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

Abstract

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 (¹⁴C or ³H) 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.