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Ellman's assay for in-solution quantification of sulfhydryl groups

Rucha Bhat , Gregory Grover, Contraline Inc.


Ellman introduced 5,5'-dithio-bis-(2-nitrobenzoic acid), also known as DTNB, as a compound used for quantitating free sulfhydryl groups in solution. A solution of this compound produces a yellow-colored product when it reacts with sulfhydryl groups. This yellow-colored product can be measured using a spectrophotometer at 412 nm.

Thiol chemistry is a rapidly expanding field in basic and applied bioscience. As a consequence, the quantitative measurement of -SH groups is a routine task in many applied disciplines where a quick and easy method is much preferred. Electrochemical and fluorimetric assays are very sensitive and accurate but they involve lengthy procedures (complete proteolysis, electrolysis, HPLC separation). Although spectrophotometric thiol assays such as Ellman's are less sensi-tive in comparison, they are rapid and simple and are therefore more popular for the quantification of sulfhydryls.

Assay Principle

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. 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 is reported to be 13,600 M-1cm-1 at 412 nm and pH 8.0.

Sulfhydryl groups may be estimated in a sample by comparison to a standard curve composed of known concentrations of a sulfhydryl-containing compound such as cysteine. Alternatively, sulfhydryl groups may be quantitated by reference to the extinction coefficient of TNB.

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