Dithiothreitol: Difference between revisions
→Properties: Ox. DTT's abs is higher and more problematic for protein work at 280. |
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'''Dithiothreitol''' ('''DTT''') is the |
'''Dithiothreitol''' ('''DTT''') is an [[organosulfur compound]] with the formula {{chem2|(CH(OH)CH2SH)2}}. A colorless compound, it is classified as a [[dithiol]] and a [[diol]]. DTT is [[redox]] reagent also known as '''Cleland's reagent''', after [[W. Wallace Cleland]].<ref>{{cite journal | vauthors = Cleland WW | title = Dithiothreitol, a New Protective Reagent for SH Groups | journal = Biochemistry | volume = 3 | issue = 4 | pages = 480–2 | date = April 1964 | pmid = 14192894 | doi = 10.1021/bi00892a002 }}</ref> The reagent is commonly used in its racemic form. Its name derives from the four-carbon [[monosaccharide|sugar]], [[threose]]. DTT has an [[epimer]]ic ('sister') compound, [[dithioerythritol]] (DTE). |
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==Synthesis== |
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The traditional route to dithiothreitol (and its isomer dithioerythritol) is sulfidation of the extremely lachrymatory {{ill|1,4-Dibromobut-2-ene|zh|1,4-二溴丁-2-烯}}. Modern industrial syntheses instead use related epoxides and hydrogen sulfide.<ref>{{cite book|pp=76-78|title=The Ageless Gergel|first=Max G.|last=Gergel|url=https://library.sciencemadness.org/library/books/the_ageless_gergel.pdf}}</ref> |
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==Reducing agent== |
==Reducing agent== |
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DTT is used as a reducing or "deprotecting" agent for thiolated DNA. The terminal sulfur atoms of thiolated [[DNA]] have a tendency to form [[Dimer (chemistry)|dimers]] in solution, especially in the presence of oxygen. [[Dimer (chemistry)|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 filtration (for the solid catalyst) or by [[chromatography]] (for the liquid form). The DTT removal procedure is often called "desalting." Generally, DTT is used as a protecting agent that prevents oxidation of [[thiol group]]s. |
DTT is used as a reducing or "deprotecting" agent for thiolated DNA. The terminal sulfur atoms of thiolated [[DNA]] have a tendency to form [[Dimer (chemistry)|dimers]] in solution, especially in the presence of oxygen. [[Dimer (chemistry)|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 filtration (for the solid catalyst) or by [[chromatography]] (for the liquid form). The DTT removal procedure is often called "desalting." Generally, DTT is used as a protecting agent that prevents oxidation of [[thiol group]]s. |
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DTT is frequently used to reduce the [[disulfide bond]]s of [[protein]]s and, more generally, to prevent ''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 [[denaturation (biochemistry)|denaturing conditions]] (e.g., at high [[temperature]]s, or in the presence of a strong denaturant such as 6 M [[guanidinium chloride]], 8 M [[urea]], or 1% [[Sodium lauryl sulfate|sodium dodecylsulfate]]). DTT is oftentimes used along with sodium dodecylsulfate in [[Polyacrylamide gel electrophoresis|SDS-PAGE]] to further denature proteins by reducing their disulfide bonds to allow for better separation of proteins during [[electrophoresis]]. Because of the ability to reduce disulfide bonds, DTT can be used to denature CD38 on red blood cells. Conversely, the solvent exposure of different disulfide bonds can be assayed by their rate of reduction in the presence of DTT. |
DTT is frequently used to reduce the [[disulfide bond]]s of [[protein]]s and, more generally, to prevent ''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 [[denaturation (biochemistry)|denaturing conditions]] (e.g., at high [[temperature]]s, or in the presence of a strong denaturant such as 6 M [[guanidinium chloride]], 8 M [[urea]], or 1% [[Sodium lauryl sulfate|sodium dodecylsulfate]]). DTT is oftentimes used along with sodium dodecylsulfate in [[Polyacrylamide gel electrophoresis|SDS-PAGE]] to further denature proteins by reducing their disulfide bonds to allow for better separation of proteins during [[electrophoresis]]. Because of the ability to reduce disulfide bonds, DTT can be used to denature CD38 on red blood cells. DTT will also denature antigens in the Kell, Lutheran, Dombrock, Cromer, Cartwright, LW and Knops blood group systems. Conversely, the solvent exposure of different disulfide bonds can be assayed by their rate of reduction in the presence of DTT. |
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DTT can also be used as an [[redox|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 bond]]s to different sulfur atoms; in such cases, DTT cannot cyclize since it has no such remaining free thiols. |
DTT can also be used as an [[redox|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 bond]]s to different sulfur atoms; in such cases, DTT cannot cyclize since it has no such remaining free thiols. |
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==Properties== |
==Properties== |
