The horseradish peroxidase enzyme gives the assay specificity for H2O2. Furthermore, H2O2 secreted to the outside of mitochondria reacts only with the H2O2 detection system, since there are no antioxidants in the incubation medium. On the other hand, addition of catalase to the incubation buffer prevents the increase in fluorescence elicited by H2O2, whereas addition of external pulses of H2O2 during a kinetic assay shows that the response of the detection system to the peroxide is instantaneous.
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