A protein was purified to homogeneity. Determination of the molecular weight by molecular exclusion chromatography yields 60 kd. Chromatography in the presence of 6 M urea yields a 30-kd species. When the chromatography is repeated in the presence of 6 M urea and 10 mM b-mercaptoethanol, a single molecular species of 15 kd results. Which of the following statement is most likely to be correct?

(a) Protein is di mer which has dissociated in presence of 6 M urea and it was further fragmented in 15 kd on treatment with p-mercaptoethanol
(b) Chromatography in presence of b-mercaptoethanol is most appropriate for validating the mass of nascent protein
(c) Protein is tetramer each unit of 60 kd which fragmented into different mass peptides on vigorous treatment with urea and b-mercaptoethanol
(d) The original 60 kd proteins made up of two 30 kd subunits associated with non-covalent interaction, when these each subunit where subsequently treated with urea and b-mercaptoethanol, a single band of 15 kd suggesting that disulphide bonds links these 30 kd subunits.

Correct Answer:

(d) The original 60 kDa protein is made up of two 30 kDa subunits associated via non-covalent interactions. When each subunit is treated with urea and β-mercaptoethanol, a single band of 15 kDa appears, suggesting that disulfide bonds link the 30 kDa subunits.

Explanation:

This experiment involves molecular exclusion chromatography (size-exclusion chromatography) in different conditions to determine the quaternary structure of the protein and its subunit interactions. Let’s analyze the given data step by step:

Step 1: Molecular Exclusion Chromatography (Native Conditions)

• The protein has an apparent molecular weight of 60 kDa, indicating that it exists as a single functional unit under native conditions.

Step 2: Chromatography in 6 M Urea (Denaturing Conditions)

• In the presence of 6 M urea, the protein appears as 30 kDa species.
• Urea disrupts non-covalent interactions, such as hydrogen bonds and hydrophobic interactions.
• This suggests that the 60 kDa protein is a dimer composed of two 30 kDa subunits held together by non-covalent interactions.

Step 3: Chromatography in 6 M Urea + 10 mM β-Mercaptoethanol

• When β-mercaptoethanol (BME) is added along with 6 M urea, the 30 kDa species further splits into 15 kDa species.
• β-mercaptoethanol reduces disulfide bonds, breaking covalent linkages.
• This suggests that each 30 kDa subunit is composed of two 15 kDa polypeptides linked by disulfide bonds.

Final Interpretation:

• The original 60 kDa protein is a dimer of 30 kDa subunits, held together by non-covalent interactions.
• Each 30 kDa subunit consists of two 15 kDa polypeptides, connected by disulfide bonds.
• The presence of urea alone dissociates the dimer into 30 kDa monomers, but full reduction with β-mercaptoethanol further breaks disulfide bonds, yielding 15 kDa fragments.

Why Other Options Are Incorrect:

• (a) Incorrect: The 30 kDa subunit was not further fragmented; rather, disulfide bonds within it were reduced to reveal two 15 kDa polypeptides.
• (b) Incorrect: β-mercaptoethanol alone does not reveal the native protein’s mass, because it only breaks disulfide bonds, whereas molecular weight determination under native conditions provides the true oligomeric state.
• (c) Incorrect: The protein is not a tetramer of 60 kDa each, as the experimental data clearly show a dimeric structure leading to 30 kDa and then 15 kDa subunits.

Conclusion:

The most likely structure of the original protein is a dimer of 30 kDa subunits, with each subunit consisting of two 15 kDa polypeptides linked by disulfide bonds. This matches option (d) as the correct choice.