DTT , also known as Cleland's Reagent, is an unusually strong reducing agent, owing to its high conformational propensity to form a six-member ring with an internal disulfide bond. It has a redox potential of -0.33 V at pH 7. The reduction of typical disulfide bond proceeds by two sequential thiol-disulfide exchange reactions. DTT is frequently used to reduce the disulfide bonds of proteins and, more generally, to prevent intramolecular and intermolecular disulfide bonds from forming between cysteine residues of proteins.
However, even DTT cannot reduce buried (solvent -inaccessible) disulfide bonds, so reduction of disulfide bonds is sometimes carried out under denaturing conditions (e.g., at higher temperatures, or in the sodium denaturant such as 6 M guanidinium hydrochloride, 8 M urea, or 1% sodium dodecyl sulfate). Conversely, the solvent exposure of different disulfide bonds can be assayed by their rate of reduction in the presence of DTT. DTT can also be used as an oxidizing agent.
Its principal advantage is that effectively no mixed disulfide species are populated, in contrast to other agents such as glutathione. In very rare cases, a DTT adduct may be formed, i.e., the two sulfur atoms of DTT may form disulfide bonds to different sulfur atoms; in such cases, DTT cannot cyclize since it has no remaining free thiols. Typically DTT is mixed with a DNA solution and allowed to react, and then is removed by filtration (for the solid catalyst) or by chromatography (for the liquid form).
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