Dithiothreitol (DTT) Application You Must Know

Dithiothreitol (DTT) Application You Must Know

Overview

Dithiothreitol (DTT), also known as Cleland’s reagent, is a small-molecule redox reagent. Its oxidized form is a disulfide-bonded 6-membered ring.  DTT has an epimeric (‘sister’) compound, dithioerythritol (DTE).

 

Fig. 1  DTT(Dithiothreitol) Chemical Structure

  • Solubility

DTT is highly soluble in water (clear solution, OD<0.05 at 0.02M), but also in ethanol, chloroform, ether and ethyl acetate.

  • Reaction

DTT participates to disulfide exchange reaction that drives its major applications. In example DTT is used typically at 1-10mM for protein SS reduction. It readily crosses biological membranes.

Reducing properties DTT is an unusually strong reducing agent, with a redox potential of -0.33 V at pH 7. The reduction of a typical disulfide bond proceeds by two sequential thiol-disulfide exchange reactions and is illustrated below. The reduction usually does not stop at the mixed-disulfide species because the second thiol of DTT has a high propensity to close the ring, forming oxidized DTT and leaving behind a reduced disulfide bond. The reducing power of DTT is limited to pH values above 7, since only the negatively charged thiolate form -S– is reactive (the protonated thiol form -SH is not); the pKa of the thiol groups is 9.2 and 10.1.

Fig. 2  Reduction of a typical disulfide bond by DTT via two sequential thiol-disulfide exchange reactions

  • Storage/Stability

DTT solutions should be prepared fresh daily. The recorded half-life (hours) of DTT solutions at various pH and temperatures are shown in Table 1 (all are in M potassium phosphate buffer).

pH Temperature Half Life (hrs)
pH 6.5 20ºC 40
pH 7.5 20 ºC 10
pH 8.5 20 ºC 1.4
pH 8.5 0 ºC 11
pH 8.5 40 ºC 0.2
pH 8.5 20 ºC (+0.1 mM Cu2+) 0.6
pH 8.5 20 ºC (+0.1 mM EDTA) 4

Reducing ability may be reduced after improper storage conditions (at room temperature, or in solution). Exposure to air should be minimized, even DTT has a lower tendency to be oxidized directly by air than other reducing agents. DTT useful life can be extended by refrigeration and handling in an inert atmosphere. Since protonated sulfurs have lowered nucleophilicities, DTT becomes less potent as the pH lowers.

Note: TCEP (Tris(2-carboxyethyl)phosphine hydrochloride) is an alternative which is more stable and works even at low pH. Learn more(DDT vs TCEP)>>

  • DTE; Epimer of Dithiothreitol(DTT)

Appli 3Fig. 3  The oxidized forms of dithioerythritol (DTE, left) dithiothreitol (DTT, right)

Dithioerythritol (DTE) is a sulfur containing sugar derived from the corresponding 4-carbon monosaccharide erythrose. It is an epimer of dithiothreitol (DTT).

Applications Like DTT, DTE makes an excellent reducing agent, although its standard reduction potential is not quite as negative, i.e., DTE is slightly less effective at reducing than DTT, presumably because steric repulsion of its OH groups makes the cyclic disulfide-bonded form of DTE less stable compared to that of DTT. In DTT, these hydroxyl groups are trans to each other, whereas they are cis to in DTE. 

  1. Reducing Agent
  • Protein

DTT is frequently used to reduce the disulfide bonds of proteins and peptides. It prevents intramolecular and intermolecular disulfide bonds from forming between cysteine residues of proteins. However, even DTT cannot reduce buried (solvent-inaccessible) disulfide bonds, so reduction of disulfide bonds is sometimes carried out under denaturing conditions (e.g., at high temperatures, or in the presence of a strong denaturant such as 6 M guanidinium hydrochloride, 8 M urea, or 1% Sodium dodecylsulfate).  Conversely, the solvent exposure of different disulfide bonds can be assayed by their rate of reduction in the presence of DTT. DTT is as a reducing or “deprotecting” agent. DTT protects notably enzyme activity loss by the oxidation of sulfhydryl groups. So DTT is widely used in biochemistry works to reduce dissulfide bridges, protect biomolecules, in sample preparation, and to denature proteins before electrophoresis analysis (SDS-PAGE). The DTT removal is performed by standard desalting procedures (dialysis, gel filtration).

  • Antioxidant

As an antioxidant, it is used as a protective agent against ionizing radiations in living cells.

