH system, all theseredox states of peroxidase coexist and that differentenzyme cycles catalyze the formation of different oxi-dation products. Hence, we propose two routes for mel-atonin oxidation catalyzed by peroxidases. The firstone involves the common HRP cycle and it is not theprincipal path responsible for AFMK formation, thesecond route involving compound III being required for Spectral change of HRP during tryptophan (0.5 mmol/L) AFMK formation. The inhibitory effect of SOD sug- oxidation under the reaction condition b; scans were recorded every gests that compound III might act as a source of O⫺• 5 min (left) and HPLC profile of this reaction determined after 120 involved in AFMK production. Superoxide anion would min (B) (right). The chromatograms also show the formation of NFK have to react with a melatonin radical since it does not and its conversion to kynurenine (Kyn) after 1 h heating at 56°C (A).
react directly with melatonin (data not shown). In this high concentration of H O (condition b) and the HPLC case, the common peroxidatic cycle generating a mela- profile observed after 120 min of reaction, where NFK tonin radical would then take place simultaneously and kynurenine are seen. As in the case of melatonin, with the cycle forming compound III. In the experi- the presence of tryptophan also inhibits p-670 forma- ments where compound III was obtained at the ex- tion (see Fig. 5).
pense of NADH, melatonin cation radical was also pro-posed to be formed (9). Although O⫺• is involved in AFMK formation, the participation of hydroxyl radicalis excluded by the absence of a mannitol effect.
The oxidation of common substrates by peroxidases Since SOD also accelerates the decomposition of usually involves the native enzyme– compound I-com- Compound III (13), another possibility is that com- pound II cycle (3). Compound I is formed from the pound III participates directly in AFMK formation. In native form by the addition of hydrogen peroxide or by this case, compound III would be acting similarly to the the presence of contaminating peroxides and is the enzyme indoleamine 2,3-dioxygenase (14). In this con- catalytically active form. Although generally less ac- text, Kettle and Winterbourn (15) have already men- tive, compound II also catalyzes the oxidation of a tioned the similarity between peroxidase compound III number of substrates. Recently, we reported that HRP and the active form of indoleamine 2,3-dioxygenase.
catalyses the oxidative cleavage of several indolic com- Since p-670 is produced from compound III (16), the pounds, via a reaction sequence in which the indolyl strong protective effect of melatonin in inhibiting the cation-radical is presumed to be the intermediate (2).
formation of the inactive p-670 form supports the sup- We also showed that melatonin is oxidized by HRP, position of a direct reaction of compound III with mel- MPO and by activated neutrophils in a reaction from atonin. The pronounced pH dependence of AFMK for- which AFMK was isolated (1). Although the classic mation may be related to the formation of protonated native-compound I-compound II-native enzyme cycle is reactive substrates or intermediates. It is indeed curi- expected to participate in the oxidation of indole com- ous to note that neutral to basic pH increases the pounds, the requirement of a large amount of hydrogen affinity of the ferrous indoleamine 2,3-dioxygenase en- peroxide indicated that HRP compound III was in same zyme for its substrates (17).
way involved. In this study, we specifically addressed Apart from the question as to the true operative the question of whether peroxidase compound IIIparticipates to the production of the kynurenine-like mechanism(s), the protection promoted by melatonin products formed in the oxidation of melatonin and and tryptophan against formation of p-670 suggests a role of these compounds in preventing the inhibition of Recently, Allegra et al. (8) clearly showed that mel- peroxidases in vivo, e.g., in neutrophils and biological atonin reacts with MPO compounds I and II. The con- fluids where peroxidases or other hemeproteins are dition utilized in the present work to produce com- present. Furthermore, as can be inferred from the re- pounds I and II (condition a) leads to melatonin sults of this and previous studies (1, 2), the formation consumption. However, the production of AFMK is in- of indole ring-opening products may also take place cipient compared to that observed under condition b, with other biological indoles and could be an alterna- where compound III prevails. It is also possible to form tive path for the production of kynurenine-like com- AFMK even in the absence of H O if compound III pounds, whose biological activity has been described formed by the HRP/NADH/catalase system is employed.
Vol. 287, No. 1, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS motes oscillations. Biochem. Biophys Res. Commun. 284, 1071–
1076.
The authors are indebted to the Fundac¸a˜o de Amparo a Pesquisa 10. Cotton, M. L., and Dunford, H. B. (1973) Studies on horseradish- do Estado de Sa˜o Paulo (FAPESP) and the Conselho Nacional de peroxidase. 11. Nature of compounds I and II as determined from Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq).
kinetics of oxidation of ferrocyanide. Can. J. Chem. 51, 582–587.
11. Ohlsson, P. J., and Paul, K. G. (1976) Molar absorptivity of horseradish-peroxidase. Acta Chem. Scand. B 30, 373–375.
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tinamide-adenine dinucleotide phosphate. Biochim. Biophys. (2000) Myeloperoxidase-catalyzed oxidation of melatonin by ac- Acta 105, 301–312.
tivated neutrophils. Biochem. Biophys. Res. Commun. 279, 657–
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oxidation of indole derivatives catalyzed by horseradish peroxi- 14. Kobayashi, K., Hayashi, K., and Sono, M. (1989) Effects of tryp- dase is highly chemiluminescent. Arch. Biochem. Biophys. 387,
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16. Adediran, S. A. (1996) Kinetics of the formation of p 670 and of 5. Odajima, T. (1971) Myeloperoxidase of leukocyte of normal the decay of compound III of horseradish peroxidase. Arch. Bio- blood. 2. Oxidation-reduction reaction mechanism of myeloper- chem. Biophys. 327, 279 –284.
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