Effects of Oxidants on Properties of Fluorescent Calcium Indicators

An increasing number of studies use calcium-sensitive fluorescent dyes to address the relationship between elevated levels of intracellular calcium and free-radical-mediated damage in a variety of pathophysiological phenomena. The present study evaluates the effects of reactive oxygen species on the spectral properties of widely used calcium probes such as Fura-2 and Fluo-3. We found that both Fura-2 and Fluo-3 are rapidly inactivated by hydroxyl radicals and enzymatically inactivated by peroxidase/H2O2. This results in a decrease in the dynamic range of sensitivity of both dyes to Ca2+, as well as in a decrease in the affinity of Fluo-3 for Ca2+. The data suggest that oxidation of the calcium probes affects the measurement of calciumin vitroand may alter the interpretation ofin vivodata since the absence of or small changes in the calcium fluorescence signal can be the result of probe deactivation by free oxygen radicals rather than the lack of actual Ca2+changes.