Fluorescence measurements of steady state peroxynitrite production upon SIN-1 decomposition: NADH versus dihydrodichlorofluorescein and dihydrorhodamine 123.
The production of peroxynitrite during 3-morpholinosydnonimine (SIN-1) decomposition can be continuously monitored, with a sensitivity < or = 0.1 microM, 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 microM SIN-1 amounted to 1.4 +/- 0.2 microM in Locke's buffer, 3.2-3.6 microM in DMEM-F12 (plus and minus phenol red) and 1.8 microM 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.
Martin-Romero
Francisco Javier
FJ
Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Ciencias and Escuela de Ingenierías Agrarias, Universidad de Extremadura, Avenida de Elvas s/n, 06080-Badajoz, Spain.
Gutiérrez-Martin
Yolanda
Y
Henao
Fernando
F
Gutiérrez-Merino
Carlos
C
D005452:Fluoresceins / Q000737:chemistry*; D008981:Molsidomine / Q000031:analogs & derivatives* / Q000737:chemistry*; D009243:NAD / Q000737:chemistry*; D030421:Peroxynitrous Acid / Q000138:chemical synthesis*; D012235:Rhodamines / Q000737:chemistry*; D013050:Spectrometry, Fluorescence
D005452:Fluoresceins; D012235:Rhodamines; D009243:NAD; C058319:dihydrorhodamine 123; D030421:Peroxynitrous Acid; C002385:linsidomine; D008981:Molsidomine