  • Thiolated DNA

The terminal sulfur atoms of thiolated DNA have a tendency to form dimers in solution, especially in the presence of oxygen. Dimerization greatly lowers the efficiency of subsequent coupling reactions such as DNA immobilization on gold in biosensors. Typically DTT is mixed with a DNA solution and allowed to react, and then is removed by filteration(for the solid catalyst) or by chromatography (for the liquid form).

  • Leading Apoptosis

Places where different agents influence the decomposition or inhibit the action of sodium nitroprusside and S-nitroso-N-acetylpenicillamine are

indicated. Dithiothreitol accelerates the decomposition of S-nitroso-N-acetylpenicillamine and sodium nitroprusside. Hemoglobin (Hb) blocks the action of S-nitroso-N-acetylpenicillamine. Superoxide dismutase (SOD), catalase (cat) and deferoxamine (DFO) inhibit the action of sodium nitroprusside.

Appli 4Fig. 4  Schematic representation of production of reactive oxygen species by sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) in the presence of cells, with and without dithiothreitol (DTT), leading to apoptosis

BIOFEUL: Effect of the reducing agent dithiothreitol on ethanol and acetic acid production by clostridium strain P11

Fig. 5 Biofuels, especially bioethanol, is already used in the transportation industry as a fuel additive. Bioethanol is produced commercially from corn and other starch rich feedstocks. In Brazil, biofuels are produced from sugar cane. There is an ongoing research on the production of bioethanol from other sugar crops

Fig.  5  Biofuels, especially bioethanol, is already used in the  transportation industry as a fuel additive. Bioethanol is produced commercially from corn and other starch rich feedstocks. In Brazil, biofuels are produced from sugar cane. There  is an ongoing research on the production of bioethanol from other sugar crops.

The objective of this research is to investigate the effect of the reducing agent dithiothreitol (DTT) on enhancing ethanol production from synthesis gas (syngas) using Clostridium strain P11 in 250-mL serum bottles. Reducing agents help in regeneration of NADH from NAD+. NADH is utilized in the production of alcohol from aldehydes. The effect of DTT concentrations from 0 to 10 g/L was studied in 1.0 g/L yeast extract (YE) and 10 g/L corn steep liquor (CSL) media and with simulated syngas and producer gas. Syngas contains mainly carbon monoxide, hydrogen, carbon dioxide and nitrogen. The fermentation process was followed for 360 h. Liquid samples were collected every 24 h to determine cell mass, pH and product concentrations. The experiment was done in quadruplets at each DTT concentration and the results were analyzed for statistical significance using SAS version 9.2 at 95% confidence level. Results showed that over 350% increase in ethanol concentration was obtained in media that contained at least 7.5 g/L of DTT in the 1.0 g/L yeast extract medium after 360 h of fermentation with simulated syngas compared to the control medium (without DTT). However, only a 35% increase in ethanol production was noticed in 10 g/L corn steep liquor media in the presence of 2.5 and 5.0 g/L of DTT compared to the control medium with simulated syngas. In addition, DTT (at a concentration of 2.5 g/L) produced about 240% more butanol in the 10 g/L CSL medium compared to the control with simulated syngas. The results suggested that the use of small concentrations of DTT in the broth enhances ethanol production from simulated syngas in YE media. When producer gas was used, DTT enhanced isopropanol production instead of ethanol production in both YE and CSL media. The electrons donated by DTT might have been utilized in the reduction of acetone to isopropanol by strain P11 instead of reduction of acetaldehyde to ethanol. The removal of acetone and other impurities from the producer gas could enhance DTT effectiveness as a reducing agent and improve ethanol production in both YE and CSL media. Full Article>> The objective of this research is to investigate the effect of the reducing agent dithiothreitol (DTT) on enhancing ethanol production from synthesis gas (syngas) using Clostridium strain P11 in 250-mL serum bottles. Reducing agents help in regeneration of NADH from NAD+. NADH is utilized in the production of alcohol from aldehydes. The effect of DTT concentrations from 0 to 10 g/L was studied in 1.0 g/L yeast extract (YE) and 10 g/L corn steep liquor (CSL) media and with simulated syngas and producer gas. Syngas contains

  1. Oxidizing Agent

DTT can also be used as an oxidizing agent. Its principal advantage is that effectively no mixed-disulfide species are populated, in contrast to other agents such as glutathione. In very rare cases, a DTT adduct may be formed, i.e., the two sulfur atoms of DTT may form disulfide bonds to different sulfur atoms; in such cases, DTT cannot cyclize since it has no remaining free thiols.

