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In class, we have studied that when running a polyacrylamide gel with our protein sample to separate the proteins by molecular size, we can use several techniques, among which we have denaturing PAGE and SDS-PAGE. Basically, we focused on the latter, in which SDS is a detergent added to negatively charge all proteins in the sample (at a constant mass:charge ratio), as well as denature them. Then, the professor mentioned that we also add reagents such as DTT or beta-mercaptoethanol to reduce the disulphide bonds in between proteins.

My question is: if SDS already denatures proteins (so we have primary structure only), why would we be interested in adding DTT?

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Proteins are thought of as long polyamide chains. This long chain of amino acids is known as the primary structure. Due to hydrogen bonding of the backbone amide groups, a protein chain will organise most of itself locally either as an α-helix or a β-sheet; which one depends on the angles to the α carbon. These secondary structure elements then cluster to form a tertiary structure in which hydrophobic residues typically cluster together, aromatic residues may form π stacks, positively and negatively charged residues come together to build up ionic interactions, cysteins come together in disulphide bridges and hydrogen bonds are also formed where applicable. This last step is repeated if a protein consists of multiple subunits to form the multi-protein complex’s quarternary structure.

Almost all of the structure elements I mentioned that explain tertiary structures are intermolecular, meaning that they do not constitute a chemical bond between the connected fragments. Therefore, these structure elements are easily broken by the action of SDS whose aliphatic chain inserts into these tertiary structures, interacts with them and practically captures them on its inside.

The only exception to the former paragraph are disulphide bridges between cysteins. These are actually a chemical bond conneting the two fragments. In a simplified reaction scheme, the synthesis of a disulphide bridge looks like this:

$$\ce{\underset{\text{cystein}}{2 R-S-H} ->[][- 2e- \ -2 H+] \underset {\text{disulphide bridge}}{\ce{R-S-S-R}}}$$

Chemically, it is an oxidation of two cysteins. Therefore, these disulphide bridges cannot be broken by the action of SDS alone. It takes a reductive agent such as DTT to resupply the electrons to reform the two cysteins. If no reducing agent is present (but there are disulphides in the protein structure), the disulphide bridge will remain intact and the protein will move not through the PA gel as it should according to its size since it is cyclic.

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  • $\begingroup$ Thank you very much. I now understand the logic behind what was stated in class. From your explanation, if we had a protein with several subunits that were brought together through weak interactions such as van Der Waals forces or hydrogen bonds, would they show up as separate bands under the sole action of SDS (assuming no disulphide bridges were present)? $\endgroup$ – Bee Oct 15 '17 at 19:50
  • $\begingroup$ @Bee If the subunits are indeed only held together by intermolecular forces, yes. $\endgroup$ – Jan Oct 16 '17 at 12:46

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