Estimation of p-Hydroxyphenylpyruvic Acid Hydroxylase Activity Based on 14CO2 Evolution from the 1-14C-labelled Substrate

The rate of evolution of 14CO2 has been measured in a modified assay for the enzyme p-hydroxyphenylpyruvic acid oxidase, utilizing chemically synthesized p-hydroxyphenyl[1-14C]-pyruvic acid. The characteristics of the enzyme system with respect to substrate concentration, protein concentration, and the ionic environment have been studied. Short incubation periods and a short post-acidification shaking period for the collection of the 14CO2 into Hyamine gave optimum conditions for the assay, and reduced the amount of nonenzymic decarboxylation of the substrate to a minimum. This assay has an increased sensitivity when compared with previous analytical methods and has enabled the Km of the enzyme to be determined (2.3 × 10−5 M) with lower substrate concentrations than those that give rise to substrate inhibition (from 50 μM). The enzyme activity of liver and kidney of the human and rat have been determined. No enzyme activity could be observed in human leukocytes and cultured fibroblasts. o-Hydroxybenzaldehyde was found to be a powerful inhibitor of the enzyme.