DTNB (Ellman’s Reagent)

Thermo Scientific Pierce DTNB (Ellman's Reagent) reacts with sulfhydryl groups to yield a colored product, providing a reliable method to measure reduced cysteines and other free sulfhydryls in solution.

Ellman's Reagent (5,5'-dithio-bis-[2-nitrobenzoic acid]) is used to estimate sulfhydryl groups in a sample by comparing to a standard curve of a sulfhydryl-containing compound such as cysteine. Alternatively, sulfhydryl groups can be assayed using the extinction coefficient of TNB (14,150M-1cm-1 at 412nm). Ellman's Reagent has been used for applications other than sulfhydryl estimation, such as for determining alkylthiols by HPLC using a pre-column derivatization procedure and to study thiols in the active site of several enzymes.

Highlights:

• Sulfhydryl assay – reacts quantitatively with free (reduced) sulfhydryl groups (–SH) to yield detectable TNB product
• Colorimetric detection – colored product enables spectrophotometric measurement in cuvettes or microplates (λmax = 412nm; ε = 14,150/M•cm)
• Proven method – well-characterized chemistry provides quantitation of peptide or protein sulfhydryl groups using extinction coefficient calculation or by comparison to a cysteine standard

Reagent Properties:

• Alternative names: DTNB, Ellman's Reagent
• Chemical name: 5,5'-dithio-bis-(2-nitrobenzoic acid)
• Reactive toward: Sulfhydryl groups (–SH)
• Chemical formula: C14H8N2O8S2
• CAS number: 69-78-3
• Molecular weight: 396.35
• Form: Yellow crystalline powder

Product Details:

DTNB reacts with a free sulfhydryl group to yield a mixed disulfide and 2-nitro-5-thiobenzoic acid (TNB). The target of DTNB in this reaction is the conjugate base (R—S-) of a free sulfhydryl group. Therefore, the rate of this reaction is dependent on several factors: (1) the reaction pH, (2) the pKa' of the sulfhydryl and (3) steric and electrostatic effects. TNB is the colored species produced in this reaction and has a high molar extinction coefficient in the visible range. The molar extinction coefficient of TNB was originally reported by Ellman (1959) to be 13,600M-1cm-1 at 412nm and pH 8.0. Consequently, this value has been cited frequently in the literature. Later studies have shown, however, that the molar extinction coefficient is more accurately reflected by a value of 14,150M-1cm-1 at 412nm. The extinction of TNB is not affected by changes in pH between 7.6 and 8.6. However, the extinction of TNB is different in other solvents.

Color-forming reaction of Ellman's reagent with sulfhydryl groups. DTNB reacts with sulfhydryl groups, such as peptide cysteines, to form mixed disulfides and TNB, whose color can be measured at 412nm with a plate reader or spectrophotometer.

General References:

1. Ellman, G.L. (1959) Tissue sulfhydryl groups. Arch Biochem Biophys 82:70-7.
2. Riddles, P.W., et al. (1983) Reassessment of Ellman’s reagent. Meth Enzymol 91:49-60.
3. Riddles, P.W., et al. (1979) Ellman’s reagent: 5, 5'-dithiobis(2-nitrobenzoic acid) - a reexamination. Anal Biochem 94:75-81.
4. Kuwata, K., et al. (1982) Liquid chromatographic determination of alkylthiols via derivatization with 5, 5’- dithiobis(2-nitrobenzoic acid). Anal Chem 54:1082-7.
5. Masamune, S, et al. (1989) Bio-Claisen condensation catalyzed by thiolase from Zoogloea ramigera. Active site cysteine residues. Chemtracts-Organic Chem 2:247-51.
6. Tsukamoto, Y. and Wakil, S.J. (1988) Isolation and mapping of the beta-hydroxyacyl dehydratase activity of chicken liver fatty acid synthase. J Biol Chem 263:16225-9.
7. Alvear, M., et al. (1989) Fractionation of chicken liver mevalonate 5-diphosphate decarboxylase by sulfhydryl-directed reagents: evidence of a functional dithiol. Biochem Biophys Acta 994:7-11.

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