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DTT is oxidized by air. So it is normally stored and handled under inert atmosphere to minimize oxidation. The rate of air-oxidation is slower at low temperatures.<ref>{{Citation|title=NLM PubChem CID Index|work=Vitamin D Handbook|year=2007|pages=239–244|publisher=John Wiley & Sons, Inc.|doi=10.1002/9780470238165.indsp1|isbn=978-0-470-23816-5|doi-access=free}}</ref> Oxidized DTT exhibits a strong absorbance peak at 280 nm. Since thiols are less nucleophilic than their conjugate bases, [[thiolate]]s, DTT becomes a less potent nucleophile as the pH falls. (2''S'')-2-Amino-1,4-dimercaptobutane ([[dithiobutylamine]] or DTBA), a related dithiol reducing agent, somewhat overcomes this limitation of DTT.<ref>{{cite journal | vauthors = Lukesh JC, Palte MJ, Raines RT | title = A potent, versatile disulfide-reducing agent from aspartic acid | journal = Journal of the American Chemical Society | volume = 134 | issue = 9 | pages = 4057–9 | date = March 2012 | pmid = 22353145 | pmc = 3353773 | doi = 10.1021/ja211931f }}</ref> Tris(2-carboxyethyl)phosphine ([[TCEP]]) is an alternative reducing agent that is more stable and effective at low pH, but it is bulky and reduces cystines in folded proteins only slowly.<ref>{{cite journal | vauthors = Cline DJ, Redding SE, Brohawn SG, Psathas JN, Schneider JP, Thorpe C | title = New water-soluble phosphines as reductants of peptide and protein disulfide bonds: reactivity and membrane permeability | journal = Biochemistry | volume = 43 | issue = 48 | pages = 15195–203 | date = December 2004 | pmid = 15568811 | doi = 10.1021/bi048329a }}</ref> |
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Due to air oxidation, DTT is a relatively unstable compound whose useful life can be extended by refrigeration and handling in an inert atmosphere. Oxidation presents further complications as oxidized DTT exhibits a strong absorbance peak at 280 nm. Since thiols are less nucleophilic than their conjugate bases, [[thiolate]]s, DTT becomes a less potent nucleophile as the pH lowers. (2''S'')-2-Amino-1,4-dimercaptobutane |
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(dithiobutylamine or DTBA) is a new dithiol reducing agent that somewhat overcomes this limitation of DTT.<ref>{{Cite journal |last1=Lukesh, III |first1=J. C. |last2=Palte |first2=M. J. |last3=Raines |first3=R. T. |title=A potent, versatile reducing agent from aspartic acid |journal=J. Am. Chem. Soc. |year=2012 |volume=134 |pmid=22353145 |pmc=3353773 |pages=4057–4059 |doi=10.1021/ja211931f |issue=9}}</ref> Tris(2-carboxyethyl)phosphine ([[TCEP]]) is an alternative reducing agent that is more stable and effective at low pH. |
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| ⚫ | DTT's half-life is 40 hours at pH 6.5 and 1.4 hours at pH 8.5 and 20 °C; its half-life decreases further as temperature increases. The presence of [[Edta|EDTA]] (ethylenediaminetetraacetic acid) to chelate divalent metal ions considerably extends the half-life of DTT in solution.<ref>{{cite journal | vauthors = Stevens R, Stevens L, Price NC | date = 1983 | title = The Stabilities of Various Thiol Compounds used in Protein Purifications. | journal = Biochemical Education | volume = 11 | issue = 2 | pages = 70 | doi = 10.1016/0307-4412(83)90048-1 }}</ref> |
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==See also== |
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| ⚫ | DTT's half-life is 40 hours at pH 6.5 and 1.4 hours at pH 8.5 and 20 °C |
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* [[Dithiobutylamine]] (DTBA) |
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* [[2-Mercaptoethanol]] (BME) |
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* [[TCEP]] |
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==References== |
== References == |
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{{Reflist}} |
{{Reflist}} |
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{{refbegin}} |
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{{refend}} |
{{refend}} |
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==External links== |
== External links == |
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*{{Commonscatinline}} |
*{{Commonscatinline}} |
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Latest revision as of 07:31, 11 November 2024
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| Names | |
|---|---|
| Preferred IUPAC name
(2S,3S)-1,4-Bis(sulfanyl)butane-2,3-diol | |
| Other names
(2S,3S)-1,4-Dimercaptobutane-2,3-diol
D-threo-1,4-Dimercaptobutane-2,3-diol D-threo-1,4-Dimercapto-2,3-butanediol 1,4-Dithio-D-threitol Cleland's reagent Reductacryl | |
| Identifiers | |
3D model (JSmol)
|
|
| ChEBI | |
| ChEMBL | |
| ChemSpider | |
| DrugBank | |
| ECHA InfoCard | 100.020.427 |
PubChem CID
|
|
| UNII | |
CompTox Dashboard (EPA)
|
|
| |
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| Properties | |
| C4H10O2S2 | |
| Molar mass | 154.253 g/mol |
| Appearance | White solid |
| Melting point | 42 to 43 °C (108 to 109 °F; 315 to 316 K) |
| Boiling point | 125 to 130 °C (257 to 266 °F; 398 to 403 K) at 2 mmHg |
| Soluble | |
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).
| |
Dithiothreitol (DTT) is an organosulfur compound with the formula (CH(OH)CH2SH)2. A colorless compound, it is classified as a dithiol and a diol. DTT is redox reagent also known as Cleland's reagent, after W. Wallace Cleland.[2] The reagent is commonly used in its racemic form. Its name derives from the four-carbon sugar, threose. DTT has an epimeric ('sister') compound, dithioerythritol (DTE).
