Dithiothreitol is a small molecule organic reducing agent which has a strong reducibility. Its reducibility is largely due to the conformational stability of its oxidized six-membered ring (containing disulfide bonds). Its redox potential is -0.33 volts at pH 7. The reduction of a typical disulfide bond by dithiothreitol consists of a two-step continuous thiol-disulfide exchange reaction.
Among them, the intermediate state formed by the first step reaction is very unstable, because the second sulfhydryl group on DTT tends to be connected to the oxidized sulfur atom, so that the intermediate state is quickly converted into the cyclic oxidized structure of DTT, thereby completing Reduction of disulfide bonds.
The reducing power of DTT is affected by the pH value and can only be reduced when the pH is greater than 7. This is because only the proton-depleted thiolate anion (-S-) is reactive, and the mercaptan (-SH) is absent; and the sulfhydryl has a pKa of typically -8.3.
One of the uses of DTT is as a reducing agent and deprotecting agent for thiolated DNA. The thiolated DNA terminal sulfur atom tends to form a dimer in solution, especially in the presence of oxygen. This dimerization greatly reduces the efficiency of some coupling reaction experiments (such as the immobilization of DNA in biosensors); while adding DTT to the DNA solution and removing it after a period of reaction, the dimerization of DNA can be reduced.
DTT is also often used for the reduction of disulfide bonds in proteins and can be used to prevent intramolecular or intermolecular disulfide bonds of proteins formed between cysteines in proteins. However, DTT often cannot reduce the disulfide bonds embedded in the protein structure (solvent unreachable). The reduction of such disulfide bonds often requires denaturation of the protein (heating at high temperature or adding a denaturing agent such as 6M guanidine hydrochloride, 8M urea or 1% SDS). On the contrary, according to the difference in the rate of reduction of disulfide bonds in the presence of DTT, the degree of embedding can be judged.
DTT is characterized by its tendency to be oxidized by air, so DTT is less stable; however, cryopreservation or treatment in inert gas can extend its useful life. Due to the low nucleophilicity of protonated sulfur, the effective reduction of DTT decreases with decreasing pH; Tris(2-carboxyethyl)phosphine HCl (TCEP hydrochloride) can be used as a low pH condition. A substitute for DTT, and it is more stable than DTT.
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