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

Autoři: Philippe Champeil aff001;  Béatrice de Foresta aff001;  Martin Picard aff001;  Carole Gauron aff001;  Dominique Georgin aff002;  Marc le Maire aff001;  Jesper V. Møller aff003;  Guillaume Lenoir aff001;  Cédric Montigny aff001
Působiště autorů: Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, France aff001;  CEA, DSV, Institut des Sciences du Vivant Frédéric Joliot, Service de Chimie Biorganique et de Marquage, Gif-sur-Yvette, France aff002;  Center for Membrane Pumps in Cells and Disease, PUMPKIN, Danish National Research Foundation, Aarhus University, Aarhus, Denmark aff003;  Department of Biomedicine, Aarhus University, Aarhus, Denmark aff004
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article


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 < 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.

Klíčová slova:

Detergents – Equipment – Lipids – Membrane electrophysiology – Vesicles – Chemical dissociation – Filtration – Light scattering


1. Jorgensen PL, Skou JC. Purification and characterization of (Na+ + K+)-ATPase. I. The influence of detergents on the activity of (Na+ + K+)-ATPase in preparations from the outer medulla of rabbit kidney. Biochim Biophys Acta. 1971;233: 366–80. doi: 10.1016/0005-2736(71)90334-8 4254310

2. Rizzolo LJ, Tanford C. Behavior of fragmented calcium (II) adenosine triphosphatase from sarcoplasmic reticulum in detergent solution. Biochemistry. 1978;17: 4049–4055. doi: 10.1021/bi00612a028 152120

3. Paternostre MT, Roux M, Rigaud JL. Mechanisms of membrane protein insertion into liposomes during reconstitution procedures involving the use of detergents. 1. Solubilization of large unilamellar liposomes (prepared by reverse-phase evaporation) by triton X-100, octyl glucoside, and sodium cholate. Biochemistry. 1988;27: 2668–77. doi: 10.1021/bi00408a006 2840945

4. Andersen JP, le Maire M, Kragh-Hansen U, Champeil P, Møller JV. Perturbation of the Structure and Function of a Membranous Ca2+ -ATPase by Non-solubilizing Concentrations of a Non-ionic Detergent. European Journal of Biochemistry. 1983;134: 205–214. doi: 10.1111/j.1432-1033.1983.tb07552.x 6307686

5. Andersen JP, le Maire M, Møller JV. Properties of detergent-solubilized and membranous (Ca2+ + Mg2+)-activated ATPase from sarcoplasmic reticulum as studied by sulfhydryl reactivity and ESR spectroscopy. Effect of protein-protein interactions. Biochim Biophys Acta. 1980;603: 84–100. doi: 10.1016/0005-2736(80)90393-4 6108784

6. Roux M, Champeil P. 31P NMR as a tool for monitoring detergent solubilization of sarcoplasmic reticulum membranes. FEBS Lett. 1984;171: 169–172. doi: 10.1016/0014-5793(84)80481-0 6723980

7. Ueno M, Tanford C, Reynolds JA. Phospholipid vesicle formation using nonionic detergents with low monomer solubility. Kinetic factors determine vesicle size and permeability. Biochemistry. 1984;23: 3070–3076. doi: 10.1021/bi00308a034 6466632

8. le Maire M, Møller JV, Champeil P. Binding of a nonionic detergent to membranes: flip-flop rate and location on the bilayer. Biochemistry. 1987;26: 4803–4810. doi: 10.1021/bi00389a030 3663628

9. de Foresta B, Legros N, Plusquellec D, Maire M, Champeil P. Brominated Detergents as Tools to Study Protein-Detergent Interactions. European Journal of Biochemistry. 1996;241: 343–354. doi: 10.1111/j.1432-1033.1996.00343.x 8917429

10. Powl AM, East JM, Lee AG. Lipid-protein interactions studied by introduction of a tryptophan residue: the mechanosensitive channel MscL. Biochemistry. 2003;42: 14306–17. doi: 10.1021/bi034995k 14640699

11. Marius P, Zagnoni M, Sandison ME, East JM, Morgan H, Lee AG. Binding of anionic lipids to at least three nonannular sites on the potassium channel KcsA is required for channel opening. Biophys J. 2008;94: 1689–1698. doi: 10.1529/biophysj.107.117507 18024500

12. East JM, Lee AG. Lipid selectivity of the calcum and magnesium ion dependent adenosinetriphosphatase, studied with fluorescence quenching by a brominated phospholipid. Biochemistry. 1982;21: 4144–4151. doi: 10.1021/bi00260a035 6127102

13. de Foresta B, le Maire M, Orlowski S, Champeil P, Lund S, Moller JV, et al. Membrane solubilization by detergent: use of brominated phospholipids to evaluate the detergent-induced changes in Ca2+-ATPase/lipid interaction. Biochemistry. 1989;28: 2558–67. doi: 10.1021/bi00432a032 2525049

