HORSERADISH-PEROXIDASE ASSAY - RADICAL INACTIVATION OR SUBSTRATE-INHIBITION - REVISION OF THE CATALYTIC SEQUENCE FOLLOWING MASS-SPECTRAL EVIDENCE

Horseradish peroxidase catalysed carbon-fluoride bond breakage of 4-fl uorophenol was studied enzymically and chemically in order to delineat e the reaction mechanism and characterize the structure of the reactio n products, The mass spectra confirmed the formation of higher relativ e molecular mass products and the unsymmetrical pairing of 4-fluorophe nol radicals. The 4-fluorophenol radicals formed were found to have a very important role in the reaction rate and explained the effect of i nterferents such as bovine serum albumin and the enzyme inhibition obs erved at high concentrations of 4-fluorophenol. The results demonstrat e that the enzyme-catalysed reaction is not suitable for homogeneous e nzyme immunoassays and precautionary measures must be taken to avoid t he undesirable effects for heterogeneous assays. Furthermore, the prop osed mechanism for inhibition of peroxidase by 4-fluorophenol could ap ply to all peroxidase catalysed reactions and could modify the current catalytic mechanism.