Radiochemical Measurement of NOS Activity by Conversion of [14C]L-Arginine to Citrulline Using HPLC Separation

Monitoring conversion of radiolabeled arginine to citrulline, the co-product of the nitric oxide synthase (NOS)-catalyzed reaction, is a widely used method to assay for NOS activity. In cases where NOS is the sole or overwhelmingly predominant arginine-metabolizing enzyme present in a cell or tissue preparation, it is convenient to separate arginine from citrulline using a Dowex cation-exchange chromatography system (see Chapter 7 , this volume). In some cells or tissues, however, other pathways of arginine metabolism are present and the products formed may fail to be separated from arginine, from each other, or from citrulline (if it is even present) using the simple Dowex method. For example, the widely distributed enzyme arginase (1 ) can hydrolyze arginine to ornithine and urea. Using the Dowex method, it is not possible to distinguish ornithine from arginine or urea from citrulline.