DithiothreitolDTT

 1,4-二硫代苏糖醇

Dithiothreitol, also known as DTT and Cleland’s reagent, is a broadly used disulfide reducing agent which finds application in the reduction of disulfide bridges in proteins. DTT is a fast, non-volatile solid, less odorous than β-ME. DTT has a short half-life in water, requiring the preparation of fresh solutions every time. DTT must be used at neutral or basic pH due to the pKa values of the thiol moieties. DTT is a slightly more powerful reducing agent than its epimer DTE, thanks to the thiols trans to each other.

Dithiothreitol (DTT) is a reducing agent that is widely used in biological and chemical research. It is commonly used to break disulfide bonds in proteins, which is essential for many biochemical and biophysical assays. DTT has been extensively studied for its physical and chemical properties, synthesis and characterization, analytical methods, biological properties, applications in scientific experiments, and toxicity and safety. In this paper, we provide an overview of DTT, its properties, and its potential implications in various fields of research and industry.

Definition and Background

DTT is a small molecule with a molecular weight of 154.25 g/mol. It is a white crystalline powder that is highly soluble in water. DTT is a reducing agent that is widely used in biochemical and biophysical assays. It is often used to break disulfide bonds in proteins, which is essential for many protein-based assays.

DTT was first synthesized in the early 1950s by Dr. L. S. B. Goldstein at the University of Pittsburg. The discovery of DTT revolutionized the field of protein chemistry by making it possible to break disulfide bonds and study the properties of individual polypeptide chains.

Physical and Chemical Properties

DTT is a small molecule with a melting point of 40-45°C. It is highly soluble in water, with a solubility of 1 M at room temperature. DTT is odorless and has a pH of 6.0-7.0. It is a strong reducing agent that can break disulfide bonds in proteins and other molecules.

DTT is a thiol compound that contains two sulfhydryl groups (-SH). The two sulfhydryl groups are able to reduce disulfide bonds (-S-S-) in proteins and other molecules, which is essential for many biochemical and biophysical assays.

Synthesis and Characterization

DTT is synthesized by reacting cystine with hydrogen sulfide in the presence of a catalyst such as Raney nickel or palladium on carbon. The reaction produces DTT and water as byproducts. The crude product is purified by recrystallization or column chromatography to obtain pure DTT.

DTT can be characterized by a variety of methods, including NMR spectroscopy, mass spectrometry, and elemental analysis. NMR spectroscopy is often used to determine the purity of DTT, while mass spectrometry can be used to confirm its molecular weight. Elemental analysis can be used to determine the presence and quantity of carbon, hydrogen, and sulfur in the molecule.

Analytical Methods

DTT is often used as a reducing agent in biochemical and biophysical assays. The concentration of DTT in these assays is critical for ensuring accurate results. DTT can be measured using a variety of methods, including UV spectroscopy, HPLC, and enzymatic assays.

UV spectroscopy is often used to measure the concentration of DTT in solutions. DTT absorbs light at a wavelength of 235 nm, which can be used to determine its concentration. HPLC can also be used to measure the concentration of DTT in solutions, while enzymatic assays can be used to determine the activity of DTT in reducing disulfide bonds in proteins and other molecules.

Biological Properties

DTT has a wide range of biological properties, including its ability to reduce disulfide bonds in proteins and other molecules. This property is essential for many biochemical and biophysical assays. DTT is also able to protect proteins from oxidation and denaturation by reducing and stabilizing disulfide bonds.

DTT has been shown to have anti-inflammatory and antioxidant properties in various animal models. It has been shown to reduce inflammation and oxidative stress in the liver, kidneys, and brain.

Toxicity and Safety in Scientific Experiments

DTT is a relatively safe and non-toxic compound when used in scientific experiments at appropriate concentrations. However, high concentrations of DTT can be hazardous to human health, especially if exposed to the skin or eyes. DTT should be handled with care and appropriate protective equipment should be used.

Applications in Scientific Experiments

DTT has a wide range of applications in scientific experiments, including its use as a reducing agent in protein-based assays. DTT is also used in the preparation of DNA and RNA samples for electrophoresis and PCR. It is often used to denature RNA secondary structures, which facilitates the binding of primers in PCR amplification.

DTT is also utilized in the generation of monoclonal and polyclonal antibodies. It can break the disulfide bridges present in the Fab regions of antibodies that impede the antigen binding affinity and specificity of the antibodies. In addition, DDT offers an attractive method of stabilizing antibodies by reduction of the overall disulfide bonds to prevent charge repulsion, misfolding or aggregation of Fab regions within fast diluting solutions.

DTT is also widely used in the synthesis of nanoparticles for biomedical and industrial applications. It is used to break disulfide bonds in proteins and peptides, which can then be used to reduce metal ions to form nanoparticles. The use of DTT helps to control the size, shape, and stability of nanoparticles, which is essential for many applications.

Current State of Research

DTT remains an active topic of research in many scientific fields, including protein chemistry, biophysics, and nanotechnology. Recent developments include the use of DTT for the preparation of protein samples for high-resolution microscopy techniques, as well as its application in the development of biosensors for the detection of disease markers.

Potential Implications in Various Fields and Limitations

DTT has potential implications in various fields of research and industry, including protein chemistry, biophysics, and nanotechnology. However, the use of DTT can also have limitations. For instance, DTT can potentially modify cysteine residues in proteins and other molecules, which can lead to unwanted side effects. Therefore, the use of DTT requires appropriate optimization and monitoring associated with the assay requirements upon its implementation.

Future Directions

There are several future directions for the research and application of DTT. One is the development of new methods for the preparation of DTT, which may include safer and environmentally friendly synthetic routes. Another is the application of DTT in the synthesis of advanced materials, such as biomaterials, catalysts and nanomaterials. Strategies for reducing DTT-limiting side effects and evaluating its long-term toxic effects in biological systems will promote its wider applicability. Furthermore, developing methodsfor the evaluation of DTT activity and detection, and new analytical methods for identifying DTT-modified cysteine resideis increasing the safety of DTT utilization in longer terms applications. Finding new applications for DTT in the fields of regenerative medicine and molecular engineering holds promise.

Title: 1,4-Dithiothreitol

CAS Registry Number: 3483-12-3

CAS Name: (2R,3R)-rel-1,4-Dimercapto-2,3-butanediol

Additional Names: Cleland's reagent; threo-2,3-dihydroxy-1,4-dithiolbutane

Molecular Formula: C4H10O2S2

Molecular Weight: 154.25

Percent Composition: C 31.15%, H 6.53%, O 20.74%, S 41.58%

Line Formula: HSCH2(CHOH)2CH2SH

Literature References: Prepn: Evans et al., J. Chem. Soc. 1949, 253.

Properties: Slightly hygroscopic needles from ether, mp 42-43°. Can be sublimed at 37° and 0.005 mm press; bp2 125-130°. Redox potential: -0.33 volts at pH 7. Freely sol in water, ethanol, acetone, ethyl acetate, chloroform, ether.

Melting point: mp 42-43°

Boiling point: bp2 125-130°

Use: Protective agent for SH groups: Cleland, Biochemistry 3, 480 (1964), see also "Cleland's Reagent" a detailed brochure and bibliography published by Calbiochem, Los Angeles.