Fluorescent Proteins Such as EGFP Catalytically Generate Superoxide Anion Free Radical and H2O2 in the Presence of NAD(P)H

Fluorescent proteins are an important tool that has become omnipresent in life sciences research. Because of its high fluorescence and light stability, enhanced green fluorescent protein (eGFP) is widely used to tag, quantify and locate biomolecules in living systems. It has been established that during the maturation of the eGFP chromophore, one molecule of the molecular oxygen-derived reactive species H2O2 is produced, reportedly with no consequences to cellular homeostasis. However, here we demonstrate that E. coli and HeLa cells expressing eGFP show signs of oxidative stress. E. coli bacteria expressing eGFP show expression of genes associated with activation of the transcription factors SoxR and OxyR, which are, in turn, directly activated by O2•– (superoxide free radical anion) and H2O2. HeLa cells expressing eGFP exhibit gene expression associated with H2O2-mediated signaling. We found that an immature species of eGFP reacts intracellularly with NAD(P)H to catalytically generate biologically significant amounts of O2●– and H2O2. This mechanism was also detected with TagRFP, a GFP-homologous red fluorescent protein. Our findings call for a careful reinterpretation of experiments using fluorescent proteins in living systems in view of the intrinsic oxidative stress induced by GFP-like fluorescent proteins.