Dithiothreitol (DTT), also known as Cleland’s Reagent, is a small molecule that plays a significant function in molecular biology and biochemistry. Its primary role is that of a potent reducing agent, preventing the formation of unwanted disulfide bonds and maintaining sulfhydryl groups in a reduced state within proteins and other biological molecules. This action is essential for preserving the native structure and activity of many proteins and enzymes during experimental procedures. DTT is highly water-soluble, making it a common additive in many aqueous buffers and solutions used in the laboratory.
The Chemical Process of DTT Oxidation
DTT’s reducing ability stems from its two sulfhydryl groups, but these groups also make it highly susceptible to degradation in solution. The primary mechanism by which DTT loses activity is through oxidation, mainly by dissolved oxygen present in the solvent. During this chemical process, the two sulfhydryl groups react to form a stable, internal, cyclic disulfide bond.
This oxidized form of DTT, which is a six-membered ring structure, is biologically inert as a reducing agent. The rate of this inactivating oxidation is heavily influenced by the pH of the solution. DTT’s reducing power relies on the presence of the negatively charged thiolate form of the molecule, which is only prevalent at higher pH values.
The oxidation rate increases significantly in alkaline conditions, particularly when the pH is above 7.0. For example, DTT’s half-life can be 40 hours at a pH of 6.5, but it drops drastically to only 1.4 hours at a pH of 8.5 when measured at room temperature. This difference demonstrates that higher pH accelerates DTT conversion into its inactive ring structure.
Short-Term Stability and Use at 4°C
DTT solution stability at 4°C is complex, as the timeframe is significantly influenced by the solution’s properties. Generally, DTT solutions are not considered stable for long periods at refrigeration temperatures, and they often lose activity rapidly. Standard stability estimates for DTT solutions stored at 4°C typically range from approximately one week to a maximum of one month.
Short-term stability depends heavily on the concentration of the DTT itself. Highly concentrated stock solutions, such as a 1 M stock, tend to be more stable than the dilute working solutions (e.g., 1–10 mM) used in experiments. The choice of solvent is also critical; DTT dissolved in simple deionized water is less stable than when dissolved in a buffered solution that contains chelating agents like EDTA. These agents bind to metal ions that catalyze the oxidation process.
For any preparation intended for short-term refrigeration, it is beneficial to use de-gassed or oxygen-free water to minimize the initial concentration of the primary oxidizing agent. Since there is often no obvious visual indicator of DTT degradation, relying solely on 4°C storage for more than a few days introduces an inherent risk of using an inactive reagent. Therefore, 4°C is best viewed as a temporary holding condition, not a reliable solution for long-term storage.
Maximizing Longevity Through Optimal Storage Practices
To maximize the functional shelf life of DTT beyond a few days, researchers must move away from 4°C storage and adopt more rigorous procedures. The gold standard for preserving DTT stock solutions is immediate and long-term storage at -20°C or colder. When stored frozen, DTT stock solutions can remain stable for a period ranging from six months up to one year without significant loss of activity.
The process of aliquoting is crucial for maximizing DTT stability. Immediately after preparing a concentrated stock solution, it should be divided into small, single-use aliquots before freezing. This measure prevents the solution from undergoing multiple freeze-thaw cycles, which introduce oxygen and accelerate degradation. It also minimizes repeated exposure to atmospheric oxygen that occurs each time a stock bottle is opened.
When preparing stock solutions, maintain the pH slightly below neutral, ideally in the range of pH 6.5 to 7.0, even before freezing. Although freezing significantly slows down chemical reactions, high pH conditions will still accelerate DTT degradation in the liquid phase during the brief periods of thawing and handling. Following these strict freezing and aliquoting protocols substantially extends the reagent’s longevity compared to simple refrigeration.