Quantitative measurement of melanin as tyrosine equivalents and as weight of purified melanin.

Abstract

Previously we reported stoichiometric studies on melanin synthesis in cultures of mouse melanoma cells (1). In this system there was no accumulation of intermediates in the Raper-Mason-Nicolaus pathway for melanin synthesis and the amount of hydroxylated tyrosine was quantitatively related to the release of hydrogen as water from positions 3 and 5 of the benzene ring of tyrosine during the series of reactions involved in melanin synthesis. Therefore, the amount of newly synthesized melanin could be expressed as tyrosine equivalents. The present paper describes a colorimetric method for determination of melanin, using protein-free melanin as standard, and values are compared with those expressed as tyrosine equivalents. For this method, a new procedure was developed to solubilize melanin. MATERIALS AND METHODS Chemicals. "Soluene 100, " a 0.5 N solution of dimethyl n-undecyl n-dodecyl ammonium hydroxide in toluene, is a product of Packard Instrument Co. L-Tyrosine-3,5-3H was purchased from the Radiochemical Centre, Amersham and L-3,4dihydroxy-phenylalanine (dopa) and mushroom tyrosinase, grade III, from Sigma Chemical Co. Eagle's minimum essential medium and calf serum were obtained from the Serum Institute, Chiba. Cells and tumors. Stable melanotic and amelanotic clonal cell lines, C2M (2) and C2W (3), respectively, of mouse melanoma B 16 were used. Culture conditions were as reported previously (1,4). C2M cells were implanted into C57BL mice and the resulting black tumors (B 16-C2M) were used for preparation of melanoma melanin.