Ellman's reagent (5,5′-dithiobis-(2-nitrobenzoic acid) or DTNB) is a

colorogenic Colorogenic or colourogenic describes a property of chemical compounds which are initially not colored (i.e. they show negligible absorbance of visible light), but become colored through a chemical reaction, often through an intermolecular covalent ...

chemical used to quantify the number or concentration of

thiol In organic chemistry, a thiol (; ), or thiol derivative, is any organosulfur compound of the form , where R represents an alkyl or other organic substituent. The functional group itself is referred to as either a thiol group or a sulfhydryl grou ...

groups in a sample. It was developed by George L. Ellman.

Preparation

In Ellman's original paper, he prepared this reagent by oxidizing 2-nitro-5-chlorobenzaldehyde to the carboxylic acid, introducing the thiol via

sodium sulfide Sodium sulfide is a chemical compound with the formula Na2 S, or more commonly its hydrate Na2S·9 H2O. Both the anhydrous and the hydrated salts are colorless solids, although technical grades of sodium sulfide are generally yellow to brick red ...

, and coupling the monomer by oxidization with

iodine Iodine is a chemical element; it has symbol I and atomic number 53. The heaviest of the stable halogens, it exists at standard conditions as a semi-lustrous, non-metallic solid that melts to form a deep violet liquid at , and boils to a vi ...

. Today, this reagent is readily available commercially.

Ellman's test

Thiols react with this compound, cleaving the

disulfide bond In chemistry, a disulfide (or disulphide in British English) is a compound containing a functional group or the anion. The linkage is also called an SS-bond or sometimes a disulfide bridge and usually derived from two thiol groups. In inor ...

to give 2-nitro-5-thiobenzoate (TNB⁻), which ionizes to the TNB²⁻ dianion in water at neutral and alkaline pH. This TNB²⁻ ion has a yellow color. : DTNB reaction with thiol

This reaction is rapid and stoichiometric, with the addition of one mole of thiol releasing one mole of TNB. The TNB²⁻ is quantified in a spectrophotometer by measuring the absorbance of visible light at 412 nm, using an extinction coefficient of 14,150 M⁻¹ cm⁻¹ for dilute buffer solutions, and a coefficient of 13,700 M⁻¹ cm⁻¹ for high salt concentrations, such as 6 M

guanidinium hydrochloride Guanidinium chloride or guanidine hydrochloride, usually abbreviated GdmCl and sometimes GdnHCl or GuHCl, is the hydrochloride salt of guanidine. Structure Guanidinium chloride on a weighing boat Guanidinium chloride crystallizes in orthorhomb ...

or 8 M urea. Ellman's original 1959 publication estimated the molar extinction at 13,600 M⁻¹ cm⁻¹, and this value can be found in some modern applications of the method despite improved determinations. Commercial DTNB may not be completely pure, so may require recrystallization to obtain completely accurate and reproducible results. Ellman's reagent can be used for measuring low-molecular mass thiols such as

glutathione Glutathione (GSH, ) is an organic compound with the chemical formula . It is an antioxidant in plants, animals, fungi, and some bacteria and archaea. Glutathione is capable of preventing damage to important cellular components caused by sources ...

in both pure solutions and biological samples, such as blood. It can also measure the number of thiol groups on proteins.

References

External links

Quantitation of sulfhydryls DTNB, Ellman’s reagent
(uses incorrect absorbance coefficient) {{DEFAULTSORT:Ellman's Reagent Biochemistry detection reactions Organic disulfides Benzoic acids Nitrobenzene derivatives Biochemistry Reagents for biochemistry Reagents