Enzymes are an integral part of life and developing skills related to their properties is critical for biochemistry undergraduates. Our project aims to map out a sequence of purification steps for horseradish peroxidase (HRP) that demonstrates a wide range of related lab techniques. HRP works as a model enzyme for this because it remains active over the course of several weeks, is cost effective, and can be easily monitored via an enzyme assay. With some initial isolation steps already in place, including an ion exchange separation as a next step was explored. Using this strategy helps students appreciate the relationship between pH, protein charges, and pI. HRP exists as multiple isoforms, so potential interactions with both anion and cation exchange columns were evaluated. A DEAE-cellulose (diethylaminoethyl) anion exchange column was tried with CAPS buffer at pH 10.0, while CM-cellulose (carboxymethyl) cation exchange was tested with MES buffer at pH 6.0. Preliminary work suggests that adjusting pH, lengthening the column, or using a different matrix might be needed to get reliable separation. Samples are saved from throughout the purification process for visualization on SDS-PAGE. Ultimately, this experiment should provide valuable student interactions with protein characteristics and behavior and could potentially be coupled to enzyme kinetics studies.

Exploration of multistage isolation of horseradish peroxidase for biochemistry lab

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