Using a five step protocol, you have purified an enzyme to the point where the s

Question

Using a five step protocol, you have purified an enzyme to the point where the specific activity remains constant when you perform any additional purification steps. When you run the purified enzyme on a gel filtration chromatography column, you find it has a molecular mass of 50 kD. However, when you run the purified enzyme on a SDS-PAGE gel, you obtain two different sized peptides one 30 kD and the other 20 kD. What could be the possible explanations for this result?
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Explanation / Answer

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Gel filtration column chromatography maintains the protein tertiary and quaternary structures. The protein stays native.

SDS-PAGE is a denaturing technique where the protein looses its tertiary and quaternary structures. SDS-PAGE involves the addition of the strong ionic detergent sodium dodecyl sulfate (SDS) that denatures the protein tertiary structure and thus that destroys the weak non-covalent interactions (H-bonds, salt-bridges and hydrophobic) between protein subunits forming the quaternary structure.

Conclusion. The 50 kDa native protein is composed of two smaller subunits, one of 30 kDa and the other of 20 kDa. On gel filtration this assembly stays native; on SDS-PAGE the two subunits dissociate and each is denatured by the detergent.

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