Ellman's reagent
Encyclopedia
Ellman's reagent is a chemical used to quantify the number or concentration of thiol
Thiol
In organic chemistry, a thiol is an organosulfur compound that contains a carbon-bonded sulfhydryl group...

 groups in a sample.

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
Sodium sulfide is the name used to refer to the chemical compound Na2S, but more commonly it refers to the hydrate Na2S·9H2O. Both are colorless water-soluble salts that give strongly alkaline solutions...

, and coupling the monomer by oxidization with iodine
Iodine
Iodine is a chemical element with the symbol I and atomic number 53. The name is pronounced , , or . The name is from the , meaning violet or purple, due to the color of elemental iodine vapor....

. Today, this reagent is readily available commercially.

Ellman's test

Thiols react with this compound, cleaving the disulfide bond
Disulfide bond
In chemistry, a disulfide bond is a covalent bond, usually derived by the coupling of two thiol groups. The linkage is also called an SS-bond or disulfide bridge. The overall connectivity is therefore R-S-S-R. The terminology is widely used in biochemistry...

 to give 2-nitro-5-thiobenzoate (NTB-), which ionizes to the NTB2- dianion in water at neutral and alkaline pH. This NTB2- ion has a yellow color.



This reaction is rapid and stoichiometric, with the addition of one mole of thiol releasing one mole of NTB. The NTB2- is quantified in a spectrophotometer by measuring the absorbance of visible light at 412 nm, using an extinction coefficient
Molar absorptivity
The molar absorption coefficient, molar extinction coefficient, or molar absorptivity, is a measurement of how strongly a chemical species absorbs light at a given wavelength...

 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
Guanidine
Guanidine is a crystalline compound of strong alkalinity formed by the oxidation of guanine. It is used in the manufacture of plastics and explosives. It is found in urine as a normal product of protein metabolism. The molecule was first synthesized in 1861 by the oxidative degradation of an...

 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
Recrystallization (chemistry)
-Chemistry:In chemistry, recrystallization is a procedure for purifying compounds. The most typical situation is that a desired "compound A" is contaminated by a small amount of "impurity B". There are various methods of purification that may be attempted , which includes recrystallization...

 to obtain completely accurate and reproducible results.

Ellman's reagent can be used for measuring low-molecular mass thiols such as glutathione
Glutathione
Glutathione is a tripeptide that contains an unusual peptide linkage between the amine group of cysteine and the carboxyl group of the glutamate side-chain...

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

External links

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