Measurement of NOS Activity by Conversion of Radiolabeled Arginine to Citrulline Using Ion-Exchange Separation

Nitric oxide synthase (NOS) catalyzes a complex reaction utilizing L -arginine, nicotinamide adenine dinucleotide phosphate (NADPH) and oxygen to synthesize NO, and with citrulline and NADP⁺ being produced as co-products (reviewed in ref. 1 ). This chapter describes the measurement of NOS-functional activity by determining the NOS inhibitor-sensitive conversion of radiolabeled (¹⁴ C or ³ H) arginine to citrulline, with separation of the labeled product from substrate by ion-exchange techniques. The earliest forms of this assay used rather slow and labor-intensive separations, e.g., on an amino-acid analysis ion-exchange column (2 ). However, a simple method of separating arginine from citrulline using the Na⁺ form of the strongly acidic-cation exchanger Dowex 50 (which has sulphonic-acid functional groups) was described by Gopalakrishna and Nagarajan (3 ), and this technique was applied by Bredt and Snyder (4 ) to assays of purified NOS. We have subsequently modified this assay (5 –7 ) to simplify it further and to make it suitable for the study of NOS activity in a wide variety of settings.