Detection system of the intracellular nitric oxide in yeast by HPLC with a fluorescence detector.

Nitric oxide (NO) is an important signaling molecule involved in various biological phenomena in many organisms. The physiological functions and metabolism of NO in yeast, a unicellular microorganism, are still unknown, mainly because it is difficult to analyze the intracellular NO levels accurately. Here, we developed a new method of more accurately measuring NO content in yeast cells with the detection limit of 6 nM, by treating the cells with an NO-specific fluorescence probe followed by high-performance liquid chromatography with fluorescence detection (HPLC/FLD). This approach successfully detected and quantified the NO content inside yeast cells treated with an NO donor. Moreover, the HPLC/FLD analysis indicates that the fluorescence induced under some environmental stress conditions, such as ethanol, vanillin, and heat-shock, was not derived from NO. The HPLC/FLD method developed in this study provides a new strategy for measuring the intracellular NO concentration with higher accuracy.