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

• Published in: Analytical biochemistry Vol. 598; p. 113707
• Main Authors: Nasuno, Ryo, Shino, Seiya, Yoshikawa, Yuki, Yoshioka, Natsuko, Sato, Yuichi, Kamiya, Kohei, Takagi, Hiroshi
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.06.2020
• Subjects: Chromatography, High Pressure Liquid; Environmental stress; Flow Cytometry; Fluorescence probe; Methodology; Nitric oxide; Nitric Oxide - analysis; Nitric Oxide - metabolism; Saccharomyces cerevisiae - chemistry; Saccharomyces cerevisiae - cytology; Saccharomyces cerevisiae - metabolism; Spectrometry, Fluorescence; Yeast; DAF-FM-T; NO; FCM; Yeast; Methodology; DAF-FM DA; DAF-FM; Nitric oxide; Fluorescence probe; HPLC; Environmental stress; FLD
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2020.113707

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. [Display omitted] •We developed an NO measurement system using HPLC and a fluorescence NO probe.•Our measurement system successfully quantified the intracellular NO level in yeast.•The previous method using flowcytometry produced false positives for NO detection.