5.10. The Analysis of Tyrosinase Activity

Spectrophotometric determination of tyrosinase activity was based on the enzymatic oxidation of L-DOPA to a colorful DOPAchrome. A solution of tyrosinase substrate (L-DOPA) was prepared in the phosphate buffer (pH = 6.8) at a concentration of 2 mg/mL. Melanocyte lysate were clarified by centrifugation at 10,000× g for 5 min. The obtained supernatants in an amount of 100 μL and 40 μL of L-DOPA solution were placed in a 96-well plate. Then, the absorbance of dopachrome was measured at 475 nm every 10 min for at least 1.5 h at 37 °C using a microplate reader Infinite 200 PRO (TECAN, Männedorf, Switzerland). The obtained results were normalized using total protein concentration and expressed as the percentage of control.