The size of the gene that codes for protein A is 900 nucleotides. After purifyin

Question

The size of the gene that codes for protein A is 900 nucleotides. After purifying protein A, a biochemist ran the protein on denaturing SDS PAGE gel, but forgot to add a reducing agent while preparing the sample for the gel. Comparing to a protein marker, she estimated the size of protein A to be 99 kDa. What is true about protein A? (Assume that one amino acid has a molecular weight of 110 Da).

A) The molecular weight of protein A monomer is 99 kDa and it cross links with other monomers to form a trimer

B) The molecular weight of protein A monomer is 99 kDa and it cross links with no other monomers

C) The molecular weight of protein A monomer is 33 kDa and it cross links with other monomers to form a trimer

D) The molecular weight of protein A monomer is 33 kDa and it cross links with no other monomers

E) None of the above

The correct answer is (C), but why? Thanks!

Explanation / Answer

Answer:

Size of the protein A = 900 nucleotides

Molecular weight of one amino acid = 110 Da

Weight of Protein A = 900 x 110 = 99000 Da or 99 Kda

Since biochemist forgot to add a reducing agent (like DTT or Beta Mercaptoethanol) to the reducing SDS PAGE therefore the disulfide bonds of the protein A will not be reduced and hence remains intact (and also free sulfhydryl groups if present will not undergo oxidation).

Protein A is a monomer of 33Kda and is associated in a trimeric form. Without the presence of reducing agent, it will run in the polyacrylamide gel as a trimeric protein of size 33 x 3 = 99 KDa.

Lets consider different scenarios:

A) The molecular weight of protein A monomer is 99 kDa and it cross links with other monomers to form a trimer - in this case, if the MW weight of protein A is 99Kda and if it crosslinks with other monomers, then without the presence of reducing agents, it would have a combined weight of more than 99 KDa and will migrate tp a lesser distance in the gel. Its band in SDS PAGE in this case would appear higher corresponding to high molecular weight (>99 KDa) when compared to marker size. Since the actual size observed in gel is 99 KDa, therefore this is not the right option.

B) The molecular weight of protein A monomer is 99 kDa and it cross links with no other monomers - In this ca

C) The molecular weight of protein A monomer is 33 kDa and it cross links with other monomers to form a trimer - Correct

D) The molecular weight of protein A monomer is 33 kDa and it cross links with no other monomers - in this case, its expected that protein A will migrate faster in the SDS PAGE and its band will correspond to size 33 KDa (based on comparison with the reference MW ladder). However, the actual size observed in gel is 99 KDa, therefore this is not a valid option.

E) None of the above

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