DTT and TCEP are two very common reducing agents used to break the disulfide bonds in proteins to help with further protein study. While both have a similar function, each has a very different chemical composition that influences the mechanism for how they work.

This may have you wondering what exactly is the difference, in terms of chemistry, between DTT and TCEP, and why does it even matter?

DTT contains two thiol groups which are important to its mechanism as a reducing agent, while TCEP contains a tertiary phosphine group that is central to its reducing activity. Their differences in chemistries and mechanisms contribute to different advantages and disadvantages for each.

The chemical differences between these two reducing agents are at the core of how they work, their stability, and what makes one better suited for some applications than the other.

We’ll dive deeper into these differences. However, if you want to know how these differences impact how they work in protein research, check out this article, which compares how things like pH, and stability differ between these two reducing agents.

 

Article Table of Contents

Crash course in disulfide bonds

Comparing the structure of DTT vs. TCEP and why it matters

The structure of DTT

How DTT works

The structure of TCEP

How TCEP works

References:

 

Crash course in disulfide bonds

In order to understand the differences between DTT and TCEP and how that impacts how they attack disulfide bonds, let’s just take a moment to do a quick refresh on disulfide bonds within the context of the amino acid cysteine.

Figure 1. (A) Cysteine with the sulfur-hydrogen bond (thiol) highlighted. (B) Two cysteines bonded together by the highlighted sulfur atoms through a disulfide bond.

Disulfide bonds between cysteines are one type of bond that help proteins fold into their 3-dimensional shape. The disulfide bond is a covalent bond between two sulfur atoms. In figure 1A, you can see the thiol group on the cysteine amino acid, which is a chemical group containing a sulfur atom bonded to a hydrogen atom.

As a protein naturally folds, regions of the peptide chain can bring distant cysteines in closer proximity, which can allow cellular enzymes to oxidize the thiol (stripping away the hydrogen), leading to the sulfur atoms from both cysteine side chains to covalently bond with each other (Figure 1 B).

While many proteins have disulfide bonds to help them fold, it’s worth keeping in mind that not all proteins do.

So now we have to understand the reducing agents that help break up the disulfide bonds.

 

Comparing the structure of DTT vs. TCEP and why it matters

DTT and TCEP differ considerably in their structure, and their respective structures control the mechanisms for how each reducing agent breaks up a disulfide bond.

The structure of DTT

Figure 2. Structure of DTT (Dithiothreitol) with the thiols (-SH) highlighted.

If you look at DTT in figure 2, you will immediately notice the two highlighted thiol groups. This is the key to how DTT works as a reducing agent for disulfide bonds. It is also why DTT is considered a thiol-based reducing agent.

How DTT works

DTT has two thiol groups, one on either end of the molecule. When it encounters a disulfide bond, it performs a thiol-disulfide exchange.

As the pH increases, a greater amount of DTT’s thiol groups becomes deprotonated, forming negatively charged thiolates. These thiolates are much stronger nucleophiles and can attack one of the sulfur atoms in the cysteine-cysteine disulfide bond (Nagy, 2013; Wingfield, 2001).

Picture yourself at a dance. Two people have each of their hands joined together. You come in, and take one of the joined hands, breaking the original pair and pairing up with one of the dancers yourself.

The problem with this picture is that you and this other person are stuck together in a dance. When it comes to DTT, we don’t want this. Fortunately, that is just an intermediate step in the reaction.

DTT’s other thiol group does the same thing, attacking the DTT-Cysteine bond, and forming a ring structure with itself. This is the oxidized form of DTT (Figure 3) (Getz et al., 1999).

In our dance analogy, it would be as if your free hand grabbed your other hand away from your new partner, and then you begin dancing with yourself.

Figure 3. The oxidized structure of DTT.

 

The structure of TCEP

Figure 4. The structure of TCEP with the phosphorus atom and its lone pair of electrons highlighted.

When you look at the structure of TCEP (Figure 4) and compare it to DTT (Figure 2) and Cysteine (Figure 1), something is missing: the thiol group.

