The effect of pH on horseradish peroxidase-catalyzed oxidation of melatonin: production of N 1 -acetyl-N 2 -formyl-5- methoxykynuramine versus radical-mediated degradation Introduction Melatonin is among the most studied endogenous molecules over the last decade [1–3]. Besides its hormonal properties, a growing body of evidence has demonstrated that melatonin has anti-inflammatory characteristics and acts as a scaven- ger of reactive oxygen species, reactive nitrogen species, reactive chlorine species, and as an inhibitor and/or modulator of cytokines, lipid mediators and enzymes involved in the inflammatory process [4–7]. The importance of melatonin as an anti-inflammatory molecule was rein- forced when its product of oxidative indole-ring cleavage, N 1 -acetyl-N 2 -formyl-5-methoxykynuramine (AFMK), was demonstrated to be a scavenger of reactive species and an inhibitor of cytokines and lipid mediators [5–7]. Several enzymes have been proposed as endogenous catalysts for the production of AFMK using hydrogen peroxide or the superoxide anion as an oxidant, including cytochrome c (cyt c), indoleamine dioxygenase and myeloperoxidase (MPO) [8–10]. Among these, the leuko- cyte peroxidase, MPO, is the most efficient catalyst for the production of AFMK and the unique for which superoxide anion has been demonstrated as a special component for the production of AFMK [11]. This property is not exclusive for the mammalian peroxidases; in fact, the plant enzyme horseradish peroxidase (HRP) is also strongly dependent on superoxide anion for the efficient production of AFMK [12]. Recently, the presence of melatonin in different plant tissues has been reported, thus pointing to its role in nutrition [13–16]. As HRP is widely present in plants and develops several physiological functions, the discovery of indoleam- ines in plants reinforces the importance of the relationship HRP and melatonin. Hence, in this investigation, our main objective was to study the effect of the pH of the reaction mixture on HRP-catalyzed oxidation of melatonin. We found that the pH is an important factor and determines the reaction rate and profile of oxidative products. Materials and methods Chemicals Melatonin, HRP (EC 1.11.1.7), and superoxide dismutase (SOD) (EC 1.15.1.1) were purchased from Sigma-Aldrich Chemical Co. (St Louis, MO, USA). The concentration of HRP was calculated from its absorption at 403 nm (e ¼ 1.03 · 10 5 /m/cm). Hydrogen peroxide (H 2 O 2 ) was prepared by diluting a 30% stock solution and calculating its concentration, using its absorption at 240 nm (240 nm ¼ Abstract: There is a growing body of evidence that melatonin and its oxidation product, N 1 -acetyl-N 2 -formyl-5-methoxykynuramine (AFMK), have anti-inflammatory properties. From a nutritional point of view, the discovery of melatonin in plant tissues emphasizes the importance of its relationship with plant peroxidases. Here we found that the pH of the reaction mixture has a profound influence in the reaction rate and products distribution when melatonin is oxidized by the plant enzyme horseradish peroxidase. At pH 5.5, 1 mm of melatonin was almost completely oxidized within 2 min, whereas only about 3% was consumed at pH 7.4. However, the relative yield of AFMK was higher in physiological pH. Radical-mediated oxidation products, including 2-hydroxymelatonin, a dimer of 2-hydroxymelatonin and O-demethylated dimer of melatonin account for the fast consumption of melatonin at pH 5.5. The higher production of AFMK at pH 7.4 was explained by the involvement of compound III of peroxidases as evidenced by spectral studies. On the other hand, the fast oxidative degradation at pH 5.5 was explained by the classic peroxidase cycle. Valdecir F. Ximenes 1,3 , Joa ˜o Roberto Fernandes 1 , Va ˆnia B. Bueno 2 , Luiz H. Catalani 2 , Georgino H. de Oliveira 3 and Rosa ˆngela G. P. Machado 3 1 Departamento de Quı ´mica, Faculdade de Cie ˆ ncias, Universidade Estadual Paulista, Bauru; 2 Instituto de Quı ´mica, Universidade de Sa ˜o Paulo, Sa ˜o Paulo; 3 Departamento de Princı ´pios Ativos e Naturais, Faculdade de Cie ˆ ncias Farmace ˆuticas, Universidade Estadual Paulista, Araraquara, Sa ˜o Paulo, Brazil Key words: 2-hydroxymelatonin, horseradish peroxidase, melatonin, melatonin dimer, N 1 - acetyl-N 2 -formyl-5-methoxykynuramine Address reprint requests to Valdecir Farias Ximenes, Departamento de Quı ´mica, Faculd- ade de Cie ˆ ncias de Bauru, Universidade do Estado de Sa ˜ o Paulo, CEP 17033-360, Brazil. E-mail: vfximenes@fc.unesp.br Received Ocober 21, 2006; accepted December 13, 2006. J. Pineal Res. 2007; 42:291–296 Doi:10.1111/j.1600-079X.2007.00419.x Ó 2007 The Authors Journal compilation Ó 2007 Blackwell Munksgaard Journal of Pineal Research 291