Formation of homovanillic acid dimer by enzymatic or Fenton system — catalyzed oxidation

Abstract

Homovanillic acid is the most extensively employed reagent for the fluorometric detection of peroxidase. However, the assays based on the determination of the oxidation product of homovanillic acid do not allow a selective detection of the enzyme, because chemical or physical factors can interfere with the fluorometric determination. The aim of this work was to verify if other enzymatic or non–enzymatic systems might catalyze the homovanillic acid oxidation. The reaction was investigated by spectrophotometric and fluorometric assays; HPLC analysis was used to separate homovanillic acid from its oxidation product and to obtain information on the oxidation process. The results obtained showed that soybean lipoxygenase in the presence of hydrogen peroxide can oxidize homovanillic acid with the formation, by an o, o′-biphenyl linkage, of the corresponding dimer as the sole reaction product. The reaction followed Michaelis–Menten kinetics, for both homovanillic acid and hydrogen peroxide. Other systems, such as cytochrome c/H 2O 2 and Fenton reagents, were also able to oxidize homovanillic acid to its dimer. It can be affirmed that possible interference by other oxidative systems — that could be present in the biological materials tested — should be considered in assays of peroxidase activity based on the detection of the dimer of homovanillic acid.