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.
50 Comments
Arushi
April 1, 2025👍✔️
Prami Masih
April 9, 2025👍👍
Gaurav
August 13, 2025Beta mercapto ethanol ,urea denatures the protein
Santosh Saini
August 14, 202560 kda Original protein is dimer st. Of 30 kda sub unit , urea and beta mercapto ethanol denature the protein so option 4th is correct
Karishma Don
August 14, 2025By MEC the mol. weight of the purified protein is 60kd which further repeated in same process but under 6 M urea and see 30kd so till here we know the protein is a dimer of 30kd and again process repeated but now in presence of 10 millimolar beta mercaptoethanol and 6 M urea and got 15kd, we know that this beta thing denatures the protein or unfolds it so the 30kda consists of 2 subunits of 15kd
Khushi Singh
August 14, 2025Done
Savita Garwa
August 14, 2025D answer correct Beta mercapto ethanol ,urea denatures the protein
Ekta yadav
August 14, 2025Beta mercapto ethanol denature protein
Parul Yadav
August 14, 2025Urea denatures proteins by disrupting non-covalent interactions and beta- mercaptoethanol further denatures proteins by reducing di sulphide bonds.
Neelam Sharma
August 15, 2025The 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.
Anil Choudhary
August 24, 2025D answer correct Beta mercapto ethanol
Manisha gujar
August 17, 2025Option d is correct urea and b mercaptethanol. Denature protein subunit
Manisha gujar
August 17, 2025Option d is correct
Manisha gujar
August 17, 2025Option d is right
Manisha gujar
August 17, 2025Urea denature the original protein into 2 subunit and b mercaptethanol divide into 4 subunit
Aafreen
August 17, 2025D is the correct answer
Sneha
August 17, 2025Right answer is D
Neeraj Sharma
August 17, 2025Urea alone will only break non covalent bonding hence the two subunits which is attached by non covalent bonding will disassociate and show 30kd where as when beta markeptoethanol is also added it will break the disulphide bonding which results into 15kd
Sheikh Aleen
August 21, 2025Option d is correct
Bhavana kankhedia
August 24, 2025the 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 b mercaptoethanol , a singal band of 15 kDa appears , suggesting that the disulfied bonds like the 30 kDa subunits.
Bharti Yadav
August 24, 2025The 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.
MOHIT AKHAND
August 24, 2025Done sir 👍🏻
Priyanka Choudhary
August 24, 2025Option d was right answer,this 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
Priya dhakad
August 24, 2025Option d is correct 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.
Anil Choudhary
August 24, 2025D answer correct Beta mercapto ethanol
Bhavana kankhedia
August 24, 2025option d is correct
Anjali
August 24, 2025Option d is correct answer
Sonam Saini
August 24, 2025Option d is right
Sakshi
August 24, 2025Option d is correct
Falguni Prakash
August 24, 2025D
Varsha Tatla
August 24, 20254th option is correct combination
Kashish yadav
August 25, 2025Done sir
Ayush Dubey
August 25, 2025The 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.
Patel Radha
August 27, 2025Done
Neelam Sharma
August 30, 2025The 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
Shriyanshi verma
August 30, 2025Beta mercapto ethanol ,urea denatures the protein
Swati Choudhary
August 30, 2025understood sir..
Meera Gurjar
August 30, 2025The 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
Gayatri Nama
August 30, 2025The original 60 kDa Protein is dimer of 30 kDa subunits,held together by non covalent interactions .
Each 30 kDa subunit consists of two 15 kDa polypeptides ,connted by disulfide bonds .
The presence of urea alone dissociates the dimer into 30 kDa monomers,but full reduction with B-mercaptoethanol further breaks disulfide bonds,yeiding 15 KDa fragments.
Anisha jakhar
August 31, 2025The 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.
Shobha Kanwar
August 31, 2025Option d is correct urea and b mercaptethanol. Denature protein subunit
Anisha jakhar
September 1, 2025D is the correct option
Aakansha sharma Sharma
September 1, 2025The 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
Muskan Yadav
September 3, 2025The 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
Komal Sharma
September 4, 2025The 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.
Soniya Shekhawat
September 6, 2025Option d is correct because beta mercaptoethanol and urea both are denature the protein.
Kirti Agarwal
September 8, 2025Opt d
Mitali saini
September 9, 2025Option d
Aakansha sharma Sharma
October 1, 2025(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
Harish Kumar
November 18, 2025Option D is correct uriya and beta mercapto ethanol donon protein ko denature karte Hain