Ellman's reagent

Ellman's reagent (5,5′-dithiobis-(2-nitrobenzoic acid) or DTNB) is a colorogenic chemical used to quantify the number or concentration of thiol groups in a sample.[3] It was developed by George L. Ellman.

Ellman's reagent[1]
Names
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)
Identifiers
3D model (JSmol)
Abbreviations DTNB
ChEBI
ChEMBL
ChemSpider
ECHA InfoCard 100.000.650
EC Number
  • 200-714-4
UNII
  • InChI=1S/C14H8N2O8S2/c17-13(18)9-5-7(1-3-11(9)15(21)22)25-26-8-2-4-12(16(23)24)10(6-8)14(19)20/h1-6H,(H,17,18)(H,19,20) checkY
    Key: KIUMMUBSPKGMOY-UHFFFAOYSA-N checkY
  • InChI=1/C14H8N2O8S2/c17-13(18)9-5-7(1-3-11(9)15(21)22)25-26-8-2-4-12(16(23)24)10(6-8)14(19)20/h1-6H,(H,17,18)(H,19,20)
    Key: KIUMMUBSPKGMOY-UHFFFAOYAD
  • c1cc(c(cc1SSc2ccc(c(c2)C(=O)O)[N+](=O)[O-])C(=O)O)[N+](=O)[O-]
Properties
C14H8N2O8S2
Molar mass 396.34 g·mol−1
Melting point 240 to 245 °C (464 to 473 °F; 513 to 518 K) (decomposes)
Hazards
GHS labelling:[2]
GHS07: Exclamation mark
Warning
H315, H319, H335
P261, P264, P271, P280, P302+P352, P304+P340, P305+P351+P338, P312, P321, P332+P313, P337+P313, P362, P403+P233, P405, P501
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

Preparation

In Ellman's original paper,[3] 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

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

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.[7] It can also measure the number of thiol groups on proteins.[7]

References
  1. 5,5′-Dithiobis(2-nitrobenzoic acid) at Sigma-Aldrich
  2. "5,5'-Dithiobis(2-nitrobenzoic acid)". pubchem.ncbi.nlm.nih.gov. Retrieved 13 December 2021.
  3. Ellman GL (1959). "Tissue sulfhydryl groups". Arch. Biochem. Biophys. 82 (1): 70–7. doi:10.1016/0003-9861(59)90090-6. PMID 13650640.
  4. 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.
  5. Riddles PW, Blakeley RL, Zerner B (1983). "[8] Reassessment of Ellman's reagent". Enzyme Structure Part I. pp. 49–60. doi:10.1016/S0076-6879(83)91010-8. ISBN 978-0-12-181991-0. PMID 6855597. {{cite book}}: |journal= ignored (help)
  6. 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. S2CID 30366479.
  7. 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. S2CID 30366479.
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