[27565-41-9]  · C4H10O2S2  · 1,4-Dithiothreitol  · (MW 154.28)

(reducing agent for disulfides;1,2 protection of thiols and proteins containing free SH groups from air oxidation3)

Alternate Names: DTT; threo-2,3-dihydroxybutane-1,4-dithiol.

Physical Data: mp 42-44 °C (racemate); redox potential -0.33 V, pH 7, varying -0.06 V per pH unit; pK 9.2 and 10.1.

Solubility: very sol water, alcohols.

Form Supplied in: white solid; widely available; normally supplied as the racemate, but chiral 1,4-dithio-L-threitol [16096-97-2],4 mp 49-51 °C, is also available. The erythro isomer (1,4-dithioerythritol, DTE, [6892-68-8], mp 82-84 °C)1 has redox properties similar to the threo isomer (E° is more positive by 0.004 V)5 and is also available. The oxidized cyclic form (trans-1,2-dithiane-4,5-diol [14193-38-5], mp 130-132 °C)1 is available; it has a UV spectrum (lmax 283 nm; ε = 273). DTT and DTE are colorless above 260 nm, but both reduced and oxidized forms absorb below 240 nm.

Analysis of Reagent Purity: the presence of monothiols can be detected by addition of arsenite (which forms a tight tridentate complex with DTT),6 followed by addition of 5,5-dithiobis(2-nitrobenzoate).7 Total thiol content is determined by omitting arsenite.

Handling, Storage, and Precautions: solid material should be stored under refrigeration in a closed bottle. Solutions will slowly oxidize in the presence of air to the cyclic disulfide, which is inert. The concentration of reduced material can be determined by assay with 5,5-dithiobis(2-nitrobenzoate).8

Reduction of Disulfides.

DTT and DTE are the reagents of choice for reducing disulfide bonds, and protecting thiols and the thiol groups on proteins from air oxidation to disulfides.1-3 DTT also reduces diselenides.9 The equilibrium constant for reduction of the disulfide of glutathione by DTT is 210 M.10 DTT undergoes disulfide interchange to liberate the thiol with the lowest pK (eq 1). The mixed disulfide then cyclizes rapidly, releasing the other monothiol (eq 2).

The high equilibrium constant for this cyclization reaction produces the low redox potential for DTT. The hydroxy groups at C-2 and C-3 convey water solubility and reduce the stench (the solid has an odor, but only at very close range). Since the active form for disulfide interchange is the thiolate anion, these reactions go faster at higher pH. With the first pK at 9.2, however, there is sufficient thiolate even at pH 7 to give a reasonable rate.

The rates of reduction of disulfides by DTT show a normal dependence on the pK of the thiol being liberated,11 and this fact has been used to determine the pK's of SH groups on proteins. For example, the pK of the active site cysteine in papain was 4.1 at pH 6 and 8.4 at pH 9, with the change resulting from a group titrating with a pK of 7.5.11b The equation used in this work was: log k = 7.03 + 0.5 pKnuc - 0.27 pKc - 0.73 pK1g, where k is the rate constant in M-1 min-1 for reaction of the monoanion of DTT with the disulfide, and the subscripts nuc, c, and 1g refer to DTT, the center sulfur in the transition state, and the leaving thiol on the protein.

A series of other dithiols have been prepared as potential reducing agents, but none of them that are water soluble have lower redox potentials than DTT.10,12 Two that have lower pK's (7.6-7.8) and thus react faster at neutral pH have higher redox potentials of -0.27 V at pH 7 and are less effective reducing agents than DTT.13 So far, none of these is commercially available.

Reagent for Studies of Protein Folding.

DTT and its oxidized form have found use in studying disulfide interchange reactions during protein folding and unfolding.14 Mixtures of these provide a redox buffer that can be adjusted to match the redox potential of individual disulfide bonds.

Other Properties and Problems.

The low redox potential of dithiothreitol interferes with its use in the presence of certain reagents. While it gives full color yield in reactions with aromatic disulfides such as 5,5-dithiobis(2-nitrobenzoate)8 or 2,2- or 4,4-dipyridyl disulfide,15 it gives only 4% the expected color in the nitroprusside assay for thiols,1,16 presumably because it reduces the iron. It reduces CrIII or CoIII, so inert complexes of nucleotides with these metal ions cannot be used in its presence. Dithiothreitol also undergoes transesterification with thioesters of coenzyme A.17

DTT and DTE chelate heavy metal ions tightly, which can be helpful at times, but deleterious with autooxidizable ions such as FeII. While they oxidize only slowly in air in the absence of heavy metal ions, DTT and DTE are readily oxidized in air in the presence of FeII, so solutions containing FeII and DTT must be kept anaerobic.18

1. Cleland, W. W. B 1964, 3, 480.
2. Jocelyn, P. C. Methods Enzymol. 1987, 143, 246.
3. Konigsberg, W. Methods Enzymol. 1972, 25, 185.
4. Carmack, M.; Kelley, C. J. JOC 1968, 33, 2171.
5. Szajewski, R. P.; Whitesides, G. M. JACS 1980, 102, 2011.
6. Cruse, W. B. T.; James, M. N. G. Acta Crystallogr. B 1972, 28, 1325.
7. Zahler, W. L.; Cleland, W. W. JBC 1968, 243, 716.
8. Ellman, G. L. Arch. Biochem. Biophys. 1959, 82, 70.
9. Gunther, W. H. H. JOC 1967, 32, 3931.
10. Lees, W. J.; Whitesides, G. M. JOC 1993, 58, 642.
11. (a) Whitesides, G. M.; Lilburn, J. E.; Szajewski, R. P. JOC 1977, 42, 332. (b) Shaked, Z.; Szajewski, R. P.; Whitesides, G. M. B 1980, 19, 4156.
12. Houk, J.; Whitesides, G. M. JACS 1987, 109, 6825. The values in this paper are in error and have been correctly given in reference 10.
13. Singh, R.; Whitesides, G. M. JOC 1991, 56, 2332. Lees, W. J.; Singh, R.; Whitesides, G. M. JOC 1991, 56, 7328.
14. Creighton, T. E. Methods Enzymol. 1986, 131, 83.
15. Grassetti, D. R.; Murray, J. F., Jr. Arch. Biochem. Biophys. 1967, 119, 41.
16. Grunert, R. R.; Phillips, P. H. Arch. Biochem. Biophys. 1951, 30, 217.
17. Stokes, G. B.; Stumpf, P. K. Arch. Biochem. Biophys. 1974, 162, 638.
18. Lambeth, D. O.; Ericson, G. R.; Yorek, M. A.; Ray, P. D. BBA 1982, 719, 501.

W. Wallace Cleland

University of Wisconsin, Madison, WI, USA

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