The electrophoretic separation of proteins

Adrenocorticotropic hormone (ACTH) is a $39$‍ amino acid polypeptide secreted by the anterior pituitary gland. ACTH is derived from the $241$‍ amino acid polypeptide pro-opiomelanocortin (POMC) via the activity of endoproteases, which catalyze the hydrolysis of peptide bonds between adjacent basic residues of polypeptide chains (see figure one). In addition to ACTH, several other polypeptides are derived from POMC. Table one lists some of these breakdown products. Of particular interest is alpha-MSH, a melanocyte-stimulating hormone consisting of the first $13$‍ amino acid residues of ACTH. Addison’s disease (primary adrenal insufficiency), resulting from the under-production of cortisol by the adrenal cortex, is associated with increased serum levels of ACTH and increased secretion of the pigment melanin by melanocytes.

Table one. Breakdown products of pro-opiomelanocortin (POMC). "Position" is measured from the N-terminus amino acid residue of POMC to the C-terminus residue. "Length" refers to the number of amino acid residues in the protein of interest.

Figure one. Simplified diagram of change in free energy during endoprotease catalyzed hydrolysis of POMC.

The development of reliable assays to assess circulating levels of hormones like ACTH is of importance in the clinic. Perhaps the most straightforward way to assess the presence or absence of a particular protein in a given sample is to use SDS-page, which spatially separates proteins in a sample according to molecular weight. In SDS-page, a given sample is heated in a solution containing sodium dodecyl sulfate (SDS) and betamercaptoethanol (BME). SDS is a negatively charged surfactant that binds polypeptides in a quantity directly proportional to the length of the polypeptide. BME is a strong reducing agent that is able to disrupt aspects of the tertiary and quaternary structure of proteins. Once treated with SDS, BME, and heat, the protein sample is loaded into a polyacrylamide gel contained in a pH buffered solution (note: polyacrylamide gels consist of a chemical matrix with regularly shaped pores through which molecules can migrate). A constant voltage is applied across the gel, which causes the movement of proteins through the gel. Assuming a constant voltage, the rate of migration of a protein in SDS-page is inversely proportional to the logarithm of its molecular weight.