A Simple HPLC-Fluorescence Detection of Nitric Oxide in Cultivated Plant Cells by in situ Derivatization with 2,3-Diaminonaphthalene
2002; Springer Nature; Volume: 18; Issue: 6 Linguagem: Inglês
10.2116/analsci.18.631
ISSN1348-2246
AutoresMitsuhiro Wada, Chieko Morinaka, Toshihiko Ikenaga, Naotaka Kuroda, Kenichiro Nakashima,
Tópico(s)Nitric Oxide and Endothelin Effects
ResumoAn HPLC method with fluorescence detection for the determination of nitric oxide (NO) in cultivated plant cells (Agave pacifica, Agavaceae) was developed. NO was derivatized in situ with 2,3-diaminonaphthalene (DAN) as a labeling reagent and converted to 1(H)-naphthotriazole. The maximum peak height of the derivative was observed by incubation for 3 h at 25°C with 0.2 mM DAN. Excess reagent in cells was removed by washing 3 times with 5 ml of water. The calibration curve for authentic standard of DAN-NO spiked to cultivated plant cells showed a good linearity (r = 0.995) in the range of 5.0 to 50 pmol/g cell. The detection limit at a signal-to-noise ratio of 3 was 3.4 pmol/g cells. The proposed method was successfully applied to the monitoring of NO concentration with cell growth. The effect of thermal treatment on the concentration of NO in plant cells was also examined. The concentration of NO in cells treated at 5°C for 1 h was significantly higher than that treated at 25°C and 35°C for 1 h (n = 3, p < 0.05).
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