Interaction of detergents with biological membranes : Comparison of fluorescence assays with filtration protocols and implications for the rates of detergent association, dissociation and flip-flop Article - 2019

Philippe Champeil, Béatrice Foresta, Martin Picard, Carole Gauron, Dominique Georgin, Marc Maire, Jesper V. Møller, Guillaume Lenoir, Cédric Montigny

Philippe Champeil, Béatrice Foresta, Martin Picard, Carole Gauron, Dominique Georgin, Marc Maire, Jesper V. Møller, Guillaume Lenoir, Cédric Montigny, « Interaction of detergents with biological membranes : Comparison of fluorescence assays with filtration protocols and implications for the rates of detergent association, dissociation and flip-flop  », PLoS ONE, 2019, e0222932. ISSN 1932-6203

Abstract

The present study mainly consists of a re-evaluation of the rate at which C12E8, a typical non-ionic detergent used for membrane studies, is able to dissociate from biological membranes, with sarcoplasmic reticulum membrane vesicles being used as an example. Utilizing a brominated derivative of C12E8 and now stopped-flow fluorescence instead of rapid filtration, we found that the rate of dissociation of this detergent from these membranes, merely perturbed with non-solubilizing concentrations of detergent, was significantly faster (t1/2 \textless 10 ms) than what had previously been determined (t1/2 300-400 ms) from experiments based on a rapid filtration protocol using 14C-labeled C12E8 and glass fiber filters (Binding of a non-ionic detergent to membranes : flip-flop rate and location on the bilayer, by Marc le Maire, Jesper Møller and Philippe Champeil, Biochemistry (1987) Vol 26, pages 4803-4810). We here pinpoint a methodological problem of the earlier rapid filtration experiments, and we suggest that the true overall dissociation rate of C12E8 is indeed much faster than previously thought. We also exemplify the case of brominated dodecyl-maltoside, whose kinetics for overall binding to and dissociation from membranes comprise both a rapid and a sower phase, the latter being presumably due to flip-flop between the two leaflets of the membrane. Consequently, equilibrium is reached only after a few seconds for DDM. This work thereby emphasizes the interest of using the fluorescence quenching associated with brominated detergents for studying the kinetics of detergent/membrane interactions, namely association, dissociation and flip-flop rates.

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