Tyrosinase is an enzyme that catalyzes the production of melanin, the pigment that gives color to skin, hair, and eyes. Inhibiting tyrosinase activity can be used to lighten skin or to reduce age spots.
To evaluate tyrosinase inhibitory activity, you would need to perform an assay. One common assay method is the mushroom tyrosinase assay, which uses the mushroom tyrosinase enzyme as a source of tyrosinase activity. The assay typically involves the following steps:
Prepare a stock solution of mushroom tyrosinase enzyme.
Prepare a series of test compounds or extracts in a range of concentrations.
Add the test compounds or extracts to a solution of L-tyrosine or L-DOPA, which are substrates for the tyrosinase enzyme.
Add the mushroom tyrosinase enzyme and measure the change in absorbance over time at a specific wavelength, typically 475 nm. The change in absorbance is directly proportional to the rate of tyrosinase activity.
Compare the rate of tyrosinase activity in the presence of the test compounds or extracts to the rate of tyrosinase activity in the absence of the test compounds or extracts.
As for the difficulties, this assay typically involves some challenges such as:
Obtaining pure and active enzyme preparation
Standardizing the assay conditions
Selecting appropriate substrate and cofactor
Interpreting the assay results
Selecting the appropriate controls and positive inhibitors
Performing this assay in a consistent and repeatable manner
Interferences from other components of the test compound that might affect the assay results.