An EPR investigation of human methaemoglobin oxidation by hydrogen peroxide: methods to quantify all paramagnetic species observed in the reaction.

Abstract

The method of Electron Paramagnetic Resonance (EPR) spectroscopy was used to study the reaction of human methaemoglobin (metHb) with hydrogen peroxide. The samples for EPR measurements were rapidly frozen in liquid nitrogen at different times after H2O2 was added at 3- and 10-fold molar excess to 100 microM metHb in 50 mM phosphate buffer, pH 7.4, 37 degrees C. Precautions were taken to remove all catalase from the haemoglobin preparation and no molecular oxygen evolution was detected during the reaction. On addition of H2O2 the EPR signals (-196 degrees C) of both high spin and low spin metHb rapidly decreased and free radicals were formed. The low temperature (-196 degrees C) EPR spectrum of the free radicals formed in the reaction has been deconvoluted into two individual EPR signals, one being an anisotropic signal (g parallel = 2.035 and g perpendicular = 2.0053), and the other an isotropic singlet (g = 2.0042, delta H = 20 G). The former signal was assigned to peroxyl radicals. As the kinetic behaviour of both peroxyl (ROO.) and non-peroxyl (P.) free radicals were similar, we concluded that ROO. radicals are not formed from P. radicals by addition of O2. The time courses for both radicals showed a steady state during the time required for H2O2 to decompose. Once all peroxide was consumed, the radical decayed with a first order rate constant of 1.42 x 10(-3) s-1 (1:3 molar ratio). The level of the steady state was higher and its duration shorter at lower initial concentration of H2O2. The formation of the rhombic Fe(III) non-haem centres with g = 4.35 was found. Their yield was proportional to the H2O2 concentration used and the centres were ascribed to haem degradation products. The reaction was also monitored by EPR spectroscopy at room temperature. The kinetics of the free radicals measured in the reaction mixture at room temperature was similar to that observed when the fast freezing method and EPR measurement at -196 degrees C were used.