L-Dithiothreitol does not have a specific biological target or receptor; instead, it acts as a chemical reducing agent that breaks disulfide bonds within and between proteins through a thiol-disulfide interchange reaction. The reducing power of DTT is derived from its two thiol groups, which can donate electrons to reduce disulfide bonds, forming a stable six-membered ring intermediate (1,2-dithiane) upon oxidation. This reaction is thermodynamically favorable and proceeds rapidly at physiological pH. The compound's ability to reduce disulfide bonds is dependent on the redox potential of the environment and the accessibility of the disulfide bonds within the protein structure. By maintaining a reducing environment, L-Dithiothreitol prevents the formation of intermolecular disulfide bonds that can lead to protein aggregation and inactivation. The compound is also used to reduce disulfide bonds in nucleic acids and other biomolecules, although its primary application is in protein biochemistry.

For life science-related study, L-dithiothreitol is a biochemical reagent that can be utilized as an organic substance or biological material.

---

In vitro, L-Dithiothreitol is used as a stereoselective reducing agent for disulfide bridges in complex molecules, maintaining enzyme activity by preserving essential cysteine residues in their reduced form. It is commonly added to protein extraction buffers, lysis buffers, and electrophoresis sample buffers to ensure complete denaturation and reduction of proteins. In SDS-PAGE, DTT is used to reduce disulfide bonds in proteins, allowing them to migrate according to their molecular weight. In enzyme assays, DTT is often included to protect the enzyme from oxidative inactivation and to maintain optimal activity. The compound can also be used to reverse the oxidation of thiol groups that occurs during protein purification or storage. In addition to its role as a reducing agent, L-Dithiothreitol can be used to study the effects of redox state on protein function and to investigate the role of disulfide bonds in protein folding and stability.

In vivo, L-Dithiothreitol has antioxidant applications, protecting cells from oxidative stress in studies related to aging and disease. The compound can be added to cell culture media to create a reducing environment, protecting cells from oxidative damage or to study the effects of redox state on cellular signaling pathways. In animal studies, L-Dithiothreitol has been used in models of oxidative stress, where it is administered to evaluate its protective effects against tissue damage caused by reactive oxygen species. However, its use in vivo is limited by its rapid oxidation and metabolism, as well as its potential toxicity at high concentrations. The compound's primary applications remain in vitro, where its reducing properties are most effectively utilized.

The non-cellular assay for L-Dithiothreitol involves incubating a protein or peptide containing disulfide bonds with L-Dithiothreitol under reducing conditions. The extent of disulfide reduction is monitored by measuring free thiol groups using Ellman's reagent (5,5'-dithiobis-(2-nitrobenzoic acid), or DTNB), which reacts with free thiols to produce a yellow chromophore that can be quantified spectrophotometrically at 412 nm. Alternatively, the reduction of disulfide bonds can be assessed by changes in protein mobility on SDS-PAGE under non-reducing versus reducing conditions. The disappearance of higher-molecular-weight bands (corresponding to disulfide-linked multimers) and the appearance of lower-molecular-weight bands (corresponding to reduced monomers) indicate successful reduction. The concentration of DTT required for complete reduction depends on the number of disulfide bonds in the protein and the reaction conditions, including temperature, pH, and incubation time. Typically, DTT is used at concentrations of 1-100 mM for protein reduction.

The cellular assay for L-Dithiothreitol involves adding the compound to cell culture media at various concentrations (typically 0.1-10 mM) and assessing its effects on cell viability, redox state, and cellular signaling. Cells are treated with L-Dithiothreitol for defined time periods, and the intracellular redox state is measured using redox-sensitive fluorescent probes, such as 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) for reactive oxygen species (ROS) or monobromobimane for thiol groups. Cell viability is assessed using standard assays such as MTT, CellTiter-Glo, or trypan blue exclusion. The effects of L-Dithiothreitol on cellular signaling pathways, such as NF-κB, MAPK, or PI3K/Akt, can be studied by Western blotting or reporter assays. In some experiments, L-Dithiothreitol is used to study the role of disulfide bond formation in protein secretion, cell adhesion, or receptor function. The compound's ability to penetrate cell membranes and create a reducing intracellular environment makes it useful for these applications.

