Optimization of conditions for preparing synthetic pheomelanin

Synthetic melanins are often used as the standards for natural melanins. Conditions for preparing synthetic pheomelanin from L-dopa and L-cysteine were studied under different temperatures (25 and 37 degrees C), different amounts of cysteine (1.5 and 2.0 eq), and different amounts of tyrosinase (10 and 20 mg). Effects of addition of catalase and of replacement of oxygen with air were also examined. The best preparation of pheomelanin was obtained in a good yield, when 1 mmol of L-dopa was oxidized at 25 degrees C under oxygen current in 100 ml of a pH 6.8 buffer by 20 mg of mushroom tyrosinase inn the presence of 1.5 mmol of L-cysteine. The pheomelanin thus obtained had an elemental composition and an absorption spectra that were very similar to those from a pheomelanin prepared by tyrosinase oxidation of 5-S-cysteinyldopa. Hydrolysis of synthetic pheomelanins and pheomelanic tissues with hydriodic acid gave aminohydroxyphenylalanines (AHP) as a major product and hydroxybenzothiazolylalanines as a minor product. It is proposed that AHP can be used as a specific indicator of purity of pheomelanins.