REDOX CYCLING OF HUMAN METHEMOGLOBIN BY H2O2 YIELDS PERSISTENT FERRYLIRON AND PROTEIN-BASED RADICALS

The formation and reactivity of ferryl haemoglobin (and myoglobin), wh ich occurs on addition of H2O2, has been proposed as a mechanism contr ibuting to oxidative stress associated with human diseases. However, r elatively little is known of the reaction between hydrogen peroxide an d human haemoglobin. We have studied the reaction between hydrogen per oxide and purified (catalase free) human metHbA. Addition of H2O2 resu lted in production of both ferryl haem iron (detected by optical spect roscopy) and an associated protein radical (detected by EPR spectrosco py). Titrating metHbA with H2O2 showed that maximum ferryl levels coul d be obtained at a 1:1 stoichiometric ratio of haem to H2O2 NO oxygen was evolved during the reaction, indicating that human metHbA does its elf not possess catalatic activity. The protein radicals obtained in t his reaction reached a steady state concentration, during hydrogen per oxide decomposition, but started to decay once the hydrogen peroxide h ad been completely exhausted. The presence of catalase, at concentrati ons around 10(4) fold lower than metHb, increased the apparent stoichi ometry of the reaction to 1 mol metHb: similar to 20 mol H2O2 and abol ished the protein radical steady state. The biological implications fo r these results are discussed.