Real-time oxygen sensing as a powerful tool to investigate tyrosinase kinetics allows revising mechanism and activity of inhibition by glabridin

A new method for studying tyrosinase kinetics and inhibition by oxygen sensing is described and matched to the conventional spectrophotometric approach. The stoichiometric ratio of O2 uptake to dopachrome formation was 1.5 ± 0.2 for substrate L-tyrosine and 1.0 ± 0.1 for L-DOPA. With both methods, we reinvestigated mushroom tyrosinase inhibition by glabridin from Glycyrrhiza glabra. The two methods agreed showing mixed-type inhibition for monophenolase and diphenolase activities, at variance with previous literature. Average KI (KSI) values for glabridin were 13.6 ± 3.5 (281 ± 89) nM and 57 ± 8 (1312 ± 550) nM, for monophenolase and diphenolase inhibition, respectively, with IC50 of 80 ± 8 nM and 294 ± 25 nM, respectively, at 1 mM substrate. For reference kojic acid KI (KSI) were 10.9 ± 8 (217 ± 55) µM and 9.9 ± 1.4 (21.0 ± 5.2) µM, for monophenolase and diphenolase, respectively, with respective IC50 of 33 ± 8 μM and 17 ± 3 μM. Glabridin's activity is among the highest in nature, being about three orders of magnitude higher than previously reported.