Measuring nitric oxide in single neurons by capillary electrophoresis with laser-induced fluorescence: use of ascorbate oxidase in diaminofluorescein measurements.

As a family of novel fluorescent indicators for nitric oxide (NO), the diaminofluoresceins (DAFs) have allowed real-time measurement of neuronal NO, an important gaseous neurotransmitter. However, the measurement of NO by the most commonly used NO sensor, 4,5-diaminofluorescein (DAF-2), is altered by two processes: the interaction of DAF-2 with intracellular dehydroascorbic acid (DHA) and the impact of ascorbic acid (AA) on the levels of N2O3, the intermediate product of the oxidation of NO that reacts with DAF-2. Similar AA/DHA effects are observed with other DAF probes, including DAF-FM and DAR-4M. To overcome these limitations, we use a specific enzymatic reaction to eliminate the confounding effect of AA on DAF quantitation of NO and then use capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection to distinguish the various reaction products. First, the enzyme ascorbate oxidase (AO) is used to catalyze the oxidation of AA to DHA. Next, CE-LIF separates the fluorescent products of the reaction of DAF-2 with NO and DHA. Control experiments, including standard mixtures and single neurons with added NO donor, successfully demonstrate the utility of this approach. This protocol is further tested with homogenates of the mouth area from the sea slug Aplysia californica, previously shown to be NO-positive, and individual nitric oxide synthase-containing buccal neurons from the freshwater snail, Lymnaea stagnalis. In each case, significant amounts of NO are detected. This AO DAF methodology is specific, effective, simple, and allows NO to be measured in single cells without detectable interference from other compounds.