AN EPR INVESTIGATION OF HUMAN METHEMOGLOBIN OXIDATION BY HYDROGEN-PEROXIDE - METHODS TO QUANTIFY ALL PARAMAGNETIC SPECIES OBSERVED IN THE REACTION

The method of Electron Paramagnetic Resonance (EPR) spectroscopy was u sed to study the reaction of human methaemoglobin (metHb) with hydroge n peroxide. The samples for EPR measurements were rapidly frozen in li quid nitrogen at different times after H2O2 was added at 3- and 10-fol d molar excess to 100 mu M metHb in 50 mM phosphate buffer, pH 7.4, 37 degrees C Precautions were taken to remove all catalase from the haem oglobin preparation and no molecular oxygen evolution was detected dur ing the reaction. On addition of H2O2 the EPR signals (- 196 degrees C ) of both high spin and low spin metHb rapidly decreased and free radi cals were formed. The low temperature (- 196 degrees C) EPR spectrum o f the free radicals formed in the reaction has been deconvoluted into two individual EPR signals, one being an anisotropic signal (g(paralle l to) = 2.035 and g(perpendicular to) = 2.0053), and the other an isot ropic singlet(g = 2.0042, Delta H = 20 G). The former signal was assig ned to peroxyl radicals. As the kinetic behaviour of both peroxyl (ROO (.)) and nonperoxyl (P-.) free radicals were similar, we concluded tha t ROO(.) radicals are not formed from P-. radicals by addition of O-2. The time courses for both radicals showed a steady state during the t ime required for H2O2 to decompose. Once all peroxide was consumed, th e 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 it s duration shorter at lower initial concentration of H2O2. The formati on of the rhombic Fe(III) non-haem centres with g = 4.35 was found. Th eir yield was proportional to the H2O2 concentration used and the cent res 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 wa s similar to that observed when the fast freezing method and EPR measu rement at - 196 degrees C were used.