Determination of nitrite by high-performance liquid chromatography system with electrochemical detector: measurement of nitric oxide synthase activity in rat cerebellum cytosol.

A simple and sensitive assay method for NO synthase activity is described. Using glassy carbon as electrode and 30% methanol solution with 10 mM NH4Cl as mobile phase, NO2- can be measured without disturbing ECD-detectable substance in NO synthase assay mixture. The NO2- production in the assay mixture of rat cerebellum: NO synthase increased with protein and in a time-dependent manner. The Km value for the substrate, L-arginine, was 1.25 microM. The enzyme activity was inhibited in a concentration-dependent manner by a NO synthase inhibitor, NNA. The Ki value for NNA was 0.166 +/- 0.060 microM. This ECD-HPLC method for determining NO synthase activity has advantages compared with the diazo-coupling method of the Greiss reagent and the isotopic method in which the conversion of the substrate, [14C]L-arginine, to the product, [14C]L-citrulline is measured; it is simple, sensitive and is convenient for studying the NO synthase activity with various compounds as the substrate.