| Common reductant |
DL-1, 4-DL-1, 4-Dithiothreitol is a common reducing agent, also known as DTT with a strong reduction capability. Its reduction capability is largely due to the conformational stability of the six-membered ring (containing disulfide bone) of its oxidation state. The reducing capability is affected by the pH value and also takes effect only in the case of a pH being greater than 7. This is because it is only the deprotonated thiolate anion (-S-) that is actively in reduction reaction while thiol (an-SH) is not; and the pKa of mercapto group is typically around~8.3.
The reduction of the disulfide bond mediated by DL-1, 4-Dithiothreitol mainly consists of a two-step continuous sulfhydryl-disulfide exchange: wherein the intermediate state formed by the first step of the reaction is very unstable because the secondary sulfhydryl group of DTT tends to connected by the oxidized sulfur atom, making the intermediate state be rapidly converted to the cyclic oxidation structure of DTT, thus completing the reduction of disulfide bonds.
Since being easily oxidized by air, the stability of DL-1, 4-Dithiothreitol (DTT) is relatively poor; frozen storage or processing in inert gas can extend its duration life. Owing to the low nucleophilicity of the protonated sulfur, with the decreased pH value, the effective reducing capability of DTT is also reduced; Moreover, Tris (2-carboxyethyl) phosphine HCl (TCEP hydrochloride) can be used as the substitute of DTT at low pH value and is also more stable than DTT.
DL-1, 4-Dithiothreitol has an antioxidant property that can protect the reductive groups on the enzyme molecules and maintain a reducing environment and stabilize the enzyme activity. DTT also contain certain RNase inhibitors which can inhibit the enzymatic activity of RNase. It has a similar effect as mercaptoethanol (b-mercaptoethanol) but the irritating odor and toxicity of DTT is much smaller and lower than the toxicity of mercaptoethanol. Moreover, DTT has a similar effect as mercaptoethanol even at a concentration of being seven times lower, but the price of DTT is slightly higher. It can be used as the protein thiol group protecting agent and the cleavage of the protein disulfide bond as well as sequence analysis.
Current implemented law regarding the poisons and toxic materials has designated β-mercaptoethanol and reagent-containing β-mercaptoethanol as poison. DL-1, 4-Dithiothreitol has similar effect as β-mercaptoethanol and can protect the SH group of protein, cutting the disulfide bond and therefore will be gradually paid attention by related applications.
The above information is edited by the chemicalbook of Dai Xiongfeng. |
