Question

A protein has a molecular mass of 400 kDa when measured by gel filtration. When subjected to gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), the protein gives three bands with molecular masses of 180, 160, and 60 kDa. When electrophoresis is carried out in the presence of SDS and b-mercaptoethanol, three bands are again formed, this time with molecular masses of 160, 90, and 60 kDa. Determine the subunit composition of the protein (how many and what sizes) and why you observe the different banding pattern under the two conditions.

Answer 1

Answer:

subunit composition

• how many: 4 subunits
• what sizes: 160 kDa, 60 kDa, 90 kDa, and 90 kDa.

Explanation:

SDS is a detergent that denaturalizes the protein and sets a relation charge size, such as that the motion of the protein in the gel is proportional to its weight. However, the detergent can not disrupt disulfide bridges, so these bonds remain together. The bigger the protein is, the stronger interaction with the polyacrylamide gel there will be. The speed of displacement in the electromagnetic field is inversely proportional to the protein size.

 On the other hand, b-mercaptoethanol acts as a reductive agent, breaking disulfide bonds. This chemical produces a complete denaturalization resulting in the unfolded protein. If disulfide bridges kept two protein subunits together, the b-mercaptoethanol separates them.

So, the most important data we need to consider here is that SDS can not disrupt disulfide bridges, while b-mercaptoethanol can.

Available data:

• A protein has a molecular mass of 400 kDa
• SDS → three bands with molecular masses of 180, 160, and 60 kDa
• SDS + b-mercaptoethanol → three bands with molecular masses of 160, 90, and 60 kDa

SDS

180 + 160 + 60 = 400 kDa

SDS + b-mercaptoethanol

160 + 90 + 60 = 310 kDa

The difference between them is 400 - 310 = 90 kDa, meaning that there is a subunit of 90 kDa that is not being detected by SDS alone.

From both measures ( SDS and SDS/b-mercaptoethanol) we can assume that subunits 160 kDa and 90kDa are the same. So the difference is in the left subunit ⇒ 180 and 90, respectively.

180 - 90 = 90 kDa

We can assume that the 180 kDa subunit is composed of two 90 kDa subunits joined by a disulfide bridge. SDS alone could not disrupt this bond, so it detected a unit of 180kDa. b-mercaptoethanol detected this bond and broke it, generating two 90 kDa units, which migrated together in the gel, so they were expressed as the same band.

This difference suggests that the protein is formed of 4 subunits. One of them is 160 kDa, another one is 90 kDa, and the last two subunits are 90 kDa each.