TCEP (Tris(2-carboxyethyl)phosphine hydrochloride) is a phosphine-based reducing agent with a central phosphorus atom bonded to three 2-carboxyethyl groups. The phosphorus atom has a free pair of electrons (shown in red in figure 4) that play a role in reducing disulfide bonds.

---

---

How TCEP works

TCEP has that lone pair of electrons on its phosphorus atom, which makes the phosphorus nucleophilic (electron donor). The phosphorus acts as a nucleophile and attacks one of the sulfur atoms in the disulfide bond through an SN2-type reaction. As the new phosphorus-sulfur bond forms, the original sulfur-sulfur bond is cleaved (Dmitrenko et al., 2007).

Just for clarity, an SN2-type reaction is where a nucleophile attacks an atom and forms a new bond as another bond breaks.

When the phosphorus attacks one sulfur, it forms a bond with that sulfur while the bond connecting the two cysteine sulfurs breaks. This releases one of the cysteines and leaves the other one temporarily attached to TCEP.

Water then hydrolyzes the TCEP-cysteine intermediate, releasing the second cysteine and converting TCEP into its phosphine oxide form. The end result is two reduced cysteines and oxidized TCEP.

Going back to that dance analogy, our dance is set up similarly to the DTT dance. You have two partners with each of their hands joined and they’re dancing. You (TCEP) come in and join your hand with one of the dance partners, bumping the other one away.

But unlike our DTT dance where you would end up joining hands with yourself, you discover another person on the dance floor (water – H₂O) and decide to dance with them instead. You pair up with the oxygen from the water molecule, forming oxidized TCEP (Figure 5). In the end, you’ve irreversibly broken up the original two partners.

Figure 5. Oxidized form of TCEP with the phosphorus-oxygen bond highlighted. The highlighted oxygen atom comes from the water molecule that disrupts the TCEP-cysteine interaction.

The major difference between DTT and TCEP comes down to their chemistry: one is thiol-based and the other is phosphine-based. These structural differences change how they interact with disulfide bonds and influence factors such as odor, pH dependence and stability.

For more details about DTT vs. TCEP you’ll want to look at this article next because it explains more about the odor, pH and stability, as well as how those factors are influenced by their chemistries.

It’s a deeper look that also examines their differences in use as well as the pros and cons of each.

 

 

References:

Dmitrenko, O., Thorpe, C., & Bach, R. D. (2007). The mechanism of S_N_2 disulfide bond cleavage by phosphorus nucleophiles: Implications for biochemical disulfide reducing agents. The Journal of Organic Chemistry, 72(22), 8298–8307. https://doi.org/10.1021/jo071271w

 

Getz, E. B., Xiao, M., Chakrabarty, T., Cooke, R., & Selvin, P. R. (1999). A comparison between the sulfhydryl reductants tris(2-carboxyethyl)phosphine and dithiothreitol for use in protein biochemistry. Analytical Biochemistry, 273(1), 73–80. https://doi.org/10.1006/abio.1999.4203

 

Kuan, S. L., Wang, T., & Weil, T. (2016). Site-selective disulfide modification of proteins: Expanding diversity beyond the proteome. Chemistry – A European Journal, 22(48), 17112–17129. https://doi.org/10.1002/chem.201602262

 

Nagy, P. (2013). Kinetics and mechanisms of thiol–disulfide exchange covering direct substitution and thiol oxidation-mediated pathways. Antioxidants & Redox Signaling, 18(13), 1623–1641. https://doi.org/10.1089/ars.2012.4973

 

Wingfield, P. T. (2001). Use of protein folding reagents. Current Protocols in Protein Science, Appendix 3, Appendix 3A. https://doi.org/10.1002/0471140864.psa03as00

 

Winther, J. R., & Thorpe, C. (2014). Quantification of thiols and disulfides. Biochimica et Biophysica Acta (BBA) – General Subjects, 1840(2), 838–846. https://doi.org/10.1016/j.bbagen.2013.03.031

 

Tags

dtt Katharine Martin protein expression protein purification reducing agents tcep

• Share :
• Deel
• X
• LinkedIn

---