Biochem/physiol Actions

Simple and specific assay for the measurement of free nitric oxide (NO) and nitric oxide synthase (NOS) activity in living cells under physiological conditions. Nitric oxide, formed from the amino acid L-arginine via the action of NOS, is a unique cell signaling molecule that functions as both an intracellular and an extracellular messenger. NO has been implicated in vasodilation, neurotransmission, cytotoxicity, and inflammation.
The standard assay for NOS activity is for solubilized cell-free enzyme preparations such as cell lysates, crude tissue extracts or purified enzymes. It measures the production of radioÂlabeled citrulline from radiolabeled arginine substrate (Sigma Product No. NOS-1). In contrast, the Fluorometric Cell-Associated NOS Detection System measures intracellular production of NO by a non-radiometric method. Radioisotopes are replaced by a cell-permeable diacetate derivative of 4,5-diaminofluorescein (DAF-2 DA). DAF-2 DA penetrates cells rapidly where it is hydrolyzed by intracellular esterase to DAF-2 that, in turn, reacts with NO produced by NOS to form a fluorescent triazolo-fluorescein. The fluorescent product can be quantitated using an excitation filter at 492 nm and an emission filter at 515 nm.