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.

Assay of superoxide dismutase activity in animal tissues.

Convenient assays for superoxide dismutase have necessarily been of the indirect type. It was observed that among the different methods used for the assay of superoxide dismutase in rat liver homogenate, namely the xanthine–xanthine oxidase ferricytochrome c, xanthine-xanthine oxidase nitroblue tetrazolium, and pyrogallol autoxidation methods, a modified pyrogallol autoxidation method appeared to be simple, rapid and reproducible. The xanthine-xanthine oxidase ferricytochrome c method was applicable only to dialysed crude tissue homogenates. The xanthine-xanthine oxidase nitroblue tetrazolium method, either with sodium carbonate solution, pH 10·2, or potassium phosphate buffer, pH 7·8, was not applicable to rat liver homogenate even after extensive dialysis. Using the modified pyrogallol autoxidation method, data have been obtained for superoxide dismutase activity in different tissues of rat. The effect of age, including neonatal and postnatal development on the activity, as well as activity in normal and cancerous human tissues were also studied. The pyrogallol method has also been used for the assay of iron-containing superoxide dismutase in Escherichia coli and for the identification of superoxide dismutase on polyacrylamide gels after electrophoresis.

Scavenging of superoxide radical by ascorbic acid.

Using acetaldehyde and xanthine oxidase as the source of superoxide radical, the second order rate constant for the reaction between ascorbic acid and superoxide radical was estimated to be 8·2 × 107 M-1 s–1. In rats, the average tissue concentration of ascorbic acid was of the order of 10–3 Μ and that of superoxide dismutase was of the order of 10–6 M. So, taking together both the rate constants and the tissue concentrations, the efficacy of ascorbic acid for scavenging superoxide radical in animal tissues appears to be better than that of superoxide dismutase. The significance of ascorbic acid as a scavenger of superoxide radical has been discussed from the point of view of the evolution of ascorbic acid synthesizing capacity of terrestrial vertebrates.