EPR spin trapping of superoxide from nitric oxide synthase

Abstract

Superoxide formation from nitric oxide synthase has not been thoroughly investigated because of the lack of specific, sensitive end artifact-free methodologies for detecting superoxide. EPR spin trapping is the only specific method for detecting superoxide in biological systems. The development of new superoxide spin traps such as 5-(diethoxyphosphoryl)-5- methyl-pyrroline N-oxide (DEPMPO) 2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (EMPO) and the [(15)N]- EMPO has significantly improved the sensitivity of superoxide detection by electron paramagnetic resonance (EPR). Unlike 5,5-dimethyl-1-pyrroline N-oxide (DMPO), a commonly used both DEPMPO and EMPO superoxide spin trap, 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 straightforward end 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 oxygenase domain of the enzyme by a calcium/calmodulin dependent mechanism. The effect of tetrahydroblopterin cotactor and NOS Inhibitors on the release of superoxide is discussed.