Quantification of reactive oxygen species production by the red fluorescent proteins KillerRed, SuperNova and mCherry

Fluorescent proteins can generate reactive oxygen species (ROS) upon absorption of photons via type I and II photosensitization mechanisms. The red fluorescent proteins KillerRed and SuperNova are phototoxic proteins engineered to generate ROS and are used in a variety of biological applications. However, their relative quantum yields and rates of ROS production are unclear, which has limited the interpretation of their effects when used in biological systems. We cloned and purified KillerRed, SuperNova, and mCherry - a related red fluorescent protein not typically considered a photosensitizer - and measured the superoxide (O₂^•-) and singlet oxygen (¹O₂) quantum yields with irradiation at 561 nm. The formation of the O₂^•--specific product 2-hydroxyethidium (2-OHE⁺) was quantified via HPLC separation with fluorescence detection. Relative to a reference photosensitizer, Rose Bengal, the O₂^•- quantum yield (ΦO₂^•-) of SuperNova was determined to be 1.5 × 10^-3, KillerRed was 0.97 × 10^-3, and mCherry 1.2 × 10^-3. At an excitation fluence of 916.5 J/cm² and matched absorption at 561 nm, SuperNova, KillerRed and mCherry made 3.81, 2.38 and 1.65 μM O₂^•-/min, respectively. Using the probe Singlet Oxygen Sensor Green (SOSG), we ascertained the ¹O₂ quantum yield (Φ¹O₂) for SuperNova to be 22.0 × 10^-3, KillerRed 7.6 × 10^-3, and mCherry 5.7 × 10^-3. These photosensitization characteristics of SuperNova, KillerRed and mCherry improve our understanding of fluorescent proteins and are pertinent for refining their use as tools to advance our knowledge of redox biology.