Question

Carl has just finished purifying a protein and analysis by gel filtration indicated that the molecular weight of the native (undenatured) protein was 130,000 dalton. His advisor wanted him to determine whether this protein had quaternary structure using gel electrophoresis. He wants to be able to describe the molecular weight of each of the subunits and the forces involved in linking these subunits together (disulfide linkages and/or electrostatic, hydrogen bonding and hydrophobic interactions).

Carl first analyzed his pure protein sample by gel electrophoresis using a procedure similar to the one we used in class. He mixed his sample with a buffer that contained mercaptoethanol and SDS and boiled the sample. SDS-PAGE analysis of this sample revealed two protein bands: one with a molecular weight of 15,000 Dalton and one at 50,000 Dalton.

Carl also analyzed his protein sample in a slightly different way - he used an SDS sample buffer that did not include mercaptoethanol. He ran this sample on the same gel as his other prepared sample. In the lane where this sample was loaded he detected two protein bands: one with a molecular weight of 30,000 Dalton and one at 50,000 Dalton.

a. Draw what you think Carl’s SDS-PAGE gel looks like with his sample containing β−mercaptoethanol in Lane 2 and without β−mercaptoethanol in Lane 3 on the gel in Figure 1.

Figure 1. SDS-PAGE analysis of a pure protein sample. Lane 1: Promega Broad Range Protein Ladder. Lane 2: sample prepared with β−mercaptoethanol. Lane 3: sample prepared without β−mercaptoethanol.

b. Help Carl and his advisor describe the structure of this protein. Does it have quaternary structure? If so, describe what the molecular weight of each subunit is, and how the various subunits are linked together.