An investigation on the role of 5-hydroxytryptophan in the biosynthesis of melanins β Tryptophan (Trp) is an essential amino acid for human nutrition and its dietary source is from naturally occurring proteins. This amino acid is bound to serum albumin to the extent of about 85-90%. 1 The free fraction (10-15%) alone can cross the blood-brain barrier (B-BB) 1,2 and influence the rate of synthesis of cerebral serotonin neurotransmitter, 3 whose tryptophan is the precursor. This amino acid, after its uptake by neurons, is hydroxylated in the 5-position by means of tryptophan 5-hydroxylase, giving rise to the formation of 5-hydroxytryptophan (5-HTP), 4 which is then decarboxylated to 5-hydroxytryptamine (5-HT, serotonin). 5 However, 5-hydroxytryptophan, when administered, can rapidly penetrate into the brain to give serotonin. As serotonin cannot cross the B-BB, tryptophan and 5-hydroxytryptophan (5-HTP) are the precursors originating the 5-HT in the brain. However, only 1% of exogenous tryptophan is transformed into 5-HT. In contrast, L-5- hydroxytryptophan crosses the B-BB and is completely converted into 5-HT in the presence of a specific enzyme, 5-hydroxytryptophan decarboxylase, in cerebral tissues. 6 Many authors assume that catecholamines are possible precursors of neuromelanin, the biosynthesis of which occurs in brain regions containing dopamine or norepinephrine. 7 -12 However, in addition to catecholamines we found that serotonin may also be considered a precursor of neuromelanin. 13 -17 In this study, we investigated the mechanism of melanogenesis of the direct precursor of serotonin, 5-HTP, utilizing two different enzymes, tyrosinase and peroxidase. The extensive melanization occurring in the brain during Parkinson's disease led us to consider the involvement of different enzymes and substrates in melanogenesis. In fact, while tyrosinase is considered to be the physiological enzyme in this process, this enzyme was not detected in the brain. 18 In a previous study, 19 investigating the role of tyrosinase and peroxidase in melanogenesis starting from tyrosine, we observed that, in the presence of H 2 O 2 , peroxidase can produce melanins in vitro. In addition, we also studied the role of peroxidase in the melanogenesis of 5-HT 20 and 5,6-and 5,7-dihydroxytryptamines (DHT) by matrix-assisted laser desorption/ionization (MALDI) 21 and found that the oligomerization of 5-HT by peroxidase requires the presence of H 2 O 2 , whereas the reaction of 5,6-and 5,7-DHT with peroxidase C H 2 O 2 showed different reactivity of the two isomers. 5,6-DHT led immediately to the formation of a black precipitate. In contrast, 5,7-DHT formed oligomers originating from the molecule itself and from its oxidation products.
Taking in consideration the paper of Rosei 22 on melanins from opioid peptides and the recent results of Jee et al., 23 we tried to subject two peptides, one containing tyrosine (L-tyrosylglycylglycine) and the other one tryptophan (Trp-Trp), to the action of tyrosinase and peroxidase to investigate whether they are substrates in vitro for these enzymes. To investigate these aspects, both electrospray (ES) and MALDI mass spectrometric (MS) techniques were employed.
In previous investigations, we studied the role of 5-HT in melanogenesis by its enzymatic reaction with tyrosinase and peroxidase. 20,24,25 The reaction environment was analyzed at different reactions times: samples were drawn, immediately ultrafiltered and lyophilized. MALDI-MS was proved to be a valid analytical tool, showing in the case of tyrosinase the formation of oligomers up to the octamer. The same approach showed that analogous results are achieved by peroxidase but, in this case, the reaction kinetics are faster.