The  protein a-chymotrypsin is a serine protease that will hydrolyze the  peptide bonds of proteins containing aromatic or hydrophobic groups such  as tyrosine, phenylalanine, tryptophan and leucine. This protein of 245  residues is comprised of three peptide chains of varying lengths  interconnected by five disulfide bonds. Using HPLC- Size Exclusion  Chromatography, a-chymotrypsin will be analyzed in its native globular  state (oxidized) and reduced state using DTT and Urea. This analysis  should show that SEC can be used to separate and determine the relative  size of complex proteins. The native species should show a single peak  on a chromatograph, where the denatured and reduced species will show  multiple peaks representing the smaller peptide chains. This  single  experiment will allow students enrolled in a typical biochemistry  laboratory course to gain experience with and working knowledge of  protein structure, disulfide bond reduction and HPLC simultaneously.

Modern techniques in biochemistry education: Protein structure analysis using size exclusion chromatography

243rd National Meeting (2012)

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