Ellman's reagent

From Wikipedia, the free encyclopedia
Jump to navigation Jump to search
Ellman's reagent[1]
Preferred IUPAC name
5,5'-Disulfanediylbis(2-nitrobenzoic acid)
Other names
3,3'-Disulfanediylbis(6-nitrobenzoic acid)
5-(3-Carboxy-4-nitrophenyl)disulfanyl-2-nitrobenzoic acid
Dithionitrobenzoic acid
5,5'-Dithiobis(2-nitrobenzoic acid)
3D model (JSmol)
Abbreviations DTNB
ECHA InfoCard 100.000.650
Molar mass 396.34 g·mol−1
Melting point 240 to 245 °C (464 to 473 °F; 513 to 518 K) (decomposes)
R-phrases (outdated) R36/37/38
S-phrases (outdated) S26 S36
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).
☑Y verify (what is ☑Y☒N ?)
Infobox references

Ellman's reagent (5,5'-dithiobis-(2-nitrobenzoic acid) or DTNB) is a chemical used to quantify the number or concentration of thiol groups in a sample.[2] It was developed by George L. Ellman.


In Ellman's original paper,[2] he prepared this reagent by oxidizing 2-nitro-5-chlorobenzaldehyde to the carboxylic acid, introducing the thiol via sodium sulfide, and coupling the monomer by oxidization with iodine. Today, this reagent is readily available commercially.

Ellman's test[edit]

Thiols react with this compound, cleaving the disulfide bond 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.

Reaction of DTNB with a thiol (R-SH).

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,[3][4] and a coefficient of 13,700 M−1 cm−1 for high salt concentrations, such as 6 M guanidinium hydrochloride or 8 M urea.[4] 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.[5] Commercial DTNB may not be completely pure, so may require recrystallization to obtain completely accurate and reproducible results.[4]

Ellman's reagent can be used for measuring low-molecular mass thiols such as glutathione in both pure solutions and biological samples, such as blood.[6] It can also measure the number of thiol groups on proteins.[6]


  1. ^ 5,5′-Dithiobis(2-nitrobenzoic acid) at Sigma-Aldrich
  2. ^ a b Ellman GL (1959). "Tissue sulfhydryl groups". Arch. Biochem. Biophys. 82 (1): 70–7. doi:10.1016/0003-9861(59)90090-6. PMID 13650640. 
  3. ^ Collier HB (1973). "Letter: A note on the molar absorptivity of reduced Ellman's reagent, 3-carboxylato-4-nitrothiophenolate". Anal. Biochem. 56 (1): 310–1. doi:10.1016/0003-2697(73)90196-6. PMID 4764694. 
  4. ^ a b c Riddles PW, Blakeley RL, Zerner B (1983). "Reassessment of Ellman's reagent". Meth. Enzymol. Methods in Enzymology. 91: 49–60. doi:10.1016/S0076-6879(83)91010-8. ISBN 978-0-12-181991-0. PMID 6855597. 
  5. ^ Riener CK, Kada G, Gruber HJ (2002). "Quick measurement of protein sulfhydryls with Ellman's reagent and with 4,4'-dithiodipyridine". Anal Bioanal Chem. 373 (4–5): 266–76. doi:10.1007/s00216-002-1347-2. PMID 12110978. 
  6. ^ a b Riener, Christian K.; Kada, Gerald; Gruber, Hermann J. (2002-07-01). "Quick measurement of protein sulfhydryls with Ellman's reagent and with 4,4′-dithiodipyridine". Analytical and Bioanalytical Chemistry. 373 (4-5): 266–276. doi:10.1007/s00216-002-1347-2. ISSN 1618-2642. 

External links[edit]