Subunit inequivalence in superoxide anion formation during photooxidation of human oxyhemoglobin.

Subunit inequivalence in the photooxidation of human oxyhemoglobin was investigated by quantitative analysis of the fraction of alpha and beta chains oxidized after low intensity flash photolysis using white light in quartz cuvettes. This reaction was previously shown to generate methemoglobin and superoxide anion as photoproducts (Demma, L.S., and Salhany, J.M. (1977) J. Biol. Chem. 252, 1226-1230). The present results indicate that superoxide anion photodissociates from the alpha chain about 3 to 4 times more extensively than from beta. This difference was observed in the presence of superoxide dismutase and catalase at both pH 7 and 9, suggesting that photolysis is directly responsible. The reaction of superoxide anion with oxyhemoglobin was also studied with the same analytical methods and no chain differences could be observed. If the electronic structure of the oxyheme complex is viewed as a spin equilibrium between a singlet ground state and a charge transfer configuration, our results may indicate that the uv component of white light perturbs this equilibrium to a greater extent in the oxygenated alpha chain, implying that the separation of energy levels may be smaller in that chain as compared with beta.

Direct generation of superoxide anions by flash photolysis of human oxyhemoglobin.

The results presented in this report suggest that human oxyhemoglobin can directly form methemoglobin and superoxide anion when flashed with low intensity (38 joules) white light. The effect only occurred in quartz but not glass (cut off lambda approximately equal to 300 nm) cuvettes. The formation of O2 was established by observing the reduction of oxidized cytochrome c concomitant with MetHb formation at pH 9, and by showing that superoxide dismultase and catalse inhibit cytochrome c reduction at that pH. The inhibition of cytochrome c reduction by catalase led us to explore the possibility that H2O2 might reduce oxidized cytochrome c at pH 9. We show that H2O2 does reduce oxidized cytochrome c at that pH but not at pH 7. Furthermore, catalase but not superoxide dismutase, almost completely inhibited this reduction process. These experiments serve to confirm our interpretation of the effect of catalase on the reduction of oxidized cytochrome c in the photolytic experiments, thus establishing that H2O2 was also formed. In addition, we were able to identify the production of O2 and H2O2 due to the photolysis of water in agreement with the results of McCord and Fridovich ((1973) Photochem. Photobiol. 17, 115-121). Production of O2 from this source was considerably less than that observed when HbO2 was present. Addition of MetHb to aerated solutions of oxidized cytochrome c did not cause additional reduction, unlike addition of HbO2. The production of MetHb was found to have at least two components. One component was the primary photolytic process, and the second was a strongly pH-dependent reattack of HbO2 by O2. Addition of superoxide dismutase inhibited this second component, but did not significantly effect the primary photolytic process.