Assay of NOS Activity by the Measurement of Conversion of Oxyhemoglobin to Methemoglobin by NO

Abstract

Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine, molecular oxygen, and nicotinamide adenine dinucleotide phosphate (NADPH) to NO, citrulline, and NADP⁺ (reviewed in ref. 1). This chapter describes the measurement of NOS activity by utilizing the conversion of oxyhemoglobin to methemoglobin by NO while monitoring the absorption difference between the wavelengths 401 and 421 nm or at just 401 nm. This assay was first described by Feelisch and Noack (2) and has since been modified to allow not only the measurement of NOS activity in vitro but also the degree of in vivo inhibition of neuronal (n) NOS (and also possibly endothelial (e) NOS) by ex vivo analysis after the administration of slowly dissociating inhibitors such as N^ω-nitro-L-arginine (L-NNA) and its methyl ester, L-NAME (3,4). Using the protocol described, this technique is sensitive (limit of detection approx 20 pmol/min per g tissue), specific for NOS in conjunction with the use of NOS inhibitors, applicable to most preparations (with the exception of samples containing large amounts of hemoglobin) and suitable for continual monitoring.