Biologic systems contain redox elements, which function in cell signaling, macromolecular trafficking and physiologic regulation. Oxidative stress includes disruption of this redox circuitry through altered functions of enzymes, receptors, transporters, transcription factors, and structural elements, in addition to the macromolecular damage, both resulting from an imbalance between pro-oxidants and antioxidants performances.
Since many proteins contain redox-sensitive free thiols, the identification and quantification of their different redox states gives us an idea of the oxidative stress level of the sample.
The present assay is based on the classic colorimetric one, first described by Ellman in 1958, and aimed at the detection of reduced thiols, but modified in order to allow the detection of those oxidized to disulfides as well.
G-Biosciences’ Thiol and Disulfide Assay Kit is recommended for estimations of oxidative stress levels in biological samples such as plasma. The assay described here measures the formation of 2-nitro-5-thiobenzoate (TNB), which is proportional to the amount of reduced thiols in the sample that are oxidized by the 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) through a non-specific reaction. The generated TNB ionizes to a dianion at alkaline pH and thus develops an intense yellow color with an absorbance maximum at 412 nm.
R-SH + DTNB → R-TNB + TNB (Yellow) (λMax = 412 nm)
The assay also allows the measurement of disulfides in the sample. Sodium borohydride (NaBH4) is used to reduce disulfides to thiols.
R-S2-R’ + NaBH4 → 2 R-SH + BH3+ Na
