Question

A protein contains three 60-kD polypeptides and six 20-kD polypeptides. Each 60-kD chain is disulfide bonded to two 20-kD chains. The 100-kD units associate noncovalently to form a protein with a molecular mass of 300 kD. Which of the following is correct?

a) Molecular mass determined by gel filtration is 300 kD
b) Molecular mass determined by SDS-PAGE without 2-mercaptoethanol is 100 kD
c) Molecular masses determined by SDS-PAGE with 2-mercaptoethanol are 20kD and 60 kD
d) PAGE shows one single band

Answer 1

The molecular mass determined by gel filtration is 300 kD, and by SDS-PAGE with 2-mercaptoethanol, the molecular masses are 20 kD and 60 kD, reflecting the separation of subunits upon disulfide bond disruption.

Step-by-step explanation:

The student's question pertains to the molecular mass determination of a protein complex consisting of disulfide-bonded 60-kD and 20-kD polypeptides and its behavior under different protein separation techniques such as gel filtration and SDS-PAGE. The protein complex has a total mass of 300 kD, with each 100-kD unit formed by one 60-kD polypeptide disulfide bonded to two 20-kD polypeptides.

Answer (a) is correct if gel filtration is used, as this technique separates proteins based on size and shape under native conditions without breaking noncovalent interactions or disulfide bonds. Answer (b) is incorrect as SDS-PAGE without 2-mercaptoethanol would still have the disulfide bonds intact, showing bands at 100 kD. Answer (c) is correct as SDS-PAGE with 2-mercaptoethanol breaks disulfide bonds, separating the subunits into their respective molecular masses of 60 kD and 20 kD. Answer (d) is incorrect because PAGE will show multiple bands corresponding to different subunits, only one band might be seen if it were native PAGE without denaturing agents and where the complex remained intact.