Detection of free radicals as intermediates in the methemoglobin formation from oxyhemoglobin induced by...

Four distinct paramagnetic intermediates could be observed in the reaction between oxyhemoglobin and hydroxylamine using ESR spectroscopy. The radical species exhibited different stability properties thus different techniques were required for their detection. Two of them were identified as the hydronitroxide radical (NH2O.) and the hemoglobin-nitric oxide complex (Hb2+-NO). The third one is a low-spin iron-(III)-complex, possibly the methemoglobin-hydroxylamine adduct. A fourth paramagnetic species was detected only in the absence of the iron chelator DETAPAC thus indicating that free iron ions were responsible for the formation of this intermediate. The same species was observed when a Fenton system was used to generate the radicals. This species was identified as being the Fe(NO)2X2 complex described in the literature (X = inorganic anions such as OH- or PO3-(4). The identification of the radical intermediates detected in the hydroxylamine-induced methemoglobin formation contributes to a more detailed understanding of the reaction sequence.