[A spectrofluorometric method of recording oxidation products of eumelanin destruction]

Dynamics of oxidative destruction of DOPA-melanin and melanin proteins was studied after treatment with 3% hydrogen peroxide within 60 min at 100 degrees. The pigments were estimated by absorption at 400 nm as well as by means of spectrofluorimetric patterns both in total preparation and in preparation obtained after fractionation on Tojapearl-55 and Tojapearl-40. Intensive fluorescence in the region 500-510 nm was detected within initial steps of oxidation while the absorption spectrum was unaltered. These fluorescent substances were heterogeneous: fluorescence was detected both in main high molecular fraction and in low molecular substances (fluorescence was absent in the initial melanin and its fractions). During incubation gradual decolorization of the pigment was observed. This process was related to depolymerization and to an increase in content of slightly coloured low molecular substances exhibiting maximal fluorescence at 430 mm. The further steps of oxidation, affecting main subunits of melanin (indol quinones), involved formation of low molecular, practically discoloured substances with fluorescence at 340 nm and 410 nm. The spectrofluorimetric procedure combined with gel chromatography may be used in evaluation of the rate and degree of eumelanins oxidative destruction.