Direct fitting improves the accuracy of the horse radish peroxidase competition assay for peroxidase activity

The peroxiredoxins are an important antioxidant protein family and their ability to neutralise oxidants is regularly investigated using horse radish peroxidase in a competition assay system. In this method, the rate constant of a peroxiredoxin is calculated from the fractional inhibition of horse radish peroxidase activity caused by competition with the peroxiredoxin for an oxidant substrate. We developed a model capable of simulating this assay and, using this model, demonstrate that the fractional inhibition calculation significantly and systematically mis-estimates the rate constant under fairly common conditions. We go on to develop a method for fitting simulated assay time-courses to experimental data directly, which significantly outperforms the fractional inhibition method yielding more accurate results. Based on our findings, we recommend using the direct fitting approach to determine peroxidase rate constants from horseradish peroxidase experiments.