Superoxide dismutase and catalase are required to detect NO from both coupled and uncoupled neuronal no synthase

Despite numerous approaches to measuring nitric oxide ( NO) formation from purified NO synthase (NOS), it is still not clear whether NO is a direct or indirect product of the NO synthase reaction. The direct detection of catalytically formed NO is complicated by side reactions with reactive oxide species like H2O2 and superoxide. The aim of the present study was therefore to reinvestigate these reactions both electrochemically and by chemiluminescence detection with particular emphasis on the requirement for cofactors and their interference with NO detection. Flavins were found to generate large amounts of H2O2 and were therefore excluded from subsequent incubations. Under conditions of both coupled and uncoupled catalysis, SOD was absolutely required to detect NO from NOS. H2O2 formation took place also in the presence of SOD and gave a smaller yet significant interfering signal. Similar data were obtained when the proposed intermediate Nω-hydroxy-l-arginine was utilized as substrate. In conclusion, standard Clark-type NO electrodes are cross-sensitive to H2O2 and therefore both SOD and catalase are absolutely required to specifically detect NO from NOS.