On the role of methylene blue in the oscillating peroxidase–oxidase reaction

Abstract

The role of methylene blue (MB) and its interaction with various enzymic and nonenzymic intermediates in the oscillating peroxidase–oxidase reaction were investigated. Time dependent spectral changes were deconvoluted into time series of the different intermediates. Ferric peroxidase, ferrous peroxidase, and peroxidase compound III account for most of the spectral changes, while the two remaining oxidation states of the enzyme (compound I and II) are only present in very low amounts during any phase of the oscillations. The presence of MB is required for the generation of sustained oscillations and complex dynamics. The amount of ferrous peroxidase formed during an oscillatory cycle decreases with increasing concentrations of MB. At ‘‘high ’’ concentrations of MB only damped oscillations are observed. These findings are interpreted in terms of either a competition between MB and other reactants for NAD• radicals, or of a reaction between MB and ferrous peroxidase. In the absence of MB only damped oscillations can be observed due to slow and irreversible degradation of the enzyme. This suggests that MB also protects the enzyme against degradation.