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.