Figure 5.

Cyt c reduction by superoxide radicals generated in xanthine/xanthine oxidase system. a) Time course of cyt c reduction by superoxide in the presence of TOCL/DOPC (curves 1,2) and DOPC (curve 3) liposomes and its absence (curve 4) monitored at 550 nm. Cyt c in the presence of DOPC (curve 3) and cyt c (curve 4) alone are shown for comparison. Arrow indicates the moment when xanthine oxidase was added; b) dependence of cyt c reduction by superoxide on lipids/cyt c ratio: TOCL/DOPC:cyt c 25:1 and 50:1 (curve 1,2 respectively), DOPC/cyt c 100:1 (curve 3) and cyt c alone (curve 4). Samples of cyt c (5 µM) were preincubated with TOCL/DOPC 1:1 liposomes at different concentrations for 15 min at room temperature in 20 mM Hepes buffer (plus 100 µM DTPA, pH 7.4). After the preincubation 25 µM xanthine (5 mM stock solution) was added and absorbance spectrum was recorded (the reference cuvette contained the same amount of liposomes). To start O₂^−• production xanthine oxidase was added (0.002 unit/ml). The time course of cyt c reduction was recorded every 10 sec measuring the absorption at 550 nm. After 5 min the total absorbance spectrum was recorded again. Differences between two spectra (ΔA₅₅₀) were calculated after alignment in 530–570 nm region.