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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.

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R-phrases (outdated) R36/37/38
S-phrases (outdated) S26 S36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).
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Infobox references



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 testEdit

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. pp. 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. PMID 12110978.

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