Journal of Fluorescence

, Volume 14, Issue 1, pp 17–23 | Cite as

Fluorescence Measurements of Steady State Peroxynitrite Production Upon SIN-1 Decomposition: NADH Versus Dihydrodichlorofluorescein and Dihydrorhodamine 123

  • Francisco Javier Martin-Romero
  • Yolanda Gutiérrez-Martin
  • Fernando Henao
  • Carlos Gutiérrez-MerinoEmail author


The production of peroxynitrite during 3-morpholinosydnonimine (SIN-1) decomposition can be continuously monitored, with a sensitivity ≤ 0.1 μM, from the kinetics of NADH fluorescence quenching in phosphate buffers, as well as in buffers commonly used with cell cultures, like Locke's buffer or Dulbecco's modified Eagle's medium (DMEM-F12). The half-time for peroxynitrite production during SIN-1 decomposition ranged from 14–18 min in DMEM-F12 (plus and minus phenol red) to 21.5 min in Locke's buffer and 26 min in DMEM-F12 supplemented with apotransferrin (0.1 mg/mL). The concentration of peroxynitrite reached a peak that was linearly dependent upon SIN-1 concentration, and that for 100 μM SIN-1 amounted to 1.4 ± 0.2 μM in Locke's buffer, 3.2–3.6 μM in DMEM-F12 (plus and minus phenol red) and 1.8 μM in DMEM-F12 supplemented with apotransferrin. Thus, the maximum concentration of peroxynitrite ranged from 1.2 to 3.6% of added SIN-1. NADH was found to be less sensitive than dihydrorhodamine 123 and 2′,7′-dichlorodihydrofluorescein diacetate to oxidation by H2O2, which is produced during SIN-1 decomposition in common buffers. It is shown that peroxynitrite concentration can be controlled (±5%) during predetermined times by using sequential SIN-1 pulses, to simulate chronic exposure of cells or subcellular components to peroxynitrite.

Peroxynitrite 3-morpholinosydnonimine NADH fluorescence 


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Copyright information

© Plenum Publishing Corporation 2004

Authors and Affiliations

  • Francisco Javier Martin-Romero
    • 1
  • Yolanda Gutiérrez-Martin
    • 1
  • Fernando Henao
    • 1
  • Carlos Gutiérrez-Merino
    • 1
    Email author
  1. 1.Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Ciencias and Escuela de Ingenierías AgrariasUniversidad de ExtremaduraBadajozSpain

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