In animal studies, L-Dithiothreitol has been used in models of oxidative stress, ischemia-reperfusion injury, and inflammation. The compound is typically administered by intraperitoneal injection or oral gavage at doses ranging from 10-100 mg/kg. In a typical oxidative stress model, animals are treated with L-Dithiothreitol prior to or concurrently with an oxidative challenge, such as exposure to a toxin or induction of ischemia. Tissue samples (e.g., liver, kidney, brain) are collected and analyzed for markers of oxidative damage, including malondialdehyde (MDA), glutathione levels, and antioxidant enzyme activities. The protective effects of L-Dithiothreitol are assessed by comparing these markers in treated versus untreated animals. However, the use of L-Dithiothreitol in vivo is limited by its rapid metabolism and potential toxicity, and it is not commonly used as a therapeutic agent.

Absorption, Distribution and Excretion
Two male patients with advanced (uremic) infantile nephrotic syndrome (INC) received oral dithiothreitol (DTT) at a dose not exceeding 25 mg/kg body weight three times daily. Both patients underwent three consecutive observation periods: during thiol administration (8.5 months); during thiol discontinuation (8–9 months); and during thiol re-administration (7 months or longer)... Although chemical methods are not reliable in detecting and measuring DTT in biological fluids, preliminary evidence suggests that silanized derivatives of oxidized DTT can be detected in the urine of patients receiving oral DTT. This finding indicates that thiols can be absorbed and excreted. Metabolism/Metabolites Two male patients with advanced (uremic) infantile nephrotic syndrome (INC) received oral dithiothreitol (DTT) at a dose not exceeding 25 mg/kg body weight three times daily. Both patients underwent three consecutive observation periods: during thiol administration (8.5 months); during thiol discontinuation (8–9 months); and during thiol re-administration (7 months or longer)... Although chemical methods are not reliable for detecting and measuring DTT in biological fluids, preliminary evidence suggests that silanized derivatives of oxidized DTT can be detected in the urine of patients taking oral DTT. This finding indicates that thiols can be absorbed and excreted.

---

L-Dithiothreitol is highly water-soluble and readily penetrates biological membranes. It is typically stored as a solid or as a concentrated stock solution (e.g., 1 M) in water or buffer at -20°C. The compound is unstable in solution, particularly at neutral or alkaline pH, and should be prepared fresh before use. Oxidation of DTT in solution can be minimized by storing it under an inert atmosphere (e.g., nitrogen or argon) and by using antioxidants such as EDTA to chelate metal ions that catalyze oxidation. The compound has a melting point of 48-53°C and a specific rotation of +22.0 to +28.0 degrees in methanol. L-Dithiothreitol should be stored desiccated at -20°C under inert atmosphere to prevent oxidation. It is commercially available as a white to off-white crystalline powder with a purity of ≥95% by iodometric titration.

Toxicity Summary
Identification and Uses: 1,4-Dithiothreitol (DTT) is commonly used in biochemical experiments involving proteins or peptides to protect sulfhydryl groups from oxidation and reduce disulfide bonds between cysteine residues. It is also used to study disulfide exchange reactions of protein disulfide bonds, and DTT can keep glutathione in a reduced state. It has been used in experimental treatments for cystinosis or diseases caused by ionic or metal toxicity. Human Studies: DTT induces apoptosis in HL-60 cells. DTT is used to thin sputum in asthmatic patients. Two male patients with late-stage (uremia) infantile nephrotic cystinosis received oral DTT at doses not exceeding 25 mg/kg body weight three times daily. No other significant toxicities were observed except for nausea and vomiting within the maximum dose range. One subject died of uremia at month 24 of the study. Animal Studies: The inhibitory effects of dithiothreitol (DTT) on the heart and intestinal tissues of rats severely limit its application as an antioxidant in pharmacological studies to protect drugs susceptible to air oxidation. Dithiothreitol treatment can mimic the intracellular activation of Clostridium difficile's potent cytotoxin B.

