Biosynthesis of melanin from dopamine. An investigation of early oligomerization products

Abstract

Matrix-assisted laser desorption/ionization (MALDI) and fast-atom bombardment (FAB) mass spectrometry experiments were applied to the study of the early stages of the oligomerization reaction of dopamine with mushroom tyrosinase. Ultrafiltration was employed to remove the enzyme at various reaction times, to prevent possible attachment of the protein to the highly reactive intermediates. Two sets of five samples each, obtained at different reaction times, in one case immediately lyophilized and in the other left to react under an oxygen stream for 24 h before lyophilization, were compared. FAB showed the presence of various species and of these, that at m/z 305 increased in abundance with reaction time in immediately lyophilized set of samples only. Accurate mass measurements and tandem mass spectrometric experiments indicated the structure of a dopamine protonated dimer for this ion. MALDI measurements showed that all samples were composed of clusters of oligomers differing in degree of oligomerization. Oligomerization increases with reaction time, resulting in the formation of species at 2643-2911 Da. These clusters in turn were formed of species with a different degree of oxidation, detected in both sets of samples.

Application of matrix-assisted laser desorption/ionization mass spectrometry to the detection of melanins formed from Dopa and dopamine

“…It is interesting that the reaction promoted by the peroxidase-H 2 O 2 system gives rise to an oligomerization pattern simpler than that observed in the case of tyrosinase. 22 In that case, in fact, complex mixtures of oligomers at various oxidation levels were obtained, proving the more oxidative action of tyrosinase.…”

“…In particular, in the case of dopamine the formation of real dimers was also proved, in early work, by FAB measurements performed with a double-focusing instrument operating in resolution conditions higher than those achieved by MALDI. 22 On performing the reaction in the presence of the peroxidase-H 2 O 2 system, oligomerization takes place. The solution becomes red after a few minutes and turns dark reddish-brown at 60 min, with the formation of a brown precipitate (Fig.…”

Enzymatic oligomerization of tyrosine by tyrosinase and peroxidase studied by matrix-assisted laser desorption/ionization mass spectrometry

“…[20][21][22][23][24][25] Briefly, 8 mg of tyrosinase or 3.7 mg of peroxidase were added to 35 mL of a 5mM solution of tyrosine in doubly distilled water saturated with oxygen.…”

“…In this context, the study of enzymatic melanogenesis promoted by tyrosinase on dopamine, 5-hydroxytryptamine (5-HT), 5,6-and 5,7-dihydroxytryptamine (5,6-and 5,7-DHT) was carried out by performing the enzymatic reaction, withdrawing samples by ultrafiltration at various reaction times, and by direct analysis of samples by matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS). [20][21][22][23] The results are very promising: in all cases, it was possible to identify oligomerization products up to the decamer level and, in some cases, the oligomerization kinetics were characterized.…”

Excitonic effects from geometric order and disorder explain broadband optical absorption in eumelanin

“…X-ray diffraction studies 21,22 and scanning tunneling microscopy measurements [23][24][25] showed that the size of eumelanin protomolecules is around 15-20 Å, which suggested that the tetramers and pentamers formed by covalently bonded DHIs are the most possible molecular structures of eumelanin. Matrix-assisted laser desorption/ionization mass spectrometry studies [26][27][28][29][30][31] showed that the mass of eumelanin protomolecules is within or below B600-1,200 amu, and large polymeric structures are not observed, with the exception of the data reported in a recent report 32 . Hence, most of the investigations performed by mass spectrometry supported that the tetramers and pentamers constitute the majority of eumelanin, while slightly larger structures, such as the hexamers and octamers, are also possible.…”