Influence of peroxidase on the oligomerization of 5,6- and 5,7-dihydroxytryptamine investigated by matrix-assisted laser desorption/ionization mass spectrometry

Abstract

The influence of peroxidase in the melanogenesis of 5,6-and 5,7-dihydroxytryptamines (DHT) was investigated by matrix-assisted laser desorption/ionization mass spectrometry. Incubation was carried out with peroxidase, in the presence or absence of hydrogen peroxide. Samples were withdrawn at various times, ultrafiltered to remove the enzyme and immediately lyophilized. Reaction of the two isomers with peroxidase alone revealed processes similar to those observed in the case of reaction with tyrosinase, leading to low molecular mass oligomers. Reaction with peroxidase and hydrogen peroxide showed very different reactivity of the two isomers. 5,6-DHT immediately led to the formation of a black precipitate, probably due to the reaction of its oxidation products with the enzyme. In contrast, 5,7-DHT formed oligomers, up to nonamers, originating from the molecule itself and from its oxidation products. Both isomers showed the same chemical reactivity with respect to hydrogen peroxide alone, with the formation of up to nonamer species from both original molecules and their oxidation products. # 1998 John Wiley & Sons, Ltd. Received 16 March 1998; Revised 17 April 1998; Accepted 20 April 1998 5,6-and 5,7-dihydroxytryptamine (5,6-DHT and 5,7-DHT) are formed in the brain from 5-hydroxytryptamine (5-HT, serotonin) either by autooxidation or enzymatically, and are known for their destructive action in vivo of both central and peripheral noradrenergic neurons.1-3 Both drugs are employed in in vivo studies, in which their neurotoxic action is of practical concern. The final process that results in neurotoxicity is similar for both drugs, i.e. covalent binding of reactive intermediates to nucleophiles in proteins. 4 In the presence of oxygen they form coloured quinones, pink with 5,7-DHT and black with 5,6-DHT, 5 which may either undergo polymerization (establishing melanin-like structures) or react with sulfydryl groups in peptides and proteins. 6 This reaction is accelerated when tyrosinase is added to the reaction medium, with the formation of insoluble black melanin-like pigments.In previous studies, 7,8 despite the usual assumption that neuromelanin originates from catecholamines, in an attempt to demonstrate that tryptamines are involved in melanogenesis we undertook an in vitro investigation of the enzymatic reaction of 5,6-DHT and 5,7-DHT with mushroom tyrosinase. Structural information on the reaction intermediates was obtained after withdrawing samples at different reaction times, and after ultrafiltration and lyophilization. The resulting samples were analysed by means of matrixassisted laser desorption/ionization (MALDI) mass spectrometry, which clearly identified several oligomer species in the reaction mixture. Using the MALDI approach it was found that, after 30 min, the reaction of 5,6-DHT with tyrosinase leads to a series of oligomers with molecular masses of 382, 572, 762, 952, 1142382, 572, 762, 952, , 1332382, 572, 762, 952, and 1522 to the oligomerization products of the precursor from dimer (382 Da)...

An investigation on the role of 3‐hydroxykynurenine in pigment formation by matrix‐assisted laser desorption/ionization mass spectrometry

“…However, some information has been gained on just the first steps of melanogenesis by using mass spectrometry, operating under different instrumental conditions. Using these approaches the first oligomerization products originated from various substrates by different enzymatic reactions have been characterized, proving the co‐occurrence of parallel reaction pathways 14,17,28–30. Using MALDI, different oligomers up to the octamer were identified, and their yields as a function of reaction time were studied.…”

“…As a result of these studies indicating the possibility that tryptophan plays a role in melanogenesis, in order to elucidate the structure of the tryptophan melanins, we exploited the analytical approach offered by modern mass spectrometry, by which we obtained interesting results 10–17…”

Cluster analysis of serum proteins in malignant cutaneous melanoma: search for disease markers

“…The matrix‐assisted laser desorption ionisation (MALDI) method has proved to be particularly highly effective for this purpose. Extensive MALDI studies13–27 have demonstrated that this approach appears particularly valid in the study of the first stages of the oligomerisation processes leading to melanins.…”