---

Interactions
Widely used thiol antioxidants (dithiothreitol, glutathione, and N-acetylcysteine) exhibit toxic activity against the human lymphocytic leukemia cell line HL60 when used in combination with hydroxycobalamin (vitamin B12). Following combined treatment with thiols and vitamin B12, early lysosomal instability and apoptosis occur. Caspase inhibitors can eliminate this cytotoxic effect. The iron chelator deferoxamine can partially prevent cell death, while lysosomal protease inhibitors and pepsin inhibitors offer no protection.
Arsenic is a naturally occurring toxic metalloid; drinking water containing As₂O₃ is believed to be associated with increased risk of neurotoxicity, liver damage, blackfoot disease, hypertension, and cancer. On the other hand, As₂O₃, as an ancient traditional Chinese medicine, has significant anticancer activity, particularly in the treatment of acute promyelocytic leukemia and in promoting chronic wound healing. However, the cytotoxicity of As₂O₃ against solid cancer cells (such as oral cancer cells) and its detailed mechanism of action remain largely unclear. In this study, we cultured four pairs of tumor and non-tumor cells from oral cancer patients and treated these cells with arsenic trioxide (As₂O₃) alone or in combination with dithiothreitol (DTT). The results showed that 0.5 μM As₂O₃ combined with 20 μM DTT significantly killed oral cancer cells but had no significant effect on non-tumor cells. Furthermore, As₂O₃ combined with DTT upregulated the expression of Bax and Bak, downregulated the expression of Bcl-2 and p53, and led to loss of mitochondrial membrane potential in oral cancer cells. On the other hand, As₂O₃ also induced endoplasmic reticulum stress and increased the expression levels of glucose regulatory protein 78, calpains 1, and 2. Our results indicate that DTT synergistically enhances the killing effect of As₂O₃ on oral cancer cells without toxicity to non-tumor cells. This combination therapy shows promise in the clinical treatment of oral cancer and warrants further investigation. Previous studies have found that vitamin B12b can significantly enhance the cytotoxic effect of ascorbic acid by catalyzing the generation of reactive oxygen species, while the antioxidant dithiothreitol (DTT), unlike catalase, cannot inhibit this cytotoxic effect. Therefore, this study investigated whether vitamin B12b could enhance the cytotoxic effect of DTT. The results showed that vitamin B12b significantly enhanced the cytotoxic effect of DTT. After the addition of vitamin B12b to DTT, the generation and accumulation of hydrogen peroxide in the culture medium rapidly reached a concentration of 260 μM within 7 minutes. The extracellular oxidative burst induced by the combined action of vitamin B12b and DTT (DTT + B12b) was accompanied by intracellular oxidative stress, lysosomal instability, and DNA damage. The accumulation of DNA damage led to the initiation of apoptosis, including the activation of caspase-3 and the release of cytochrome c. The antioxidants pyruvate and catalase completely inhibited DTT+vitamin B12b-induced oxidative stress and cell death. While the iron chelators deferoxamine and phenanthrene-1,000 did not reduce exogenous oxidative burst, they inhibited the genotoxic and cytotoxic effects of this combination, indicating that intracellular iron plays a crucial role in the cytotoxicity of this combination. Therefore, vitamin B12b significantly enhanced the cytotoxicity of DTT, catalyzing hydrogen peroxide production and inducing intracellular and extracellular oxidative stress, early lysosomal instability, and iron-dependent DNA damage. Inorganic trivalent arsenic compounds are ortho-thiol reactants, and dithiothreitol (DTT) is a well-known dithiol reactant. Interestingly, it has been reported that dithiothreitol (DTT) has both inhibitory and promoting effects on arsenic trioxide-induced apoptosis. The data we present now indicate that at high concentrations, DTT, dimercaptosuccinic acid (DMSA), and dimercaptopropanesulfonic acid (DMPS) all reduce arsenic trioxide-induced apoptosis in NB4 cells (a human promyelocytic leukemia cell line). Conversely, at low concentrations, DTT, DMSA, and DMPS increase arsenic trioxide-induced apoptosis. High concentrations (3 mM) of DTT reduce the growth-inhibiting effects of arsenic trioxide, methylarsenic acid (MMA(III)), and dimethylarsenic acid (DMA(III)) on NB4 cells, while low concentrations (0.1 mM) of DTT enhance this inhibitory effect. DMSA and DMPS are currently used as antidotes for acute arsenic poisoning. In experiments using human epithelial cell lines derived from arsenic target tissues (such as the kidneys and bladder), these two dithiol compounds exhibit a reverse excitatory effect against arsenic toxicity, specifically manifested in DNA damage, micronucleus induction, apoptosis, and colony formation. Following oral administration of dithiols, the concentrations of these dithiols in the human body may be low. Therefore, current findings suggest the need to reassess the therapeutic effects of these dithiols on arsenic poisoning. For more complete data on interactions of 1,4-dithiothreitols (7 compounds in total), please visit the HSDB record page. Non-human toxicity values: Mouse intramuscular LD50: 108 mg/kg; Mouse intraperitoneal LD50: 154 mg/kg

