Calibration of nitric oxide flux generation from diazeniumdiolate *NO donors

The 1-(secondary amino) diazen-1-ium-1,2-diolates (NONOates) are the most commonly utilized nitric oxide (*NO, nitrogen monoxide) donor because of the ability of different NONOates to spontaneously break down liberating *NO at different rates, which can be utilized to control *NO fluxes. However, the parameters that determine these fluxes of *NO generation, half-lives and stoichiometry of *NO per donor, can vary significantly with specific experimental conditions in addition to the donor chosen. Here we report straightforward methods that can be used to determine these parameters. For donors of intermediate half-life (10-80 min) a real-time oxymyoglobin (oxyMb) assay can be analyzed to simultaneously determine both the half-life and the total amount of *NO liberated, from which the *NO flux can be obtained for any given donor concentration. The half-lives obtained by oxyMb assay are very similar to those obtained by following NONOate decomposition kinetics spectrophotometrically, and a survey of several NONOates from different commercial sources show consistent results. These data provide validation for the methodologies employed. In addition, procedures are described for calibration of donors with shorter (<10 min) and longer (>80 min) half-lives. These procedures can be used to reproducibly and routinely calibrate *NO fluxes for a variety of donors under any specific condition.