Fig. 3.

Guanidino nitro groups are essential to the production of NO from reduction by sodium L-ascorbate (NaAscH). n=3. A) Structures of the guanidino compounds tested with the nitro group highlighted in red. B) Representative EPR spectra showing that only guanidines with nitro groups in their structure contribute to the production of NO from reduction by NaAscH as captured by (MGD)₂Fe²⁺. Twenty-five mmol/L guanidine was reacted with 25 mmol/L NaAscH in 10 mmol/L HEPES for 15 min, and then mixed at 1:1 ratio with 10:0.5 mmol/L (MGD)₂Fe²⁺. Spectra were obtained after standing for l.5 h at room temperature. C) L-NAME or L-NNA with ¹⁵N nitro group produced a doublet EPR signal, characteristic of ¹⁵N (MGD)₂FeNO, whereas that with ¹⁴N nitro group produced a triplet signal, characteristic of ¹⁴N (MGD)₂FeNO, regardless of the isotopic type of L-arginine backbone nitrogen atoms. Experiments were performed in the same manner as (B). D) Representative direct measurement of authentic NO from reaction of L-NAME and NaAscH. HEPES buffer containing 25 mmol/L L-NAME and 25 mmol/L NaAscH (pH=7.42±0.03) was deoxygenated by argon in a purge vessel, never previously used for triiodide reagent, for 0.5 h, and then allowed to react for 12 h at room temperature, in the dark, and while anaerobic (sealed; to avoid reaction of NO with atmosphere O₂). To measure NO the headspace gas was carried by a stream of argon to the chemiluminescence NO analyzer. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)