Introduction:
Dithiothreitol is a commonly used reducing agent, also known as dimercaptothreitol (DTT), which has strong reducing ability. Its reducing ability is largely due to the conformational stability of its oxidized state six-membered ring (containing disulfide bonds). Reducing power is affected by pH and is only able to play a reducing role when pH is greater than 7. This is because only the negative ion of the thiol salt (-s -) without the proton is active, while the thiol (-SH) is not. However, sulfhydryl groups generally have a pKa of ~ 8.3.
Chemical properties:
The reduction of a typical disulfide bond by dithiothreitol is composed of two successive sulfhydryl-disulfide bond exchange reactions. The intermediate state formed by the first reaction is very unstable, because the second sulfhydryl group on DTT tends to connect with the oxidized sulfur atom, so that the intermediate state is quickly converted to the cyclic oxidation structure of DTT to complete the reduction of disulfide bonds. Dithiothreitol (DTT) has poor stability because it is easily oxidized by air. But it can be kept frozen or treated in inert gas to extend its life. Due to the low nucleophilicity of the protonated sulfur, the effective reduction of DTT decreases with decreasing pH. Tris(2-carboxyethyl)phosphineHCl (TCEP hydrochloride) can be used as a substitute for DTT at low pH and is more stable than DTT.
Role of:
Dithiothreitol has antioxidant effect, which can protect the reducing group on the enzyme molecule, maintain the reducing environment, and stabilize the activity of the enzyme. DTT also contains certain ribonuclease inhibitors, which can inhibit the activity of ribonuclease. "It has similar effects as mercaptoethanol (dimercaptoethanol), but DTT has a much less irritating odor and much less toxicity than mercaptoethanol." When the concentration of DTT was 7 times lower than that of mercaptoethanol, the two effects were similar. But DTT is slightly more expensive. It is used as a protein thiol protector and for cleavage and sequential analysis of protein disulfide bonds.
Application:
Dithiothreitol (DTT) is a small molecule organic reducing agent with the chemical formula C4H10O2S2, which is mainly used in the field of biotechnology as a protein cleavage reagent. At present, the most widely used method for the synthesis of dithiothreitol is to oxidize threitol with potassium permangate, and then to obtain thiointermediate under the action of thioacetic acid, and finally hydrolyze the thiointermediate to obtain dithiothreitol.
Purpose
Used to use? An excellent reagent for maintaining the SH base in the reduced state; Quantitative reduction of disulphide; DTT was effective in reducing protein disulfide bonds in sample buffer before SDS-PAGE. DTT can also be used to reduce the disulfide bridge of the cross-linking agent N,N '-bis (acryloyl) cystamine to break down the polyacrylamide gel matrix; In protein sequence analysis, it is used to clear disulfide links; To investigate increasing as well as dynamic reduction reactions; It prevents the oxidative reaction that occurs during the extraction of cholecystokinin from brain and duodenal tissue
| Point of melting: |
41-44 °C(lit.) |
| Boiling point: |
125 °C |
| Specific optical rotation: |
-0.2~+0.2°(20ºC/D)(c=5,H2O) |
| Density of density: |
1.04 g/mL at 20 °C |
| Density of vapor: |
5.3 (vs air) |
| Pressure of vapor: |
0.019-0.29Pa at 20-50ºC |
| Index of refraction: |
1.5200 (estimate) |
| Point of flash: |
>230 °F |
| Storage conditions: |
-20°C (or 4°C short term). |
| Degree of solubility: |
H2O: 50 mg/mL, clear and colorless |
| form: |
White powder crystallization |
| Coefficient of acidity (pKa): |
pK1:8.9 (25°C) |
| PH value: |
4.0-6.0 (20-25ºC, 0.1m in H2O) |
| Odor: |
A bad smell |
| Acid-base indicator discoloration ph range: |
4 - 6 at 15,4 g/l at 25 °C |
| Solubility of water: |
freely soluble |
| Sensitivity: |
Air Sensitive |
| Maximum wavelength (λmax): |
λ: 260 nm Amax: 0.400λ: 280 nm Amax: 0.100 |
| stability: |
Stable, but sensitive to heat. Incompatible with strong oxidants. The freezing temperature was maintained at -20 to -10 ° C. |
| LogP: |
0.07 at 25ºC and pH5 |
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