Assay of neuronal nitric oxide synthase by HPLC determination of citrulline

Biological membranes from different tissue sources were incubated for nitric oxide synthase (NOS) activity under standard conditions at 20 degrees C and compared with rat cerebellar cytosol. NOS activity was monitored as the formation of L-citrulline from L-arginine. Samples were purified on Amprep CBA cation-exchange minicolumns prior to derivatization with o-phthaldialdehyde (OPA) and HPLC analysis. The OPA derivatives of L-citrulline and L-arginine eluted well separated within 15 min during isocratic elution at room temperature. A linear relation between peak height and quantity of L-citrulline was seen down to the detection limit at 0.1 pmol L-citrulline. Formation of L-citrulline was measurable in rat cerebellar cytosol as well as in preparations not previously assayed for NOS activity, including rat colon, cat oesophagus and crayfish (Pacifastacus leniusculus) brain. The method provides a sensitive and non-radioactive method for assaying NOS activity in small tissue samples and in tissues with low to moderate levels of NOS activity.