---

L-Dithiothreitol has low toxicity at typical working concentrations (1-10 mM), but at high concentrations it can be toxic to cells due to its reducing properties, which can disrupt the cellular redox balance and cause oxidative stress. The compound is considered to be a skin and eye irritant and should be handled with appropriate personal protective equipment, including gloves and safety goggles. Inhalation of DTT dust should be avoided. In animal studies, high doses of L-Dithiothreitol can cause gastrointestinal disturbances, hemolysis, and other adverse effects. The compound is not approved for human therapeutic use and is strictly a research reagent.

L-1,4-Dithiothreitol is a 1,4-dithiothreitol, the enantiomer of D-1,4-dithiothreitol. It is a commonly used reagent in biochemical research, acting as a protective agent to prevent the oxidation of thiol (SH) groups and to reduce disulfide bonds to dithiols. See also: D-1,4-Dithiothreitol (note moved here).

---

L-Dithiothreitol is one of the most commonly used reducing agents in molecular biology and biochemistry. It is preferred over β-mercaptoethanol for many applications due to its lower odor, greater stability, and higher reducing potential at physiological pH. The compound is used in a wide range of applications, including protein extraction, enzyme assays, SDS-PAGE, Western blotting, and protein folding studies. Its ability to reduce disulfide bonds and maintain proteins in their reduced state has made it an indispensable tool in the life sciences. In addition to its role as a reducing agent, L-Dithiothreitol can be used to study the redox regulation of proteins and signaling pathways. The compound's L-enantiomer is the biologically active form and is preferred over the racemic mixture for certain applications.

C4H10O2S2

154.24

154.012

16096-97-2

DL-dithiothreitol;3483-12-3;DL-dithiothreitol-d6;850153-85-4;DL-dithiothreitol-d10;302912-05-6;DL-dithiothreitol-d10-1;203633-21-0

439196

White to off-white solid powder

1.3±0.1 g/cm3

364.5±42.0 °C at 760 mmHg

42-44ºC

174.2±27.9 °C

0.0±1.8 mmHg at 25°C

1.579

0.07

4

4

3

8

52

2

C([C@@H]([C@H](CS)O)O)S

VHJLVAABSRFDPM-IMJSIDKUSA-N

InChI=1S/C4H10O2S2/c5-3(1-7)4(6)2-8/h3-8H,1-2H2/t3-,4-/m0/s1

(2R,3R)-1,4-bis(sulfanyl)butane-2,3-diol

L-Dtt L-Dithiothreitol

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

 (Please use freshly prepared in vivo formulations for optimal results.)