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

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Abstract
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, NO can be measured without disturbing ECD-detectable substance in NO synthase assay mixture. The NO 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 μM. 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 μM. 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.

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