Ellman's reagent

Ellman's reagent[1]
IUPAC name 5-(3-Carboxy-4-nitrophenyl)disulfanyl-2-nitrobenzoic acid
Other names Dithionitrobenzoic acid; 5,5'-Dithiobis(2-nitrobenzoic acid)
Identifiers
Abbreviations	DTNB
CAS number	69-78-3
PubChem	6254
ChemSpider	6018 Y
ChEMBL	CHEMBL395814 Y
Jmol-3D images	Image 1
SMILES c1cc(c(cc1SSc2ccc(c(c2)C(=O)O)[N+](=O)[O-])C(=O)O)[N+](=O)[O-]
InChI 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) Y Key: KIUMMUBSPKGMOY-UHFFFAOYSA-N Y 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
Properties
Molecular formula	C14H8N2O8S2
Molar mass	396.35 g mol−1
Melting point	240-245 °C (dec.)
Hazards
R-phrases	R36/37/38
S-phrases	S26 S36
Y (verify) (what is: Y/N?) Except where noted otherwise, data are given for materials in their standard state (at 25 °C, 100 kPa)
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]

Preparation

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

Thiols react with this compound, cleaving the disulfide bond to give 2-nitro-5-thiobenzoate (NTB^-), which ionizes to the NTB^2- dianion in water at neutral and alkaline pH. This NTB^2- 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 NTB. The NTB^2- 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.^[5]

References

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 9780121819910. PMID 6855597. 
5. ^ ^{a b} 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. ^ Sedlak J, Lindsay RH (1968). "Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent". Anal. Biochem. 25 (1): 192–205. doi:10.1016/0003-2697(68)90092-4. PMID 4973948. 

External links

• Quantitation of sulfhydryls DTNB, Ellman’s reagent (uses incorrect absorbance coefficient)