Fig. 2. Fluorometric analysis of formation of HNO from peroxidation of NH₂OH by HRP.

DAF (5 μM) was incorporated into MCF-7 cells during a 15 min incubation period at 37°C. Following centrifugation and wash steps to remove unincorporated DAF, signal intensity was monitored in a suspension of 10⁶ cells/mL in 10 mM phosphate buffer (pH 7.4) containing DTPA (50 μM), HRP (5 μM) and H₂O₂ (2.5 μM) at 37°C. Intracellular triazole fluorescence was detected at 515 nm following 488 nm excitation. A) Dose response to NH₂OH; B) intensity after 5 min following addition of H₂O₂ as indicated in the absence or presence of NH₂OH. The last panel shows the effect of depletion of GSH by overnight incubation with BSO (5 mM). Data are representative A) or the mean B) of at least four individual experiments. C) Comparison of intracellular fluorescence intensity during peroxidation of NH₂OH or decomposition of PROLI/NO (1.25 μM) in phosphate buffer alone.