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31 Laboratoire de Spectrome´trie de Masse des Prote´ines, Institut de Biologie Structurale (CEA, CNRS, UJF, UMR 5075), Grenoble, France2 Laboratory of Molecular Structure Characterization, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vi´denˇska´ 1083,Prague 4, Czech Republic3 CEA; DSV; iRTSV; Laboratoire de Chimie et Biologie des Me´taux (UMR 5249); CEA-Grenoble, Grenoble, France4 Commissariat a` l’Energie Atomique (CEA), Institut de Biologie et Technologies de Saclay (iBiTecS), Service d’Inge´nierie Mole´culaire desProte´ines (SIMOPRO), F-91191 Gif sur Yvette, FranceKeywordsdiphtheriatoxin;hydrogen⁄deuteriumexchanges;massspectrometry;protein⁄membraneinteractions;translocationdomainCorrespondenceD. Gillet, Commissariat a` l’Energie Atomique(CEA), Institut de Biologie et Technologiesde Saclay (iBiTecS), Service d’Inge´nierieMole´culaire des Prote´ines (SIMOPRO),F-91191 Gif sur Yvette, FranceFax: +33 1 69 08 94 30Tel: +33 1 69 08 76 46E-mail: daniel.gillet@cea.frE. Forest, Laboratoire de Spectrome´trie deMasse des Prote´ines, Institut de BiologieStructurale (CEA-CNRS-UJF), 41 rue JulesHorowitz, 38027 Grenoble, FranceFax: +33 4 38 78 54 94Tel: +33 4 38 78 34 03E-mail: eric.forest@ibs.frV. Forge, CEA; DSV; iRTSV; Laboratoire deChimie et Biologie des Me´taux (UMR 5249);CEA-Grenoble, 17 rue des martyrs, 38054Grenoble, FranceFax: +33 4 38 78 54 87Tel: +33 4 38 78 94 05E-mail: vincent.forge@cea.fr*These authors contributed equally to this work(Received 7 August 2009, revised 6November2009,accepted23November2009)doi:10.1111/j.1742-4658.2009.07511.x

This study assessed the location of melatonin (N-acetyl-5-methoxytryptamine) and of a pinoline derivative (GWC22) [6-ethyl-1-(3-methoxyphenyl)-2-propyl-1,2,3,4-tetrahydro-beta-carboline], when present in lipid assemblies such as linoleate micelles, phosphatidylcholine liposomes or low density lipoproteins (LDL). The efficiency of radical scavenging by these compounds is highly dependent on their partitioning between the lipidic and aqueous phases. We determined the proportion of melatonin or GWC22 in the aqueous and lipid phases of each system (concentrations of the antioxidants ranging between 3 x 10(-5) and 10(-4) m) by assaying melatonin or GWC22 by HPLC/UV detection, or by fluorescence for melatonin in micelles. Our results show that melatonin and GWC22 were preferentially located in the aqueous phase of micelles (68.4% and 59.0%, respectively), whereas only 30.5% of melatonin and 39.0% of GWC22 were found in the lipid phase. By contrast, in phosphatidylcholine liposomes, both compounds were essentially present in the lipid phase (73.5% for melatonin and 79.1% for GWC22, versus 25.9% and 19.5% in the aqueous phase, respectively). In the case of LDL, 99.9% of the melatonin added was found in the methanol/water extracting phase containing phospholipids, unesterified cholesterol and apolipoprotein B100. The partitioning of melatonin and GWC22 in linoleate micelles gave new insights on the marked protective effect of GWC22 towards radiation-induced lipid peroxidation and allowed us to determine more accurately the lower limit values of the reaction rate constants of the two molecules studied with lipid peroxyl radicals, i.e. k(LOO.+melatonin)) >or= 9.0 x 10(4)m(-1)s(-1) and k(LOO.+GWC22) >or= 3.5 x 10(5)m(-1)s(-1).