Horseradish peroxidase (HRP) is an enzyme extracted from horseradish roots that is classified as an oxidoreductase. HRP has proved to be a robust enzyme that maintains activity over an extended time, making it quite suitable for study in an undergraduate teaching lab. The focus of this project overall is to isolate HRP using multiple protein purification techniques, such as gel filtration, ion exchange, and hydrophobic interaction chromatography as a biochemistry lab experiment. Prior work found that some purification could be achieved using a hydrophobic interaction column (HIC) after an ammonium sulfate precipitation step. Purity of the sample through this stage was evaluated using SDS-PAGE though improved visibility is needed. We are working to further purify HRP eluted from the HIC column using ion exchange chromatography. Since various HRP isozymes of HRP exist with a wide range of isoelectric points, our study will compare anion exchange using diethylaminoethyl cellulose (DEAE) versus cation exchange using carboxymethyl cellulose (CM). We will evaluate which column best separates detectable HRP from protein contaminants remaining after the HIC column step. The HRP will be followed enzymatically and its purification demonstrated by SDS-PAGE analysis of samples throughout the purification process.

Purification of horseradish peroxidase for biochemistry lab

253rd National Meeting (2017)

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