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. 2000 Aug 22;97(18):9943–9948. doi: 10.1073/pnas.180155397

Figure 2.

Figure 2

Sensitivity and specificity of I3−/ozone-based chemiluminescence assay, with and without KCN/K3Fe(CN)6, for the quantification of total nitrosylated hemoglobin, SNO-Hb, and Hb(FeII)NO. A demonstrates the reaction of standards of pure SNO-Hb (stippled bars) and Hb(FeII)NO (slashed bar) with and without KCN and K3Fe(CN)6 (indicated as + and −KCN, respectively), which selectively removes the NO from heme while preserving the S-nitrosothiol bond. Within 5 min, treatment with a large molar excess of KCN/K3Fe(CN)6 (0.2 M) eliminates 90% of the signal of Hb(FeII)NO in the chemiluminescent I3− reaction, while preserving the signal from SNO-Hb (n = 5). Results are expressed as the percentage of the original signal from the standards reacted without KCN/K3Fe(CN)6 pretreatment. Similar results were observed up to 120 min (data not shown). B demonstrates the NO signal released from three injections of water from the Sephadex G25 column (background control), followed by increasing standard dilutions of SNO-Hb, pretreated with KCN/K3Fe(CN)6 (control hemoglobin, 0.002%, 0.004%, 0.008%, 0.016%, 0.032%), measured by I3−/ozone-based chemiluminescence. C demonstrates measurement linearity of four serial dilutions of SNO-Hb, pretreated with KCN/K3Fe(CN)6, from 0.002% to 0.032% (r2 = 0.996; P < 0.001; n = 4). Similar results were obtained for standard curves of Hb(FeII)NO (r2 = 0.999, P < 0.001, n = 5).