Horseradish peroxidase assay—radical inactivation or substrate inhibition? Revision of the catalytic sequence following mass spectral evidence

Abstract

Horseradish peroxidase catalysed carbon–fluoride bond breakage of 4-fluorophenol was studied enzymically and chemically in order to delineate the reaction mechanism and characterize the structure of the reaction products. The mass spectra confirmed the formation of higher relative molecular mass products and the unsymmetrical pairing of 4-fluorophenol radicals. The 4-fluorophenol radicals formed were found to have a very important role in the reaction rate and explained the effect of interferents such as bovine serum albumin and the enzyme inhibition observed at high concentrations of 4-fluorophenol. The results demonstrate that the enzyme-catalysed reaction is not suitable for homogeneous enzyme immunoassays and precautionary measures must be taken to avoid the undesirable effects for heterogeneous assays. Furthermore, the proposed mechanism for inhibition of peroxidase by 4-fluorophenol could apply to all peroxidase catalysed reactions and could modify the current catalytic mechanism.