Question

4. Give the most likely explanation for each of the following experiment observations prior to sequencing.

a) Your proteins has an apparent molecular mass (Mr) of 50 KDa as determined by native SDS-PAGE. After treatment of protein with DTT or beta-mercaptoethanol, using the same technique now reveals 2 proteins of Mr 15 KDa and 35 KDa. Explain these observations.

b) Size exclusion chromatography experiments indicate the native protein/enzyme has an apparent Mr of 200 KDa. Explain this observation.

c) Describe potential approach you would take to sequence this native, pure protein.

d) How would you determine if the protein, which you now find to be an enzyme, actually has active domains after you had to re-purify in a urea buffering system?

Answer 1

4. a. The beta-mercaptoethanol breaks the disulfide bonds within the protein, which results in the visualization of the two protein bands (in the given protein) in the SDS PAGE. The protein has been broken down into its two component part with size of Mr 15 KDa and 35 KDa.

b. The size exclusion chromatography separates the proteins based on their size/molecular weight. The size of the native protein is indicated to be 200 KDa, which means that the size of the protein (pure and in its natural state) is 200 KDa.

c. The mass spectroscopy may be used to sequence this native pure protein. As it occurs in its naturally folded state, mass spectrometrywill be the best approach to sequence the protein. The peptide de novo sequencing can also be utilized.

d. In order to determine the catalytic domains, the catalytic activity of the enzyme could be tested.