Mushroom tyrosinase oxidizes tyrosine-rich sequences to allow selective protein functionalization.

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.