4.3. Tyrosinase Enzymatic Assay

The tyrosinase enzymatic assay was performed as described in our previous papers [20,21] using l-3,4-dihydroxyphenylalanine (l-DOPA) (Sigma-Aldrich, Taufkirchen, Germany) as a substrate. l-DOPA was dissolved in a 0.15 mM phosphoric (V) acid solution to a concentration of 5 mM. All TSCs were dissolved in DMSO to a concentration of 50 mM and then diluted in sodium phosphate buffer (0.1 M, pH 6.8) to test concentrations (DMSO concentrations in final reaction mixtures did not exceeded 1%).

The isolated tyrosinase solution (0.5 mg/mL, 40,000 U/mg) was diluted 10 times in sodium phosphate buffer (0,1 M, pH 6.8). First, 10 µL of the diluted enzyme solution was preincubated with TSC solutions (5 min at 25 °C). The optimal ionic strength and pH for tyrosinase activity were provided by 0.1 M sodium phosphate buffer (pH 6.8).

After the solution of l-DOPA was added, monitoring of the reaction was immediately started by measuring the change in absorbance of the color product (dopachrome) for 10 min (λ = 475 nm, 25 °C) using Molecular Devices SpectraMax Plus 384 Microplate Reader. The control sample contained the same reagents as the test samples except for the substrate. Kojic acid (Sigma-Aldrich, Taufkirchen, Germany), a positive control, was treated the same way as the other inhibitors.