Electron spin resonance study of peroxidase activity and kinetics

Abstract

An electron spin resonance (ESR) assay has been developed for peroxidase activity. The assay measures the formation of the paramagnetic nitroxide Tempol from the oxidation of its hydroxylamine derivative (TOLH) by short-lived radicals produced by peroxidase cycle intermediates, Compounds I and II. Using phenol as a peroxidase electron donor, the ESR approach is suitable for measurements of peroxidase activity (⩾0.003 U/ml) and micromolar quantities of H 2O 2 in sample sizes as small as 2 μl. In addition, the ESR method can be used to continuously monitor activity in cell suspensions and other media that are susceptible to optical artifacts. The high membrane permeability of TOLH also makes it possible to estimate peroxidase activity in membrane-enclosed compartments, provided that TOLH oxidation rates can be stimulated with exogenous peroxidase reductants, e.g., phenol. Analysis of TOLH oxidation rates under conditions of low electron donor concentrations and high concentrations of H 2O 2 also shows clear indications of substrate-dependent inhibition and increased catalatic activity. Computer simulations indicate that the results obtained are consistent with the peroxidase reaction scheme proposed by Kohler et al. (1988, Arch. Biochem. Biophys. 264, 438–449) modified to correct for a nitroxide dependent stimulation of peroxidase catalytic activity.