Synthesis
[edit]The traditional route to dithiothreitol (and its isomer dithioerythritol) is sulfidation of the extremely lachrymatory 1,4-Dibromobut-2-ene. Modern industrial syntheses instead use related epoxides and hydrogen sulfide.[3]
Reducing agent
[edit]DTT is a reducing agent; once oxidized, it forms a stable six-membered ring with an internal disulfide bond. It has a redox potential of −0.33 V at pH 7.[1] 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.
Applications
[edit]DTT is used as a reducing or "deprotecting" agent for 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 filtration (for the solid catalyst) or by chromatography (for the liquid form). The DTT removal procedure is often called "desalting." Generally, DTT is used as a protecting agent that prevents oxidation of thiol groups.
DTT is frequently used to reduce the disulfide bonds of proteins and, more generally, to prevent 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 chloride, 8 M urea, or 1% sodium dodecylsulfate). DTT is oftentimes used along with sodium dodecylsulfate in SDS-PAGE to further denature proteins by reducing their disulfide bonds to allow for better separation of proteins during electrophoresis. Because of the ability to reduce disulfide bonds, DTT can be used to denature CD38 on red blood cells. DTT will also denature antigens in the Kell, Lutheran, Dombrock, Cromer, Cartwright, LW and Knops blood group systems. Conversely, the solvent exposure of different disulfide bonds can be assayed by their rate of reduction in the presence of DTT.
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 such remaining free thiols.
Properties
[edit]DTT is oxidized by air. So it is normally stored and handled under inert atmosphere to minimize oxidation. The rate of air-oxidation is slower at low temperatures.[4] Oxidized DTT exhibits a strong absorbance peak at 280 nm. Since thiols are less nucleophilic than their conjugate bases, thiolates, DTT becomes a less potent nucleophile as the pH falls. (2S)-2-Amino-1,4-dimercaptobutane (dithiobutylamine or DTBA), a related dithiol reducing agent, somewhat overcomes this limitation of DTT.[5] Tris(2-carboxyethyl)phosphine (TCEP) is an alternative reducing agent that is more stable and effective at low pH, but it is bulky and reduces cystines in folded proteins only slowly.[6]
DTT's half-life is 40 hours at pH 6.5 and 1.4 hours at pH 8.5 and 20 °C; its half-life decreases further as temperature increases. The presence of EDTA (ethylenediaminetetraacetic acid) to chelate divalent metal ions considerably extends the half-life of DTT in solution.[7]
See also
[edit]- Dithiobutylamine (DTBA)
- 2-Mercaptoethanol (BME)
- TCEP
References
[edit]- ^ a b O'Neil MJ, ed. (2001). Merck Index : an encyclopedia of chemicals, drugs, & biologicals (13th ed.). United States: Merck & Co, Inc. ISBN 0-911910-13-1.
- ^ Cleland WW (April 1964). "Dithiothreitol, a New Protective Reagent for SH Groups". Biochemistry. 3 (4): 480–2. doi:10.1021/bi00892a002. PMID 14192894.
- ^ Gergel, Max G. The Ageless Gergel (PDF). pp. 76–78.
- ^ "NLM PubChem CID Index", Vitamin D Handbook, John Wiley & Sons, Inc., pp. 239–244, 2007, doi:10.1002/9780470238165.indsp1, ISBN 978-0-470-23816-5
- ^ Lukesh JC, Palte MJ, Raines RT (March 2012). "A potent, versatile disulfide-reducing agent from aspartic acid". Journal of the American Chemical Society. 134 (9): 4057–9. doi:10.1021/ja211931f. PMC 3353773. PMID 22353145.
- ^ Cline DJ, Redding SE, Brohawn SG, Psathas JN, Schneider JP, Thorpe C (December 2004). "New water-soluble phosphines as reductants of peptide and protein disulfide bonds: reactivity and membrane permeability". Biochemistry. 43 (48): 15195–203. doi:10.1021/bi048329a. PMID 15568811.
- ^ Stevens R, Stevens L, Price NC (1983). "The Stabilities of Various Thiol Compounds used in Protein Purifications". Biochemical Education. 11 (2): 70. doi:10.1016/0307-4412(83)90048-1.
External links
[edit]
Media related to Dithiothreitol at Wikimedia Commons
