Supplementary Discussion

Experiments that demonstrate temporal H2O2 increase upon growth factor stimulation allow us to compare HyPer with two other probes – H2DCFDA, the widely used dye for detection of intracellular ROS production, and roGFPs, recently developed redox sensitive mutants of GFP1,2. While both H2DCFDA and roGFP are nonselective, e.g. could be oxidized by various oxidants1,3, HyPer demonstrates high selectivity for H2O2. Besides, DCF derivatives have some more disadvantages, firstly, they can produce ROS upon exposure to light4,5 and, secondly, can not be targetedto different cell compartments. In contrast, HyPer and roGFP are genetically encoded and therefore could be localized to various subcellular compartments. Both HyPer and roGFP change fluorescence upon formation of disulfide bond between two cystein residues but mechanisms of these reactions are different in thesesensors. In roGFP redox active cysteins are exposed to water phase and available for various oxidants. Therefore roGFP acts mostly as a sensor of intracellular redox state. In HyPer Cys199 of OxyR-RD is hidden in hydrophobic pocket and could be oxidized only by amphiphilic molecule of H2O2 and insensitive to other oxidants such as superoxide and GSSG. Theoretically, Cys199 could be nitrosylated by NO, but our experiments did not reveal any sensitivity of HyPer to NO neither in vitro nor in the cytoplasm of bacterial cells exposed to NO donor.

In contrast to roGFP1 HyPer is able todetect low-level H2O2 produced in PC-12 cells upon NGF stimulation. Probably, during physiological stimulation redox potential in the cytoplasm does not change because the amount of H2O2 produced is not enough to perturb intracellular redox state. HyPer can be oxidized by submicromolar H2O2 even in highly reduced environment. In vitro tests showed that roGFPs require long incubation with 0.1-1 mM H2O2 to be sufficiently oxidized1 whereas HyPer is completely oxidized in seconds by submicromolar H2O2.

Like in roGFP2, fluorescence of HyPer depends on pH. In our experiments with HyPer expressed in mammalian cells (Fig. 3 and 4 in the main text), we observed an increase in the fluorescence of the deprotonated chromophore form (excitation at 500 nm). Under specific conditions (apoptosis in the case of cytosolic HyPer; drop in the case of mitochondrial HyPer) acidification may occur6,7. This indicates that changes in thefluorescence of HyPer in our experiments are not due to pH variations. Moreover, in the experiments with NGF stimulation of PC-12 cells we used pH-sensitive dye BCECF-AM to ensure that no pH changes occurred during stimulation.

DCF has greater dynamic range than HyPer, probably due to accumulation of oxidized fluorescent form. For example, upon cell stimulation with a growth factor DCF and HyPer demonstrate 100-200%1and 15% fluorescence increase, respectively. However, in contrast to DCF, HyPer can be reduced and thus provides opportunity to follow both increase and decrease in H2O2level (Fig. 1d, Fig. 5b in the main text).

References

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