Fig 3. Characterization of TYR variants by gel filtration and enzyme activity assays.

Overlap of the analytical gel filtration profiles of TYR456 (peak B, in red) and TYR469 (peak A, in blue) on a Superdex 200 10/300 GL column (GE Healthcare). Colorimetric activity assays with the corresponding eluted fractions using L-DOPA as substrate generated a pink or a dark pink pigment product (i.e. quinone-MBTH adduct), indicating that both variants are enzymatically active (10 μL of each elution fraction containing TYR was added in a 80 μL reaction well containing L-DOPA. 10 μL gel filtration buffer solution was used as a negative control). The apparent gradient of light pink to dark pink, and back to light pink in the reaction well indicates different protein concentrations of each elution fraction.