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DTT, also known as Clelands Reagent, is an unusually strong reducing agent, owing to its high conformational proprensity to form a six member ring with an internal disulfide bond.
DTT is frequently used to reduce the dissulfide bonds of proteins and, more generally, to prevent intramolecular and intermolecular disulfide bonds from forming between cysteine residues of proteins.
Figure 1. Dithiothreitol (DTT) chemical structure
- Solubility
DTT is highly soluble in water (clear solution, OD<0.05 at 0.02M), but also in ethanol, chloroform, ether and ethyl acetate.
- Reaction
DTT participates to disulfide exchange reaction that drives its major applications. In example DTT is used typically at 1-10mM for protein SS reduction. It readily crosses biological membranes.
Reducing properties DTT is an unusually strong reducing agent, with a redox potential of -0.33 V at pH 7. The reduction of a typical disulfide bond proceeds by two sequential thiol-disulfide exchange reactions and is illustrated below. The reduction usually does not stop at the mixed-disulfide species because the second thiol of DTT has a high propensity to close the ring, forming oxidized DTT and leaving behind a reduced disulfide bond. The reducing power of DTT is limited to pH values above 7, since only the negatively charged thiolate form -S– is reactive (the protonated thiol form -SH is not); the pKa of the thiol groups is 9.2 and 10.1.
Fig. 2 Reduction of a typical disulfide bond by DTT via two sequential thiol-disulfide exchange reactions.
- Applications
As a reducing agent, it is used to reduce the disulfide bonds of proteins and peptides. It prevents 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 high temperatures, or in the presence of a strong denaturant such as 6 M guanidinium hydrochloride, 8 M urea, or 1% Sodium dodecylsulfate).
Dithiothreitol (DTT) reduces disulfides to dithiols, allowing release of the DNA from its protective proteins and further degradation of the proteins by Proteinase K. DTT is an essential component for sperm cell lysis because the cell membrane contains a high concentration of disulfides.
It can be antioxidant agent, be used to produce biofuel, and lead apoptosis
As a 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 thils.
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DTT replaces in most applications the very pungent 2-mercaptoethanol. The optimal pH range for DTT is between 7.1 and 8.0, but the reagent can be used effectively at pH 6.5-9.0. DTT is well stable (longer shelf life as a powder than 2-mercaptoethanol), however stock solutions must be used immediately and any remaining solution discarded.
