Validation of the tri-iodide method. (A) A SNO-Hb standard sample that was 125 μM in heme and 59 μM in nitrosated thiol was serially diluted 1:1 reaching a final concentration of 0.9 μM (SNO). For each concentration, the sample was split in 2 and assayed by the tri-iodide method (○) or the 3C method (•). The measured concentration of SNO is plotted against the expected amount based on the amount that the initial sample was diluted. The solid line shows the predicted amount. The initial concentration of SNO-Hb was taken as the average of that using the 3C and tri-iodide method (57 μM and 60 μM). (B) A SNO-Hb standard sample (57 μM in SNO and 1 mM in heme) was spiked into high concentrations of NO-free Hb with a heme concentration 5 mM to give expected final concentrations of SNO between 1 and 12 μM (SNO/heme between 0.00025 and 0.003). The amount of SNO-Hb in the spiked samples was measured using the tri-iodide method and plotted against the expected values.