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 H(2)O(2) 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 H(2)O(2) and were therefore excluded from subsequent incubations. Under conditions of both coupled and uncoupled catalysis, SOD was absolutely required to detect (.-)NO from NOS. H(2)O(2) 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 N(omega)-hydroxy-l-arginine was utilized as substrate. In conclusion, standard Clark-type ()NO electrodes are cross-sensitive to H(2)O(2) and therefore both SOD and catalase are absolutely required to specifically detect (.-)NO from NOS.