Radiolytic yield of cardiolipin peroxidation by gamma rays in large unilamellar vesicles of phosphatidylcholine.

Large unilamellar vesicles of 1-hexanoyl-2-(9Z-12Z-octadecadienoyl)-sn-glycero-3-phosphocholine (PLPC) have been used as model membrane to investigate the effect of increasing amount of cardiolipin (1',3'-bis-[1,2-Di-(9Z-12Z-octadecadienoyl)-sn-glycero-3-phospho]-sn-glycerol, CL) on the peroxidizability of the lipid phase. Hydroxyl radicals generated by gamma radiolysis of water initiated the lipid peroxidation. Both peroxidation products (conjugated dienes and hydroperoxides of PLPC, mono- and dihydroperoxides of CL) and disappearance of CL and PLPC were assessed as a function of the radiation dose (25 to 400 Gy, I = 10 Gy min(-1)). Our results show that the addition of 5% to 15% CL to large unilamellar vesicles (concentration ratio) produces almost complete inhibition of PLPC peroxidation. Thus, for 15% CL (known to be the proportion of CL in the inner mitochondrial membrane), the radiolytic yield of formation of PLPC hydroperoxides is reduced to zero, whereas it is equal to (3.1 +/- 0.2) x 10(-7) mol J(-1) for CL hydroperoxides, showing the importance of the targeted CL. For this concentration ratio (CL/ PLPC 15%), we have established the balance equation between the consumption of CL [G(-CL) = (2.8 +/- 0.1) x 10(-7) mol J(-1)] and the formation of CL hydroperoxides [G(CLOOH(T)) = (3.1 +/- 0.2) x 10(-7) mol J(-1)]. In addition, the radiolytic yields of disappearance of PLPC and CL have been determined [(1.5 +/- 0.1) x 10(-7) mol J(-1) and (2.8 +/- 0.1) x 10(-7) mol J(-1), respectively], their sum [(4.3 +/- 0.2) x 10(-7) mol J(-1)] being higher than G(HO.) (2.8 x 10(-7) mol J(-1)). However, there is no balance between the radiolytic yield of formation of PLPC hydroperoxides [G (PCOOH(T)) approximately 0] and the yield of disappearance of PLPC [(1.5 +/- 0.1) x 10(-7) mol J(-1)], likely because lipid fragments (not measured in this work) could be generated from HO(.) reaction on the polar head of PLPC. These results have been interpreted by assuming that the hydroxyl radicals attack in competition both lipid targets, i.e. PLPC and CL, with a higher sensitivity to CL oxidation. It can be concluded that a little amount of CL (10-15% CL/ PLPC concentration ratio) may exert a strong protective effect against the HO(.)-induced peroxidation of PLPC.

Marked difference in cytochrome c oxidation mediated by HO(*) and/or O(2)(*-) free radicals in vitro.

Cytochrome c (cyt c) is an electron carrier involved in the mitochondrial respiratory chain and a critical protein in apoptosis. The oxidation of cytochrome c can therefore be relevant biologically. We studied whether cytochrome c underwent the attack of reactive oxygen species (ROS) during ionizing irradiation-induced oxidative stress. ROS were generated via water radiolysis under ionizing radiation (IR) in vitro. Characterization of oxidation was performed by mass spectrometry, after tryptic digestion, and UV-visible spectrophotometry. When both hydroxyl and superoxide free radicals were generated during water radiolysis, only five tryptic peptides of cyt c were reproducibly identified as oxidized according to a relation that was dependent of the dose of ionizing radiation. The same behavior was observed when hydroxyl free radicals were specifically generated (N(2)O-saturated solutions). Specific oxidation of cyt c by superoxide free radicals was performed and has shown that only one oxidized peptide (MIFAGIK+16), corresponding to the oxidation of Met80 into methionine sulfoxide, exhibited a radiation dose-dependent formation. In addition, the enzymatic site of cytochrome c was sensitive to the attack of both superoxide and hydroxyl radicals as observed through the reduction of Fe(3+), the degradation of the protoporphyrin IX and the oxidative disruption of the Met80-Fe(3+) bond. Noteworthy, the latter has been involved in the conversion of cyt c to a peroxidase. Finally, Met80 appears as the most sensitive residue towards hydroxyl but also superoxide free radicals mediated oxidation.