DTNB 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 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.
Modification of Enzyme with DTNB:
Modification of the SH groups of the enzyme is carried out by reacting 10 μL of 10 mM DTNB solution (about a 20-fold molar excess) at room temperature with 0.6 mL of enzyme solution (0.807 mg/mL) in 80 mM phosphate buffer, pH 8.0, containing 1 mM EDTA, which has been dialyzed previously against the same buffer solution for 24 h. The number of SH groups is estimated from the increase of absorbance at 410 nm using a molar extinction coefficient of 13,600 M ^-1 cm ^-1 for thionitrobenzoate ions liberated.