A new protein of unknown structure has been purified. Size-exclusion chromatogra
ID: 788768 • Letter: A
Question
A new protein of unknown structure has been purified. Size-exclusion chromatography under varying conditions produced the elution results below:
In buffer one peak of MW 180,000
In buffer with 8 M urea two peaks of MW 50,000 and 40,000
In buffer with 8 M urea and 10 mMb-mercaptoethanol two peaks of MW 25,000 and 20,000
(a)What is the total number of N-termini in the protein?
(b)How many different types of subunits are there?
(c) What are the molecular weights of the subunits?
(d) What kinds of interactions are present among the subunits?
(e)Describe the quaternary structure using the nomenclature
Explanation / Answer
A. 8
B. 2
C. 25,000 and 20,000
D. Ionic or hydrophobic interactions (disrupted by the urea) and covalent di-sulfide bonds (disrupted by the mercaptoethanol)
E. Octomer
There is a single complex that is MW 180,000. 8M urea will disrupt non-covalent protein-protein interactions. The fact that you get two peaks suggests that there are two different, non-covalently-linked subunits. The mercaptoethanol disrupts covalent di-sulfide bonds by acting as a reducing agent. Each of the two peaks observed from urea alone are now cut in half when adding mercaptoethanol. This suggests that two individual peptides at MW 25,000 and 20,000 are covalently bound through the di-sulfide bonds to make the two peaks at MW 50,000 and 40,000 respectively, totalling four peptides (N-termini) that make up the two peaks observed in urea alone. However this only gives a MW of 90,000, which is half of what is observed in the buffer without urea or mercaptoethanol. This means there is double of everything, totalling 8 peptides. Eight subunits to a protein complex is called an octomer.
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