Cleland`s Reagent - DTT
Cleland’s reagent, also known as DL-Dithiothreitol or DTT is a water soluble protective reagent for sulfhydryl groups. It reduces disulfide linkages to free sulfhydryl groups in proteins and enzymes. It is a component of buffers used in protocols for the isolation and purification of proteins. DTT is a very strong reducing agent, due to the property to form a six-membered ring with an internal disulfide bond in oxidized form. The redox potential is -0.33 V at pH 7. The pKa values of the thiol groups are 9.2 and 10.1 respectively. The reduction of a typical disulfide bond proceeds by two sequential
thiol-disulfide exchange reactions as illustrated below. The reducing power of DTT is limited to pH values above 7, since only the negatively charged thiolate form is the
reactive agent in opening disulfide bonds.
DTT is also used as a reducing agent for thiolated DNA. The terminal sulfurs of thiolated DNA have a tendency to oxidize and form dimers in solution, especially in the presence of oxygen. Dimerization significantly lowers the efficiency of subsequent coupling reactions such as DNA immobilization on gold surfaces in biosensors. Normally DTT is mixed with a DNA solution and allowed to react, and then is removed by filtration (solid catalyst) or by chromatography (liquid form). DTT is frequently used to reduce the disulfide bonds of proteins and in order to prevent intramolecular (cyclization) and intermolecular (oligomerisation, polymerization) disulfide bonds from cysteine residues of proteins. However, DTT cannot reduce 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 strong denaturating agents such as 6 M guanidinium hydrochloride, 8 M urea, or 1% sodium dodecylsulfate). 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 inherent advantage is that effectively no mixed-disulfide species will be formed, which can occur with other agents such as glutathione.
- Cleland W.W., Dithiothreitol, A New Protective Reagent for SH Groups. Biochemistry; 1964; 3: 480-2. doi:10.1021/bi00892a002.
- Ruegg U.T., Rudinger J., Cleavage of disulfide bonds in proteins; Methods Enzymol. 1977; 47: 111.