Peroxidase Active Site Activity Assay

Abstract

Cyclooxygenase enzymes house spatially distinct cyclooxygenase- and peroxidase-active sites. The two-electron reduction of peroxides to their corresponding alcohols by the heme bound in the peroxidase-active site converts the heme to a ferryloxoprotoporyphrin cation radical, with a reductant providing the two electrons necessary to bring the heme back to its resting state. The ferryloxoprotoporyphrin cation radical can abstract a hydrogen atom from a tyrosine residue in the cyclooxygenase-active site, activating the oxygenase functionality. The tyrosyl radical subsequently abstracts a hydrogen atom from the cyclooxygenase substrate, arachidonic acid, leading to its oxygenation and the formation of a hydroperoxy endoperoxide intermediate, PGG₂. The peroxidase functionality reduces PGG₂ to the hydroxy endoperoxide, PGH₂, which serves as the precursor to downstream prostaglandins and thromboxane. The peroxidase activity of cycloxygenase enzymes can be assayed by quantifying the oxidation of a peroxidase reductant or the reduction of a hydroperoxide substrate. Here we describe a spectrophotometric assay used to measure the oxidation of a reductant, 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), as well as a high-performance liquid chromatography method for the measurement of the conversion of 5-phenyl-4-pentyl hydroperoxide (PPHP) to its corresponding alcohol. The first provides a continuous but indirect assay of peroxidase activity, whereas the second provides a discontinuous but direct assay.