A Simple, Quantitative Peroxidase Assay Demonstrating Enzyme Inhibition with L-cysteine

Abstract

We present a simple enzyme assay using the robust horseradish peroxidase and the indicator guaiacol which, when oxidized to form tetraguaiacol, can be detected and quantified using a visible spectrophotometer at 470nm. This assay uses buffers (NaPO4), substrates (H2O2) and equipment already widely used and available in most first-year laboratories and is a cost-effective addition to current experiments. In addition to determining and comparing specific activity under a variety of traditional variables (pH, temperature) participants will use L-cysteine, reported in the literature to be a non-competitive inhibitor of peroxidase. L-cysteine demonstrates a unique inhibition pattern in our assay losing its inhibitory characteristics in a concentration and time dependent manner. This gives the instructor a range of options to demonstrate enzyme inhibition as well as an opportunity for investigations and self-directed experimentation by the students with a more challenging result to consider and analyze. Firstpage