The molecular weight of an unspecified protein, at physiological conditions, is

Question

The molecular weight of an unspecified protein, at physiological conditions, is 70,000 Dalton as determined by gel filtration chromatography. The SDS-polyacrylamide gel electrophoresis (SDS PAGE) of the protein yields a single band corresponding to molecular weight of 70,000 Dalton. However, in the presence of the reducing agent, -mercaptoethanol, the SDS PAGE shows two bands, corresponding to molecular weights of 30,000 and 20,000 Dalton.

1: From these data, describe the native protein in terms of the number of subunits present, their molecular weight, stoichiometry of subunits, and the kinds of bonding (covalent, noncovalent) existing between the subunits.

2: How would your answers to problem (a) change if the molecular weight of the protein was 100,000 Dalton?

3: You synthesized two 200-aminoacid long polypeptides, one being poly-glutamic acid and another one poly-lysine. You accidentally mixed the peptides and need to separate them. Suggest schematics for their separation. Can you use IEC? GEC?

Explanation / Answer

1)The protein consists of 3 subunits with molecular weights: 30,000 Da (one subunit) and 20,000 Da (2 subunits), connected with each other by disulfide bonds.

2)100,000 = 2 x 30,000 + 2 x 20,000 This protein consists of 4 subunits, 2 with molecular weight of 30,000 Da and 2 with 20,000 Da.

3)we can use IEC for  synthesis two 200-aminoacid long polypeptides, one being poly-glutamic acid and another one poly-lysine. You accidentally mixed the peptides and need to separate them.

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