Fig 1.
Chemical structures of α-arbutin and β-arbutin.
Fig 2.
A. Representation of iM (degree of inhibition of the monophenolase activity) vs. the concentration of α-arbutin. The experimental conditions were [E]0 = 80 nM, [L-tyrosine]0 = 0.25 mM and [L-dopa]0 = 0.01 mM. Inset. Spectrophotometric recordings of the effect of different concentrations of α-arbutin on the monophenolase activity of tyrosinase, using L-tyrosine as substrate. The experimental conditions were [E]0 = 80 nM, [L-tyrosine]0 = 0.25 mM, [L-dopa]0 = 0.01 mM and α-arbutin (mM): a) 0, b) 1.5, c) 3, d) 6.5, e) 13, f) 20, g) 32 and h) 41. B. Representation of iM (degree of inhibition of the monophenolase activity) vs. the concentration of β-arbutin. The experimental conditions were [E]0 = 80 nM, [L-tyrosine]0 = 0.25 mM and [L-dopa]0 = 0.01 mM. Inset. Spectrophotometric recordings of the effect of different concentrations of β-arbutin on the monophenolase activity of tyrosinase, using L-tyrosine as substrate. The experimental conditions were [E]0 = 80 nM, [L-tyrosine]0 = 0.25 mM, [L-dopa]0 = 0.01 mM and β-arbutin (mM): a) 0, b) 0.5, c) 1, d) 2, e) 5, f) 10 and g) 20.
Table 1.
Kinetic constants for the apparent inhibition of α-arbutin and β-arbutin on tyrosinase.
Fig 3.
A. Representation of iD (degree of inhibition of the diphenolase activity) vs. the concentration of α-arbutin. The experimental conditions were [E]0 = 30 nM and [L-dopa]0 = 0.5 mM. Inset. Spectrophotometric recordings of the effect of different concentrations of α-arbutin on the diphenolase activity of tyrosinase, using L-dopa as substrate. The experimental conditions were [E]0 = 30 nM, [L-dopa]0 = 0.5 mM and α-arbutin (mM): a) 0, b) 2.5, c) 5, d) 8, e) 13, f) 20 and g) 30. B. Representation of iD (degree of inhibition of the diphenolase activity) vs. the concentration of β-arbutin. The experimental conditions were [E]0 = 30 nM and [L-dopa]0 = 0.5 mM. Inset. Spectrophotometric recordings of the effect of different concentrations of β-arbutin on the diphenolase activity of tyrosinase, using L-dopa as substrate. The experimental conditions were [E]0 = 30 nM, [L-dopa]0 = 0.5 mM and β-arbutin (mM): a) 0, b) 0.5, c) 2.5, d) 5, e) 12, f) 20 and g) 30.
Fig 4.
Inhibition of monophenolase activity by arbutins.
Graphical representation of the Lineweaver–Burk equation to show the inhibition of the monophenolase activity of tyrosinase in the presence of 3 mM α-arbutin. The experimental conditions were [E]0 = 50 nM and R = [L-dopa]0 / [L-tyrosine]0 = 0.042. Inset. Graphical representation of the Lineweaver–Burk equation showing the inhibition of the monophenolase activity of tyrosinase in the presence of β-arbutin 3 mM. The experimental conditions were [E]0 = 50 nM and R = [L-dopa]0 / [L-tyrosine]0 = 0.042.
Fig 5.
Total oxygen consumption test (TBC).
A total oxygen consumption test was carried out in the presence of tert-butylcatechol and different concentrations of α-arbutin (mM): a) 0, b) 5, c) 10 and d) 20. The rest of the experimental conditions were [E]0 = 50 nM and [TBC]0 = 1 mM. Inset. Total oxygen consumption test in the presence of tert-butylcatechol and different concentrations of β- arbutin (mM): a) 0, b) 5, c) 10 and d) 20. The rest of the experimental conditions were [E]0 = 50 nM and [TBC]0 = 1 mM.
Fig 6.
Action of tyrosinase on α-arbutin in the presence of MBTH.
The experimental conditions were [E]0 = 300 nM, [MBTH]0 = 0.2 mM, [α-arbutin]0 = 10 μM and DMF 2%. The spectrophotometric recordings were made every 60 seconds. Inset. Determination of the MBTH saturation concentration. The experimental conditions were [E]0 = 100 nM, [α-arbutin]0 = 20 mM and DMF 2%.
Fig 7.
Kinetic characterization of the action of tyrosinase on arbutins.
Representation of the initial rate values obtained for the action of tyrosinase on α-arbutin. The experimental conditions were [E]0 = 100 nM, [MBTH]0 = 0.2 mM and DMF 2%. Inset. Representation of the initial rate values obtained for the action of tyrosinase on β-arbutin. The experimental conditions were the same as Fig 7.
Table 2.
Kinetic constants for the characterization of the activity of tyrosinase on α-arbutin and β-arbutin and chemical shift values of the carbon with the phenolic hydroxyl group.
Fig 8.
Computational docking of α-arbutin.
Docking poses obtained with AutoDock of α-arbutin in the active site of the oxy form of mushroom tyrosinase are shown as sticks. The atom colors are as follows: red = oxygen, blue = nitrogen, brown = copper, green = carbon, and white = hydrogen. Polar interactions and hydrogen bonds are shown as black dotted lines. The distance from the ortho carbon of the phenolic ring to the oxygen atom of the peroxide ion is shown in blue lines.