Colorimetric Thiol/Disulfide Detection Assay Kit (-SH/ S-S)
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 result 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 bases on the classic colorimetric one, first described by Ellman in 1958. It was aimed to detect reduced thiols, but modified in order to allow the detection of those oxidated to disulfides as well.
Bioquochem’s Thiol and Disulfide Assay Kit is recommended for estimations of oxidative stress levels in biological samples such as plasma.
Specifically, this assay measures the formation of 2-nitro-5-thiobenzoate (TNB). It is proportional to the amount of reduced thiols in the sample that the 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) oxidize through a non-specific reaction. The generated TNB ionizes to a dianion at alkaline pH. Then it develops an intense yellow color with an absorbance maximum at 412 nm (Scheme 1).
In addittion, the assay also allows the measurement of disulfides in the sample, thanks to NaBH4, which reduces them to thiols (Scheme 2).
R-SH + DTNB → R-TNB + TNB (Yellow) (λMax = 412 nm)
Scheme 1. Reaction of DTNB with thiol groups
R-S_S-R’ + NaBH4 → 2 R-SH + BH3+ Na
Scheme 2. Reduction of disulfides by Na〖BH〗4