Detection of S-Nitroso Compounds by Use of Midinfrared Cavity Ring-Down Spectroscopy

Abstract

S-nitrosocompounds have received much attention in biological research. In addition to their role as nitric oxide donors, there is a growing evidence that these compounds are involved in signaling processes in biological systems. Determination of S-nitrosylated proteins is of great importance for fundamental biological research and medical applications. The most common method to assay biological S-nitrosocompounds is to chemically or photochemically reduce SNO- functional groups to release nitric oxide that is then entrained in an inert gas stream and detected, usually through chemiluminescence. We report a method of S-nitrosocompounds detection using cavity ring-down measurements of gaseous NO absorbance at 5.2μ. The proposed method, in contrast to the chemiluminescence-based approach, can be used to distinguish isotopic forms of NO. We demonstrated sensitivity down to ~2 pmole of S-14NO groups and ~5 pmole of S-15NO groups for S-nitrosocompounds in aqueous solutions. The wide dynamic range of cavity ring-down detection allows the measurement of S-nitrosocompounds levels from pico- to nanomole amounts.

Photolytic Measurement of Tissue S-Nitrosothiols in Rats and Humans In Vivo

“…The assay uses focused UV light to cleave the S-N bond in the S-nitrosothiol molecules (photolysis) and release nitric oxide (NO). The NO, released from S-nitrosothiols [17], can diffuse through skin and other tissue to be recorded by the chemiluminescence device (here, the NOA280). (B).…”

“…We studied two biologic low mass S-nitrosothiols, GSNO (stable) and L-CSNO (more labile; Figure 1 [17]). We performed initial time course and dose-response studies with GSNO [17,18].…”

“…We studied two biologic low mass S-nitrosothiols, GSNO (stable) and L-CSNO (more labile; Figure 1 [17]). We performed initial time course and dose-response studies with GSNO [17,18]. GSNO standards were pipetted into 96-well opaque polystyrene microplates that prevented the passage of UV light from one well to another (Corning Incorporated, Corning, NY, USA).…”

“…Several chemical methods have been developed to measure S-nitrosothiols [15]. Colorimetric assays are simple and relatively easy to use, but their limit of sensitivity (except using cavity ring-down [16]) can be out of range for most biological S-nitrosothiols [15,17]. Methods involving mass spectrometry require liquid chromatography to separate S-nitrosothiols, during which the S-NO bond can be broken.…”