Mechanism of autoxidation of oxyhaemoglobin.

Oxyhaemoglobins from erythrocytes of different animals including fish, amphibians, reptiles, birds, mammals and human beings have been isolated by ionexchange chromatography over phosphocellulose and the comparative rates of autoxidation of oxyhaemoglobin studied. The mechanism of autoxidation in vitro has been elucidated using toad as well as human oxyhaemoglobin. Autoxidation is markedly inhibited by carbon monoxide as well as by anion ligands, namely, potassium cyanide, sodium azide and potassium thiocyanate. The inhibition by anions is in the same order as their strength as nucleophiles, indicating that it is the oxyhaemoglobin and not the ligandbound deoxy species which undergoes autoxidation. The structure of oxyhaemoglobin is considered to be mainly Hb3+O and determination of the rate of autoxidation with or without using superoxide dismutase and catalase indicates that the initial process of autoxidation takes place by dissociation of Hb3+O to methaemoglobin and superoxide to the extent of 24%. The superoxide thus produced reattacks oxyhaemoglobin to produce further methaemoglobin and hydrogen peroxide. H2O2 is a major oxidant of oxyhaemoglobin producing methaemoglobin to the extent of 53%. A tentative mechanism of autoxidation showing the sequence of reactions involving superoxide, H2O2 and OH has been presented.

Haemoglobin: A scavenger of superoxide radical.

Superoxide is continuously generated in the erythrocytes, and oxyhaemoglobin from different animals including fish, amphibians, reptiles, birds, flying mammals, mammals and human beings acts as a scavenger of superoxide. The approximate rate constants of the reaction between superoxide and oxyhaemoglobin of different animals are 0·32–1·6 × 107M–1 s–1. Results obtained with anion ligands like CN–- and N indicate that superoxide preferentially reacts with anion ligand-bound deoxyhaemoglobin. Carbonmonoxyhaemoglobin and methaemoglobin are ineffective. Work with photochemically generated oxyradical indicate that oxyhaemoglobin may also act as a scavenger of singlet oxygen. The rate constant of the reaction between superoxide and human oxyhaemoglobin is Kapp= 6·5×106 M–1 s–1, which is about three orders less than KSOD (2× 109 M–1 s–1). Thus, in the erythrocytes, oxyhaemoglobin would appear to act as a second line of defence. Oxyhaemoglobin appears to be as effective as superoxide dismutase for scavenging superoxide in the erythrocytes.