QUESTIONS; 1. A protein has a molecular weight of 400 K Daltons when measured by
ID: 220420 • Letter: Q
Question
QUESTIONS; 1. A protein has a molecular weight of 400 K Daltons when measured by molecular exclusion chromatography. When subjected to gel electrophoresis in the presence of SDS, the protein gives 3 bands with molecular weights 180, 160, and 60 K Daltons. When electrophoresis was carried out in the presence of SDS and beta mercaptoethanol, 3 bands are again visible with molecular weights 160, 90 and 60 K Daltons. Explain these observations and determine the subunit composition of the protein. (3 points) 9,90, 16oExplanation / Answer
Ans. Given- Molar mass of protein = 400 kDa
#I. Bands obtained in presence of SDS: SDS provides uniform negative charge along the length of peptide chain and disrupts non-covalent interactions (H-bonds, van der Waal’s interactions, etc.). The peptide chains linked by non-covalent interactions are separated. The proteins linked by covalent interactions (disulfide bond) remain attached together.
For clarity, let label peptide of each molar mass with different lattes-
Mass of a-subunit = 180 kDa
Mass of b-subunit = 160 kDa
Mass of y-subunit = 60 kDa
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Total = (180 + 160 + 60) kDa = 400 kDa
#II. Bands obtained in presence of SDS + b-mercaptoethanol: Treatment with b-mercaptoethanol (reducing agent) hydrolyzes the disulfide bonds in the protein. If b-mercaptoethanol treatment and subsequent SDS-PAGE electrophoresis gives 2 bands, the two peptide chains in the protein are covalently linked by one or more disulfide bonds.
Observation:
Mass of a-subunit = 90 kDa
Mass of b-subunit = 160 kDa
Mass of y-subunit = 60 kDa
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Total = (90 + 160 + 60) kDa = 310 kDa
To get a total mass of 400 kDa, there must be another fragment of 90 kDa (= 400 kDa – 310 kDa). It means that there are 2 subunits of 90 kDa linked together by disulphide bond (which could NOT be separated by SDS alone).
# Conclusion: The overall composition of protein is-
2 a-subunits of 90 kDa each
1 b-subunit of 160 kDa
1 y-subunit of 60 kDa
That is, the protein is: a2by