Direct chemiluminescence detection of nitric oxide in aqueous solutions using the natural nitric oxide target soluble guanylyl cyclase

Abstract

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.

Measurement of Reactive Oxygen Species, Reactive Nitrogen Species, and Redox-Dependent Signaling in the Cardiovascular System

“…128 This approach is based on NO-mediated activation of soluble guanylyl cyclase, which catalyzes the conversion of GTP to cGMP and inorganic pyrophosphate. The assay measures the pyrophosphate, which is converted into ATP with the help of ATP sulfurylase.…”

Nitric Oxide Sensors for Biological Applications

“…Chemiluminescent probes can be used to detect NO in both liquid and gaseous samples [74][75][76][77]. The general scheme for the detection of NO in fluid samples via chemiluminescence is through the activation of guanylyl cyclase by NO exposure, causing guanosine triphosphate (GTP) to be converted to guanosine 3 ,5 -cyclic monophosphate (cGMP) which results in the release of pyrophosphate, which can then interact with adenosine triphosphate sulfurylase (ATP-sulfurylase) to form ATP [76,77].…”

“…The general scheme for the detection of NO in fluid samples via chemiluminescence is through the activation of guanylyl cyclase by NO exposure, causing guanosine triphosphate (GTP) to be converted to guanosine 3 ,5 -cyclic monophosphate (cGMP) which results in the release of pyrophosphate, which can then interact with adenosine triphosphate sulfurylase (ATP-sulfurylase) to form ATP [76,77]. The ATP that is formed through this series of reactions is then able to interact with a luciferin-luciferase system to emit light at 560 nm [76,77]. An advantage of the chemiluminescent system is that it is able to detect NO at nM concentrations, since the initial guanylyl cyclase interaction with NO leads to a 200-fold linear increase [76,77].…”

“…The ATP that is formed through this series of reactions is then able to interact with a luciferin-luciferase system to emit light at 560 nm [76,77]. An advantage of the chemiluminescent system is that it is able to detect NO at nM concentrations, since the initial guanylyl cyclase interaction with NO leads to a 200-fold linear increase [76,77]. Chemiluminescent probes have some disadvantages, including the inability to detect NO in vivo.…”

Detection of nitric oxide production in cell cultures by luciferin–luciferase chemiluminescence

“…Enzyme-linked NO detection suggested in our and other works [15–17] is based on the changing of catalytic activity of an enzyme in the presence of NO. The concept, first suggested in our publication [17], is outlined on Scheme 1.…”