becomes less potent as the pH lowers. Tris(2-carboxyethyl)phosphine HCl (TCEP hydrochloride) is an alternative which is more stable and works even at low pH.(repeated part) Due to air oxidation, DTT is a relatively unstable compound whose useful life can be extended by refrigeration and handling in an inert atmosphere. Since protonated sulfurs have lowered nucleophilicities, DTT

  1. Directions for use / reducing proteins and peptides

DTT replaces in most applications the very pungent 2-mercaptoethanol. The optimal pH range for DTT is between 7.1 and 8.0, but the reagent can be used effectively at pH 6.5-9.0. DTT is well stable (longer shelf life as a powder than 2-mercaptoethanol), however stock solutions must be used immediately and any remaining solution discarded.

Concentrations to use Application
1-10 mM to maintain reduced proteins in solution
50-100 mM for complete reduction for electrophoresis

After reduction is complete, excess reducing reagents should be removed from the sample to prevent reformation of the disulfide and oxidation. Effectively, soluble reducing agents may interfere with downstream assays (determination of free thiol groups in the sample, coupling of free thiol to a sulfhydryl reactive reagent such as SMCC/MAL-PEO-NHS)or various other applications. Desalting can be performed by desalting columns for direct downstream use of reduced protein/peptides (labeling, conjugation, bioassays). Alternatively this can be done by dialysis (but an long process, and sample is recover more or less diluted). These techniques do not suit well for small molecular weight samples, such as peptides (difficult to separate from the reducing agent), unless using reverse phase chromatography or immobilized reducing agents.

  1. Hints / reducing proteins for electrophoresis

Some proteins get on running SDS PAGE analysis an extra band with twice mol. wt , even when using 2-mercaptoethanol reduction. Switch to DTT reduction, or even better to DTT will achieve more complete reduction.

Dithiothreitol is a chemical reagent wiwth a wide actuation spectrum not only from a laboratorial view but also from a therapeutic standpoint, more clinical and practical. 1. DTT is frequently used in a variety of experiences that involve proteins or peptides, protecting sulfhydryl groups from oxidation and reducing disulfide bonds between cysteines; 2. Is also used in the study of disulfide exchange reactions of protein disulfides; 3. Is able to keep glutathione in the reduced state; 4. Acts as an “antidote” enabling the activity of detoxification systems; 5. Participates in cellular mechanisms such as vesiculation, cell morphology, signal transduction pathways (hormone-‘like’ role), etc.; 6. Can be used in the treatment approach of diseases like cystinosis or medical conditions resulting from ion or metal toxicity. In erythrocytes, there’s literature pointing that DTT may trigger changes on the normal discoid shape following metabolic depletion, and additionally modulate the exovesiculation kinetics as demonstrated by us. The present article dissects in detail recent findings in our Unit concerning the DTT influence on human erythrocytes. A standard loading buffer contains 1% SDS, 10% glycerol, 10 mM Tris-Cl, pH 6.8, 1 mM EDTA, bromophenol blue tracking dye ~0.05 mg/ml and 10mM dithiothreitol (DTT) as reducing agent.

 

Protocols; Reducing Proteins or Peptides with Dithiothreitol(DTT)

General Protocol to Reducing the Cysteines in a Protein or Peptide Solution

  1. Make a 1M diothreitol DTT stock solution in water, best to make fresh. (Try not to inhale the DTT.)
  2. Add DTT to the protein or peptide solution(which is in a buffer) to a final concentration of 1mM to 10mM DTT. Incubate for 10min. to 30 min. The reduction may work slightly better if incubating at higher temperature, such as at 37 degrees C or at 56 degrees C. However, room temperature or ice should work too.

It may be necessary to first denature the protein or peptide first prior to DTT reduction. For example, it is possible to use urea(1-8M concentration) or SDS(0.1-4%) to first expose the cysteins for a few minutes on ice or at higher temperature. Alternatively, it may be possible to denature and reduce at the same time.

Reducing Proteins or Peptides in Solution Prior to Alkylation

  1. Reduce the protein as above.
  2. Incubate the disulfide-reduced protein or peptide samples at an NEM molarity of at least 3-fold higher than that used for DTT. Incubate for 30 min. to 1 h.
  3. Dialyze away the DTT or NEM if desired.

 

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References

 

 

 

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