Quantitative analysis of superoxide anion radicals photosensitized by hypericin in a model membrane using the cytochrome c reduction method

Abstract

The O 2 ▪ production by photoactivated hypericin (hyp) in egg yolk lecithin (EYL) liposomes was examined by using the cytochrome c reduction method in order to elucidate the mechanism of formation of superoxide and to quantify this formation. Specifically, production of O 2 ▪ was demonstrated unequivocally by reaction with superoxide dismutase. The rate of reduction of cytochrome c was dependent not only on the concentration of cytochrome c, hyp and oxygen but also on the pH of the system. The rate constant k c for the reaction of O 2 ▪ with ferricytochrome c (Cyt.Fe 3+) to form ferrocytochrome c (Cyt.Fe 2+) was found to be 1.5 × 10 5 M −1 s −1 and 2.4 × 10 3 M −1 s −1 at pH 7.0 and at pH 9.7 respectively. In contrast, the relative quantum yield of superoxide anion radicals increased considerably in alkaline solution: Φ O2 ▪=7.5×10 −3 at pH 9.7 as compared with Φ o2 ▪=4.7×10 −4 at pH 7.0 In anaerobic conditions, the semiquinone radical (hyp ▪ ) was predominantly photoproduced via self-electron transfer between excited and ground state hyp. In aerobic conditions around neutral pH, the formation of O 2 ▪ was competitively quenched by known 1O 2 quenchers. Furthermore, there was a large increase in O 2 ▪ formation in D 2O, relative to H 2O, as would be expected for 1O 2 reaction. Thus, hyp undergoes a one-electron transfer to 1O 2 in EYL liposomes at pH 7.0. However, the contribution of the singlet oxygen pathway decreased when the pH was increased. At pH 9.7, the O 2 ▪ production proceeded mainly via electron transfer between the excited hyp and its ground state, with little 1O 2 participation.