DTNB

DTNB, or 5,5'-dithiobis-(2-nitrobenzoic acid), commonly known as Ellman's reagent, is a colorimetric reagent used to quantify free thiol groups in proteins, peptides, and small molecules. When it reacts with a thiol (R-SH), it yields 2-nitro-5-thiobenzoate (TNB−), a yellow anion that absorbs light at 412 nm. The amount of TNB− formed is proportional to the thiol concentration, enabling spectrophotometric quantification.

In typical assays, DTNB is dissolved in a buffered solution near neutral pH, and a sample is mixed with the reagent. After a brief incubation (about 5–15 minutes at room temperature), the absorbance at 412 nm is measured. The thiol concentration is calculated from the absorbance using the molar extinction coefficient of TNB−, commonly cited around 13,600–14,000 M−1 cm−1. The method is adaptable to microplate readers and standard spectrophotometers.

DTNB is widely used to determine thiol content in proteins, such as estimating accessible cysteine residues, and to measure reduced glutathione (GSH) levels in biological samples. It is often used to assess changes in thiol status under various treatments or conditions.

Limitations include its reliance on accessible free thiols; disulfide bonds or buried cysteines may not react readily without prior reduction. The assay can be interfered with by substances that absorb at similar wavelengths, suspensions that cause turbidity, or by reducing agents (e.g., DTT, beta-mercaptoethanol) that must be removed or masked prior to measurement. Care should be taken to maintain appropriate pH and to handle DTNB as a hazardous chemical, using protective equipment and proper disposal.