Nonsteroidal antiinflammatory drugs interact with horseradish peroxidase in an in vitro assay system for hydrogen peroxide scavenging

The established horseradish peroxidase/guaiacol in an in vitro assay system was used for investigation of the reactivity of nonsteroidal antiinflammatory drugs with hydrogen peroxide. Although the drugs rapidly seemed to react in the selected conditions, difficulties were encountered in attempts to quantify the reaction and an interaction with horseradish peroxidase was suspected, A more specific assay system based on the absolute specificity of the enzyme glutathione peroxidase for glutathione was subsequently used which demonstrated that none of the investigated nonsteroidal antiinflammatory drugs was able to scavenge hydrogen peroxide. An original procedure to further evidence the interaction was developed thereafter, based on the reaction of 5-aminosalicylic acid with similar hemoproteins. This led to the demonstration that nonsteroidal antiinflammatory drugs were substrates for horseradish peroxidase and explained their reactivity in the horseradish peroxidase/guaiacol assay system. The compound 5-aminosalicylic acid showed an unusual behaviour that was attributed to its ability to both scavenge hydrogen peroxide and interact with horseradish peroxidase. It was concluded that the lack of specificity of horseradish peroxidase for its donor substrate may lead to erroneous results in assays for hydrogen peroxide scavenging of some drugs. An alternative method is however available and a simple spectroscopic assay can evidence the interaction with horseradish peroxidase.