Protein Protocols Protein Protocols

Modification of Sulfhydryl Groups
70
Modification of Sulfhydryl Groups with DTNB
Dan S. Tawfik
1. Introduction
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
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5-5'-Dithio-bis (2-nitrobenzoic acid) (DTNB or Ellman’s reagent; ref. 1) reacts with
the free sulfhydryl side chain of cysteine to form an S–S bond between the protein and
a thionitrobenzoic acid (TNB) residue. The modification is generally rapid and selective.
The main advantage of DTNB over alternative reagents (e.g., N-ethylmaleimide or
iodoacetamide) is in the selectivity of this reagent and in the ability to follow the course
of the reaction spectrophotometrically. The reaction is usually performed at pH 7.0–8.0
and the modification is stable under oxidative conditions. The TNB group can be
released from modified protein by treatment with reagents that are routinely used to
reduce S–S bonds, for example, mercaptoethanol, or by potassium cyanide (2) (see
Note 1). In addition, the often highly pronounced differences in reactivity of different
cysteine side chains in the same protein or even active site, and the availability of a
variety of thiol-modifying reagents can be exploited to selectively modify cysteine
side chains in proteins in the presence of other, more reactive cysteine residues (3).
2. Materials
1. DTNB.
2. 0.1 M Tris-HCl, pH 8.0.
3. Protein for modification: approx 5 µM in 0.1 M Tris-HCl, pH 8.0.
3. Method
1. Prepare fresh solutions of DTNB (0.5–5 mM; see Note 2) in 0.1 M Tris-HCl, pH 8.0.
2. Add 20-µL aliquots of the DTNB solution to 180 µL of the protein solution and incubate
for 30 min.
3. Determine the number of modified cysteines by measuring the absorbance of the released
TNB anion at 412 nm (ε = 14,150 M –1 cm –1 ) (see Note 3).
4. Dialyze against an appropriate buffer (see Note 4).
5. Determine the activity of the protein.