Direct Chemiluminescent Detection of Nitric Oxide in Aqueous Solutions Using the Natural Nitric Oxide Target Soluble Guanylyl Cyclase

摘要

Nitric oxide (NO) is a free radical involved in many physiological processes including regulation of blood pressure, immune response, and neurotransmission. However, the measurement of extremely low, in some cases sub-nanomolar physiological concentrations of nitric oxide presents an analytical challenge. The purpose of this methods article is to introduce a new highly sensitive chemiluminescent approach for direct NO detection in aqueous solutions using a natural nitric oxide target, soluble guanylyl cyclase (sGC), which catalyzes the conversion of guanosine triphopshate to guanosine 3’, 5’-cyclic monophosphate and inorganic pyrophosphate. The suggested enzymatic assay uses the fact that the rate of the reaction increases about 200 times when NO binds with sGC, and in so doing provides a sensor for nitric oxide. Luminescent detection of the above reaction is accomplished by converting inorganic pyrophosphate into ATP with the help of ATP sulfurylase followed by light emission from ATP-dependent luciferin-luciferase reaction. Detailed protocols for NO quantification in aqueous samples are provided. The examples of applications include measurements of NO generated by nitric oxide donor (PAPA-NONOate), nitric oxide synthase and NO gas dissolved in buffer. The method allows for the measurement of NO concentrations in the nanomolar range and NO generation rates as low as 100 pM/min.