Novel fluorescence method for real-time monitoring of nitric oxide dynamics in nanoscale concentration

A novel assay was developed for the measurement of nitric oxide. The proposed method is based on fluorescence, using a fluorophore-heme dual functionality probe (FHP). The heme group can serve as an effective NO-trap, due to its very fast reaction with NO and the high stability of the resulting complex. Since the heme is connected with a fluorophore as a part of the FHP dual-functionality probe, the heme can quench the fluorophore fluorescence, under certain conditions, by a singlet-singlet energy transfer mechanism. The proposed method was tested using myoglobin covalently modified by a stilbene label. The change in emission intensity of the stilbene fragment, versus an increasing concentration of NO precursors, clearly demonstrated the spectral sensitivity required to monitor the formation of a heme-NO complex in a concentration range of 10 nM-2 microM. Furthermore, the new methodology for NO measurement was also found to be an effective assay using tissues from rabbit and porcine trachea epithelium. The measured NO flux (in an initial time interval) in tissue sample from rabbit trachea epithelia and porcine trachea epithelia is approximately 7.9x10(-12) mol/sxg and approximately 3.0x10(-12) mol/sxg respectively.