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Interfaces électrochimiques appliquées à l'étude de composés d'intérêt biologique : application à l'étude de l'interaction entre cytochrome c et cardiolipide

Abstract : The main goal of this thesis was to set up an electrochemical interface in order to characterize interactions between cytochrome c (cyt c), a mitochondrial protein, and cardiolipin (CL), a phospholipid localized to the mitochondrial membrane.The cyt c, whereas the main function is to carrying electrons along the respiratory chain, is known to interact with CL.Previously, a mechanism of cyt c retention onto a CL containing membrane was highlighted by the “extended lipid anchorage” theory. This theory imply, with electrostatic interactions between CL (negatively charged) and cyt c (positively charged), the presence of hydrophobic interaction likely emanating from the insertion of an acyl chain of CL into the cyt c interior. The specificity of the hydrophobic moiety of CL, constituted of four similar chains lead us to make hypothesis about specific interactions between cyt c and acyl chains of CL. Attention have been made to the bivalves CL acyl chains carrying unique composition of 8O% DHA chains.Electrochemicals techniques have been used to study these interactions. A glassy carbon has been modified with a deposit by spin-coating of phosphatidylcholine (PC) and CL in 80/20 ratio (mol/mol). This CL modified electrode allows the study of the cyt c electrochemistry. Firstly, we showed that electroactivity of cyt c require the presence of CL onto the modified electrode meaning cyt c isn't electroactive on a modified electrode with PC only. ln add, CL allows retaining cyt c onto the electrode surface. lt is the first time that this lipid anchorage has been identified on a modified electrode.Electrochemical methods adapted to the study of adsorbed protein (AC voltametry) have been used in order to characterize the electrode transfer kinetics of cyt c. With these methods, two sub-populations of adsorbed cyt c have been characterized. The major part of cyt c, called sub-population 1, has a redox potential close to the formal potential of the native protein (around 0Vvs SCE). Electron transfer rate is in the range of 20s-1. The subpopulation 2, counting for ~10% of the total adsorbed cyt c, hold a negative shifted potential around -0.15V vs SCE and a faster electron transfer rate (~500s-1).To understand the nature of the interaction for the two subpopulations, the effect of an high ionic strength solution (0.5M KCI), pH, calcium, phospholipids classes or cyt c sources have been assayed. These tests shows that subpopulation t have electrostatics interactions and require the presence of a phospholipid holding a terminal glycerol pattern like PG or CL.The nature of the protein-lipid interaction in the case of the subpopulation 2 is more complex. lt is more sensitive to the presence of divalent cation or pH but high ionic strength solution doesn't affect it. This could be explained by hydrophobic interactions between lipid and the cyt c.The assays realized with cyt c from yeast let foresee specificity between these protein and the phospholipids carried by the same organism. The purification of bivalve cyt c could be a progress in order to validate this hypothesis.
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Antoine Perhirin. Interfaces électrochimiques appliquées à l'étude de composés d'intérêt biologique : application à l'étude de l'interaction entre cytochrome c et cardiolipide. Chimie analytique. Université de Bretagne occidentale - Brest, 2012. Français. ⟨NNT : 2012BRES0094⟩. ⟨tel-01636044⟩

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