Cellular applications of a sensitive and selective fiber optic nitric oxide biosensor based on a dye-labeled heme domain of soluble guanylate cyclase

Nitric oxide-selective sensors have been prepared with the heme domain of s oluble guanylate cyclase (sGC), the only known receptor for signal transduc tion involving nitric oxide. Expressed in and purified from E. coli, the he me domain contains a stoichiometric amount of heme that has electronic and resonance Raman spectra almost identical to those of heterodimeric (native) sGC purified from bovine lung. The small size of the heme domain, its: ina bility to bind oxygen, and its high affinity for nitric oxide make it well- suited for sensor applications. The heme domain has been labeled with a flu orescent reporter dye and changes in this dye's intensity are observed base d on the sGC heme domain's characteristic binding of nitric oxide. The curr ent sensors are prepared with 100-mu m optical fiber but could also be prep ared using submicrometer fiber tips. These sensors have fast, linear, and r eversible responses to nitric oxide and are unaffected by numerous common i nterferents, such as oxygen, nitrite and nitrate. The sensor limit of detec tion is 1 mu M nitric oxide. Glutathione has been shown to decrease the: se nsitivity of the sensor; however, the sensor response: remains linear and c an be calibrated on the basis of the glutathione concentration present in t he biological environment of interest. The sensors have been used to measur e extracellular nitric oxide production by BALB/c mouse macrophages, Minima l nitric oxide was produced by untreated cells, while high levels of nitric oxide were released from activated cells, e.g., 111 +/- 2 mu M in a given cell culture.