Visible Photolysis and Amperometric Detection of S-Nitrosothiols

Abstract

The concentration of S-nitrosothiols (RSNOs), endogenous transporters of the signaling molecule nitric oxide (NO), fluctuate greatly in physiology often as a function of disease state. RSNOs may be measured indirectly by cleaving the S–N bond and monitoring the liberated NO. While ultraviolet photolysis and reductive-based cleavage both decompose RSNOs to NO, poor selectivity and the need for additional reagents preclude their utility clinically. Herein, we report the coupling of visible photolysis (i.e., 500–550 nm) and amperometric NO detection to quantify RSNOs with greater selectivity and sensitivity. Enhanced sensitivity (up to 1.56 nA µM−1) and lowered theoretical detection limits (down to 30 nM) were achieved for low molecular weight RSNOs (i.e., S-nitrosoglutathione, S-nitrosocysteine) by tuning the irradiation exposure. Detection of nitrosated proteins (i.e., S-nitrosoalbumin) was also possible, albeit at a decreased sensitivity (0.11 nA µM−1). This detection scheme was used to measure RSNOs in plasma and illustrate the potential of this method for future physiological studies.

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

“…Photolysis-chemiluminescence is based on photosensitive homolytic cleavage of RSNO after exposure to light; however, ultraviolet photolysis has been criticized for overestimating RSNO values because of the presence of endogenous nitrite and nitrate that can also be converted to NO by ultraviolet irradiation. 209 The Cu-Cys assay is not useful for solutions with high protein content (plasma, hemoglobin) and has been replaced by triodiode (KI 3 )-based reductive chemiluminescence, which provides a more specific assay of RSNO levels. 210,211 Triodiode-based assays have become the most accepted and widely used method for RSNO measurement, providing sensitive and specific measurements in complex systems.…”

S-Nitrosothiol Analysis via Photolysis and Amperometric Nitric Oxide Detection in a Microfluidic Device

“…47–50 Riccio et al previously reported on the use of visible photolysis and amperometric detection for the quantification of RSNOs in both phosphate buffered saline (PBS) and blood plasma. 51 To achieve adequate limits of detection, this detection scheme required deoxygenation of samples to avoid the reaction of NO with oxygen. The requirement of large sample volumes (40 mL), further limited the clinical utility of the setup.…”

Detection of S-nitrosothiols

“…Typically, the range of shorter wavelength is used for irradiation, although recently visible-wavelength photolysis was also suggested [36] to avoid possible interference with nitrite [21] and nitrate [37]. The light intensity must not be higher than 100 W, otherwise thermal effects might cause signal distortion.…”