Making and Working with Peroxynitrite

Peroxynitrite (ONOO⁻ ) is formed by the reaction of nitric oxide (NO) with superoxide (O₂ ⁻ ) (1 ). Under normal circumstances, these two free radicals are not formed at similar rates in the same cellular or extracellular compartment; however, under some pathological conditions, such as inflammation, this does appear to occur (2 –3 ). Peroxynitrite is a reactive compound that has the potential to modify biomolecules through several different mechanisms, and is a good candidate as a mediator of NO-dependent inhibition of key physiological or biochemical processes (4 ). There are, of course, several steps in testing the role of ONOO⁻ in an experimental model, including assessing whether the process is both sensitive to NOS inhibitors and scavengers of O₂ ⁻ , as well as examining the sample for specific evidence of nitration reactions. One example, the nitration of tyrosine, may be used as a marker, and this will be covered in Chapter 28 , this volume. Here, we focus on the practical aspects of determining the effects of ONOO⁻ addition on biochemical, cell culture, or tissue systems. Testing the biochemical basis of the hypothesis that ONOO⁻ plays an important role in the pathophysiology of human disease must be approached with a keen awareness of its physicochemical properties.