Chemical characterization of pheomelanogenesis starting from dihydroxyphenylalanine or tyrosine and cysteine. Effects of tyrosinase and cysteine concentrations and reaction time.

Two types of melanin pigment are produced in mammals; the brown-to-black eumelanins and the yellow-to-reddish-brown pheomelanins. The switch from one type of melanin to the other appears to be regulated by the levels of tyrosinase and thiols, such as cysteine and glutathione. This study examines the process of pheomelanin formation starting from dihydroxyphenylalanine (dopa) or tyrosine and cysteine. We prepared pheomelanins by tyrosinase oxidation of dopa or tyrosine in the presence of cysteine. Experimental variables were reaction time, tyrosinase concentration, and dopa or tyrosine to cysteine ratio. Following the reactions, we measured concentrations of tyrosine, dopa, cysteine and cysteinyldopas, amounts of total melanin (TM) by Soluene-350 solubilization and aminohydroxyphenylalanine (AHP), a specific indicator of pheomelanin, formed by hydriodic acid hydrolysis, and absorbance ratio, A650/A500. It was found that (1) mixed melanogenesis is a heterogeneous process in which pheomelanogenesis proceeds first, followed by eumelanogenesis, as shown by changes in the tyrosine and cysteinyldopa concentrations, the AHP/TM ratio, and the A650/A500 ratio during the course of melanogenesis and (2) lower tyrosinase concentration favors pheomelanogenesis even when the availability of cysteine is limited, as shown by AHP/TM ratios that were higher than the corresponding tyrosine to cysteine ratios. These results indicate that the switch from eumelanogenesis to pheomelanogenesis can be achieved by lowering the tyrosinase activity, which conforms to our proposal that tyrosinase activity is the major factor controlling the course of melanogenesis.