In the undergraduate curriculum, the use of High Performance Liquid Chromatography (HPLC) is often used for purification purposes; however, there is potential for more. We are in the process of creating a novel biochemistry laboratory experiment that will investigate the structural modifications of Bovine Pancreatic Trypsin Inhibitor (BPTI) and α-chymotrypsin using Reverse Phase HPLC (HPLC-RP). BPTI is a 6.5 KDa monomer protein (58 amino acids) that contains three intramolecular disulfide bonds. HPLC has been employed to distinguish the following: native BPTI (completely folded), denatured BPTI (partially unfolded) and denatured/reduced BPTI (completely unfolded). For these experiments, Mass Spectrometry (MS) is the primary detection method. It was found that a clear delineation between folding states can be observed. We are currently investigating a larger protein, α-chymotrypsin which is a tri-mer protein (245 amino acids) that contains 3 intramolecular and 2 intermolecular disulfide bonds. Once these protocols are formally established, this project will be developed into an upper level undergraduate biochemistry laboratory experiment that provides students with a deeper understanding of protein structure, oxidation/reduction, and a hands-on experience with HPLC.<br/> <br/>

Modern techniques in biochemistry education: Analysis of bovine pancreatic trypsin inhibitor using HPLC

254th National Meeting (2017)

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