I want to conduct phenol oxidase assays (in soil slurries) using L-DOPA (L-3,4-Dihydroxyphenylalanine). There doesn't seem to be a commercial standard and it is suggested to oxidize DOPA using tyrosinase (assuming it completely oxidizes all of the substrate) and use the product as standard. Does anyone has experience with that? Does anyone know how much tyrosinase is needed for different volumes/concentrations of DOPA solutions?
Hi Peter,
Initialy, I would check the properties of comercial tyrosinase preparations. You need to undertake a series of initial exploratory studies. Taking a fixed concentration (micromolar) of the substrate, find out the effect of protein concentration (different quantities of the enzyme) on the extent of enzyme activity. In this way, you can find out the optimal concentration of the protein required. Now, use this optimal protein concentration and run assays using different concentrations of the substrate. The next experiment would be determining the time course of the enzyme reaction.
Best!
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