EPR spin trapping of superoxide from nitric oxide synthase

Abstract

15 N)- EMPO has significantly improved the sen- sitivity of superoxide detection by electron para- magnetic resonance (EPR). Unlike 5,5-dimethyl-1- pyrroline N-oxide (DMPO), a commonly used superoxide spin trap, both DEPMPO and EMPO form superoxide adducts that are persistent, which do not decay to the corresponding hydroxyl adduct. Using these new spin traps, the EPR-detection of superoxide is now straightfor- ward and more sensitive. These new superoxide traps in combination with the loop gap resonator technology have enabled us to demonstrate unequivocally the formation of superoxide from the endothelial and neuronal isoforms of nitric oxide synthase. Superoxide is formed at the oxy- genase domain of the enzyme by a calcium/ calmodulin dependent mechanism. The effect of tetrahydrobiopterin cofactor and NOS inhibitors on the release of superoxide is discussed.