Kinetic analysis of the inhibition of tyrosinase

A series of experiments was performed to calculate the inhibitory kinetics of compounds 4c, 6a, 6b and 6d following the previously reported method.30 The compounds’ concentrations were as follows: 0, 3.125, 6.25 and 12.5 µM for 4c; 0, 0.3915 and 0.783 µM for 6a; 0, 0.453, 0.965, 1.813 and 3.626 µM for 6b; and 0, 0.08, 0.16 and 0.32 µM for 6d. Substrate l-DOPA concentration was between 0.0625 and 2 mM in all kinetic studies. Preincubation and measurement time was the same as discussed in mushroom tyrosinase inhibition assay protocol. Maximal initial velocity was determined from initial linear portion of absorbance up to 5 min after addition of enzyme at a 30 s interval. The type of inhibition was determined by using Lineweaver–Burk plots. The enzyme inhibitor (EI) dissociation constant Ki was determined by secondary plot of 1/V versus inhibitor concentrations, while enzyme–substrate–inhibitor (ESI)-dissociation constant Ki′ was determined by intercept versus inhibitor concentrations. The reversible kinetics of the EI complex was also determined for different concentrations of compound 6d versus the enzyme concentration (4, 6, 8, 10, 15 and 20 µg/mL).