Abstract
Ferritin was found to promote the peroxidation of phospholipid liposomes, as evidenced by malondialdehyde formation, when incubated with xanthine oxidase, xanthine, and ADP. Activity was inhibited by superoxide dismutase but markedly stimulated by the addition of catalase. Xanthine oxidase-dependent iron release from ferritin, measured spectrophotometrically using the ferrous iron chelator 2,2'-dipyridyl, was also inhibited by superoxide dismutase, suggesting that superoxide can mediate the reductive release of iron from ferritin. Potassium superoxide in crown ether also promoted superoxide dismutase-inhibitable release of iron from ferritin. Catalase had little effect on the rate of iron release from ferritin; thus hydrogen peroxide appears to inhibit lipid peroxidation by preventing the formation of an initiating species rather than by inhibiting iron release from ferritin. EPR spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide was used to observe free radical production in this system. Addition of ferritin to the xanthine oxidase system resulted in loss of the superoxide spin trap adduct suggesting an interaction between superoxide and ferritin. The resultant spectrum was that of a hydroxyl radical spin trap adduct which was abolished by the addition of catalase. These data suggest that ferritin may function in vivo as a source of iron for promotion of superoxide-dependent lipid peroxidation. Stimulation of lipid peroxidation but inhibition of hydroxyl radical formation by catalase suggests that, in this system, initiation is not via an iron-catalyzed Haber-Weiss reaction.
Highlights
Releasefrom ferritin, measured spectrophotometri- .OH is highly reactive, its in vivo formation is cally using the ferrous iron chelator 2,2’-dipyridyl, contingent upon the availability of physiological iron
The resultant spectrum was that of a hydroxyl the existence of low molecular weight complexes radical spin trap adduct which was abolished by the remains controversial it is known that themajority of intraaddition of catalase
Peroxidation was initiated by the addition of xanthine oxidase, and aliquots from the reaction mixtures were assayed for MDA content
Summary
Materials-Xanthine, cytochrome c(TypeVI), 2-thiobarbituric acid, ADP, butylated hydroxytoluene, mannitol, 4,7-diphenyl-l,10phenantholine, potassium superoxide, and crown ether were purchased from Sigma. The pellet obtained after centrifugation at 1,500 X g for 30 min was resuspended in PBS (0.02 M phosphate buffer, pH 7.4, containing 0.1 M sodium chloride) and dialyzed for 24 h against 4 litersof the same buffer with one change of buffer. EDTA was addedto theisolated ferritin to a final concentration of 10 mM and incubated on ice for 1 h. This was done to ensure that no iron was looselyassociated on the protein. Care was taken to extract endogenous iron from the saturated sodium acetate with the phenanthroline/isoamyl alcohol mixture prior to use. Incubations in a final volume of 1ml contained 0.025 unit of xanthine oxidase, 0.33 mM xanthine, 500 PM ferritin iron, and 5.12 mM 2,2'-dipyridyl in 50 mM NaCl, pH 7.0. Spectrometer settings were:3329.4 G magnetic field, 15 milliwatts microwave power,9.4232 GHz, 1000kHz modulation frequency, 0.63 modulation amplitude, 2.0-5 time constant, and 8-min scan time
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