The giant extracellular hemoglobin from earthworm Glossoscolex paulistus (HbGp) reacts with hydrogen peroxide, displaying peroxidase activity in the presence of guaiacol. The formation of ferryl-HbGp (compound II) from the peroxidase cycle was studied in the present work. The hypervalent ferryl-HbGp species was formed directly by the reaction of oxy-HbGp and hydrogen peroxide. The oxy-HbGp heme groups (144) under different excess of H2O2, relative to heme, showed an influence in the total amount of ferryl-HbGp at the end of the reaction. The ferryl-HbGp was formed with second order rate constant of 27.1±0.5M-1s-1, at pH7.0 and 25°C. The increase of the pH value to 8.0 induces both faster formation and decay of ferryl-HbGp, together with oligomeric dissociation induced by the presence of H2O2, as observed by DLS. This effect of dissociation increases the heme exposure and decreases the ferryl-HbGp stability, affecting the rate constant as a parallel reaction. At pH7.0, high excess of H2O2, above 1:5 oxy-HbGp heme: H2O2, produces the aggregation of the protein. Our results show for the first time, for an extracellular giant hemoglobin, the possible effects of oxidative stress induced by hydrogen peroxide.
Keywords: Extracellular hemoglobin; Ferryl-HbGp; Glossoscolex paulistus; Hydrogen peroxide; Optical absorption spectroscopy; Rate constant.
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