Abstract
We show that mushroom tyrosinase catalyzes the formation of reactive o‐quinones on unstructured, tyrosine‐rich sequences such as hemagglutinin (HA) tags (YPYDVPDYA). In the absence of exogenous nucleophiles and at low protein concentrations, the o‐quinone decomposes with fragmentation of the HA tag. At higher protein concentrations (>5 mg mL−1), crosslinking is observed. Besthorn's reagent intercepts the o‐quinone to give a characteristic pink complex that can be observed directly on a denaturing SDS‐PAGE gel. Similar labeled species can be formed by using other nucleophiles such as Cy5‐hydrazide. These reactions are selective for proteins bearing HA and other unstructured poly‐tyrosine‐containing tags and can be performed in lysates to create specifically tagged proteins.
Mushroom tyrosinase is a multifaceted chemical biology tool: This enzyme catalyzes the formation of o‐quinones in tyrosine‐rich peptide sequences such as the HA tag. These o‐quinones can be selectively functionalized with hydrazine dyes to create labeled proteins. Under different reaction conditions, the o‐quinones induce protein cleavage or crosslinking.