Melatonin directly scavenges free radicals generated in red blood cells and a cell-free system: Chemiluminescence measurements and theoretical calculations

Melatonin, a pineal secretory product, has properties of both direct and indirect powerful antioxidant. The aim of the present study was to compare the radical-scavenging, structural and electronic properties of melatonin and tryptophan, precursor of melatonin. Using the alkoxyl- and peroxyl radical-generating systems [the organic peroxide-treated human erythrocytes and a cell-free system containing the azo-initiator 2,2′-azobis(2-amidinopropane)dihydrochloride], we evaluated the radical-scavenging effects of melatonin and tryptophan. Melatonin rather than tryptophan at concentrations of 100-2000 μM markedly inhibited membrane lipid peroxidation in human erythrocytes treated with organic hydroperoxide as well as radical-induced generation of luminol-dependent chemiluminescence. The apparent Stern-Volmer constants for inhibition of membrane lipid peroxidation by melatonin and tryptophan were estimated to be (0.23 ± 0.05) × 10⁴ M^{- 1} and (0.02 ± 0.005) × 10⁴ M^{- 1}, respectively. The apparent Stern-Volmer constants for inhibition of azo-initiator-derived peroxyl radical generation by melatonin and tryptophan were determined to be (0.42 ± 0.05) × 10⁴ M^{- 1} and (0.04 ± 0.01) × 10⁴ M^{- 1}, respectively. The structural and electronic properties of melatonin and its precursor, tryptophan, were determined theoretically by performing semi-empirical and ab initio calculations. The high radical-scavenging properties of melatonin may be explained by the high surface area value and high dipole moment value. From the thermodynamic standpoint, based on our calculations, N¹-acetyl-N²-formyl-5-methoxykynuramine (AFMK), was the most stable end oxidative product of melatonin.