Is there a reliable source for storage and stability of reducing agents like DTT?

Question

Reading the literature on DTT, one is confronted with a confusing mass of papers; some claim that a 1M solution in water is stable, other papers say it is not. I use the reaction with DTNB to show that the DTT is still good, but that is cumbersome.

Is there some source of reliable (as in data driven) information on how to store compounds like DTT and other reducing agents?

Answer 1

Thiol-Based Reducing Agents

The instability of thiol-based reducing agents in solution is due to their propensity to form disulfide bonds by the following half reaction:

$$\ce{2RSH -> RSSR + 2H+ + 2e-}$$

It is clear that disulfide formation requires:

• Deprotonation of the thiols to form thiolates (RS^-).
• An electron acceptor.

Therefore, solutions of thiol-based reducing agents would be most stable when:

• The pH is buffered to where the thiol form predominates. The pKa of the thiol in β-mercaptoethanol is 9.5 and in dithiothreitol are 9.2 and 10.1 (Whitesides et al., 1977). Buffering the pH lower than this should increase stability.
• Electron acceptors are removed. In aqueous solution, the electron acceptor is molecular oxygen. Degassing the buffer and storing the solutions under an inert gas such as argon should increase stability.
• Reaction catalysts are removed. Divalent metal cations, especially copper (II) (Cu²⁺), catalyze oxygen dependent disulfide formation, even in trace amounts (Smith et al., 1994). Therefore a chelator such as EDTA should increase stability.
• Temperature is low. As with most chemical reactions, rate increases with temperature.

The stabilities of the thiol-based reducing agents β-mercaptoethanol (β-ME), dithiothreitol (DTT) and glutathione (GSH) have been determined in solution as a function of pH at 20°C (Stevens et al., 1983):

...and temperature at pH 8.5:

...as well as in the presence of copper (II) and EDTA at pH 8.5 and 20°C:

Below is the tabulated raw data (this is the actual data from the paper which I used to create the above graphs, but take note that I found it difficult to plot “>100”):

                               Half-life (h)        
pH    Temp (°C)    Additive    β-ME    DTT    GSH    
6.5   20           -           >100    40     16
7.5   20           -           10      10     9
8.5   20           -           4.0     1.4    1.3
8.5   0            -           21      11     8
8.5   40           -           1.0     0.2    0.2
8.5   20           Cu(II)      0.6     0.6    1.2
8.5   20           EDTA        >100    4      70

As expected, stability decreased with increasing pH and temperature, and also with the addition of copper. Stability increased with the addition of EDTA.

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Non-Thiol-Based Reducing Agents

Han and Han (1994) studied the effect of pH and buffer composition on the solution stability of tris(2-carboxyethyl)phosphine (TCEP). Though this is not a thiol-based reducing agent, it is still subject to oxidation. However, it is quite stable in solution:

pH    Solute/Buffer     % TCEP Oxidized After 3 Weeks
7.5   Tris-HCl          18.7 ± 0.6
8.5   Tris-HCl          16.8 ± 0.5
9.5   Tris-HCl          14.5 ± 0.5
8.2   borate             6.6 ± 0.2
10.2  borate             5.3 ± 0.1
6.8   HEPES             14.8 ± 0.4
8.2   HEPES             13.6 ± 0.3
9.7   CAPS               9.8 ± 0.3
11.1  CAPS               4.3 ± 0.1

Stability increased with increasing pH, though the effect was not large. Interestingly, the buffer composition had a larger effect on stability which was especially pronounced with phosphate:

        % TCEP Oxidized After 72 Hours
 pH     0.15 M phosphate    0.35 M phosphate
 6.0    not determined      11.2 ± 0.3
 6.8     8.1 ± 0.4          56.8 ± 2.3
 7.0    23.5 ± 0.9           100
 7.2    33.4 ± 1.0           100
 7.4    56.5 ± 1.4           100
 7.6    56.8 ± 2.1           100
 7.8    48.9 ± 2.5           100
 8.0    36.6 ± 1.7           100
10.6    not determined      11.7 ± 0.5
11.6    not determined       9.1 ± 0.4

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References

Han JC, Han GY. 1994. A Procedure for Quantitative Determination of Tris(2-Carboxyethyl)phosphine, an Odorless Reducing Agent More Stable and Effective Than Dithiothreitol. Anal Biochem 220(1):5-10.

Smith RC, Reed VD, Hill WE. 1994. Oxidation Of Thiols By Copper(II). Phosphorus Sulfur Silicon Relat Elem 90(1-4):147-154.

Stevens R, Stevens L, Price NC. 1983. The stabilities of various thiol compounds used in protein purifications. Biochemical Education 11(2):70.

Whitesides GM, Lilburn JE, Szajewski RP. 1977. Rates of thiol-disulfide interchange reactions between mono- and dithiols and Ellman's reagent. J Org Chem 42(2):332-338.