A practical method for preparing peroxynitrite solutions of low ionic strength and free of hydrogen peroxide.
The reaction of ozone (approximately 5% in oxygen) with sodium azide (0.02-0.2 M in water) at pH 12 and 0-4 degrees C is shown to yield concentrated, stable peroxynitrite solutions of up to 80 mM. The product of this reaction is identified based on a broad absorption spectrum with a maximum around 302 nm and by its first-order rate of decomposition (k = 0.40 +/- 0.01 s-1 at pH 7.05 and 25 degrees C). These peroxynitrite solutions can be obtained essentially free of hydrogen peroxide (detection limit 1 microM) and only traces of azide (detection limit 0.1 mM). They are low in ionic strength and have a pH of about 12 but without buffering capacity; therefore, they can be adjusted to any pH by addition of buffer. These preparations of peroxynitrite frozen at -20 degrees C show negligible decomposition for about 3 weeks of storage and follow a first-order decomposition with a halflife of about 7 days at refrigerator temperatures (approximately 5 degrees C). These preparations give reactions that are characteristic of peroxynitrite. For example, at pH 7.0, they react with L-tyrosine to give a 7.3 mol % yield of nitrotyrosine(s), and with dimethyl sulfoxide to give a 8.2 mol % yield of formaldehyde, based on starting peroxynitrite concentration.
Pryor
W A
WA
Biodynamics Institute, Louisiana State University, Baton Rouge 70803-1800.
Cueto
R
R
Jin
X
X
Koppenol
W H
WH
Ngu-Schwemlein
M
M
Squadrito
G L
GL
Uppu
P L
PL
Uppu
R M
RM
D001386:Azides / Q000737:chemistry; D002021:Buffers; D004340:Drug Contamination / Q000517:prevention & control; D004355:Drug Stability; D005609:Free Radicals; D005615:Freezing; D006861:Hydrogen Peroxide; D006863:Hydrogen-Ion Concentration; D009566:Nitrates / Q000737:chemistry*; D009994:Osmolar Concentration; D010126:Ozone / Q000737:chemistry; D019810:Sodium Azide; D012996:Solutions; D013053:Spectrophotometry; D014443:Tyrosine / Q000737:chemistry
D001386:Azides; D002021:Buffers; D005609:Free Radicals; D009566:Nitrates; D012996:Solutions; C039642:peroxynitric acid; D014443:Tyrosine; D010126:Ozone; D019810:Sodium Azide; D006861:Hydrogen Peroxide