A protein has a molecular mass of 400 kDa when measured by...

A protein has a molecular mass of $400 \mathrm{kDa}$ when measured by size-exclusion chromatography. When subjected to gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), the protein gives three bands with molecular masses of $180,160,$ and 60 kDa. When electrophoresis is carried out in the presence of SDS and dithiothreitol, three bands are again formed, this time with molecular masses of $160,90,$ and $60 \mathrm{kDa} .$ Determine the sub-unit composition of the protein.

Key Concepts

Size-Exclusion Chromatography

Size-exclusion chromatography is a technique that separates molecules based on their hydrodynamic volume (or effective size in solution). It provides an estimate of the overall molecular mass of a protein complex in its native, undenatured state, which is critical for understanding the quaternary structure of multi-subunit proteins.

SDS-PAGE

SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) is a method that separates proteins based on their molecular weight under denaturing conditions. SDS binds uniformly to proteins, giving them a similar charge-to-mass ratio. When combined with reducing agents, it can further resolve proteins by breaking disulfide bonds, revealing the individual polypeptide subunits.

Disulfide Bonds

Disulfide bonds are covalent bonds formed between cysteine residues in proteins, often stabilizing the structure or linking subunits together. In electrophoresis, the use of reducing agents such as dithiothreitol (DTT) breaks these bonds, allowing for the detection of individual subunits that may have been covalently bonded in the native complex.

Subunit Composition Determination

Subunit composition determination involves combining data from different analytical techniques (such as size-exclusion chromatography and SDS-PAGE with and without reducing agents) to deduce the number, size, and arrangement of individual protein subunits within a larger complex. This process is essential for understanding the architecture and function of multi-subunit proteins.

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