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

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 in pure crystalline form are colorless solids, although technical grades of sodium sulfide are gener ...

, and coupling the monomer by oxidization with

iodine Iodine is a chemical element with the symbol I and atomic number 53. The heaviest of the stable halogens, it exists as a semi-lustrous, non-metallic solid at standard conditions 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 biochemistry, a disulfide (or disulphide in British English) refers to a functional group with the structure . The linkage is also called an SS-bond or sometimes a disulfide bridge and is usually derived by the coupling of two thiol groups. In ...

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 crystallizes in orthorhombic space group ''Pbca''. The crystal str ...

or 8 M urea. Unfortunately the extinction coefficient for dilute solutions was underestimated in the original 1959 publication, as 13,600 M⁻¹ cm⁻¹, and as noted in a recent article, this mistake has persisted in the literature. 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 antioxidant in plants, animals, fungi, and some bacteria and archaea. Glutathione is capable of preventing damage to important cellular components caused by sources such as reactive oxygen species, free radicals, pero ...

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 Nitrobenzenes Biochemistry Reagents for biochemistry Reagents