14. Norimatsu Y, Hasegawa K, Shimizu N, Toyoshima C. Protein-phospholipid interplay revealed with crystals of a calcium pump. Nature. 2017;545: 193–198. doi: 10.1038/nature22357 28467821

15. de Foresta B, Champeil P, le Maire M. Different classes of tryptophan residues involved in the conformational changes characteristic of the sarcoplasmic reticulum Ca2+-ATPase cycle. European Journal of Biochemistry. 1990;194: 383–388. doi: 10.1111/j.1432-1033.1990.tb15631.x 2148514

16. Georgin D, le Maire M, Noël JP. Syntheses of [14C]-detergents: octaethylene-glycol-[1-14C]-dodecylether, [1-14C]-dodecyl-β-D-maltoside and dibromo-analogues. Journal of Labelled Compounds and Radiopharmaceuticals. 2001;44: 575–585. doi: 10.1002/jlcr.485

17. Brown E, Hayes T. The Absorptiometric Determination of Polyethyleneglycol Mono-Oleate. Analyst. 1955;80: 755–767. doi: 10.1039/an9558000755

18. Garewal HS. A procedure for the estimation of microgram quantities of triton X-100. Anal Biochem. 1973;54: 319–324. doi: 10.1016/0003-2697(73)90359-x 4724542

19. Goldstein S, Blecher M. The spectrophotometric assay for polyethoxy nonionic detergents in membrane extracts: a critique. Anal Biochem. 1975;64: 130–135. doi: 10.1016/0003-2697(75)90414-5 1169888

20. Montigny C, Dieudonné T, Orlowski S, Vázquez-Ibar JL, Gauron C, Georgin D, et al. Slow Phospholipid Exchange between a Detergent-Solubilized Membrane Protein and Lipid-Detergent Mixed Micelles: Brominated Phospholipids as Tools to Follow Its Kinetics. PLoS ONE. 2017;12: e0170481. doi: 10.1371/journal.pone.0170481 28118404

21. Møller JV, Olesen C. Preparation of Ca(2+)-ATPase1a Enzyme from Rabbit Sarcoplasmic Reticulum. Methods Mol Biol. 2016;1377: 11–17. doi: 10.1007/978-1-4939-3179-8_3 26695018

22. Benzonana G. Study of bile salts micelles: properties of mixed oleate-deoxycholate solutions at pH 9.0. Biochim Biophys Acta. 1969;176: 836–848. doi: 10.1016/0005-2760(69)90265-3 5797094

23. Lenoir G, Dieudonné T, Lamy A, Lejeune M, Vazquez-Ibar J-L, Montigny C. Screening of Detergents for Stabilization of Functional Membrane Proteins. Curr Protoc Protein Sci. 2018;93: e59. doi: 10.1002/cpps.59 30021058

24. Paternostre MT, Roux M, Rigaud JL. Mechanisms of membrane protein insertion into liposomes during reconstitution procedures involving the use of detergents. 1. Solubilization of large unilamellar liposomes (prepared by reverse-phase evaporation) by triton X-100, octyl glucoside, and sodium cholate. Biochemistry. 1988;27: 2668–2677. doi: 10.1021/bi00408a006 2840945

25. Champeil P, Henao F, de Foresta B. Dissociation of Ca2+ from Sarcoplasmic Reticulum Ca2+-ATPase and Changes in Fluorescence of Optically Selected Trp Residues. Effects of KCl and NaCl and Implications for Substeps in Ca2+ Dissociation. Biochemistry. 1997;36: 12383–12393. doi: 10.1021/bi9709699 9315879

26. le Maire M, Champeil P, Moller JV. Interaction of membrane proteins and lipids with solubilizing detergents. Biochim Biophys Acta. 2000;1508: 86–111. doi: 10.1016/s0304-4157(00)00010-1 11090820

27. Lichtenberg D. Characterization of the solubilization of lipid bilayers by surfactants. Biochim Biophys Acta. 1985;821: 470–8. doi: 10.1016/0005-2736(85)90052-5 4074739

28. de Foresta B, Merah Z, le Maire M, Champeil P. How to evaluate the distribution of an “invisible” amphiphile between biological membranes and water. Anal Biochem. 1990;189: 59–67. doi: 10.1016/0003-2697(90)90044-a 2278392

29. de Foresta B, Henao F, Champeil P. Kinetic characterization of the perturbation by dodecylmaltoside of sarcoplasmic reticulum Ca(2+)-ATPase. Eur J Biochem. 1992;209: 1023–1034. 1425684

30. Kragh-Hansen U, le Maire M, Møller JV. The mechanism of detergent solubilization of liposomes and protein-containing membranes. Biophys J. 1998;75: 2932–2946. doi: 10.1016/S0006-3495(98)77735-5 9826614

31. Fang F, Satulovsky J, Szleifer I. Kinetics of protein adsorption and desorption on surfaces with grafted polymers. Biophys J. 2005;89: 1516–1533. doi: 10.1529/biophysj.104.055079 15994887

32. Rice DM, Meadows MD, Scheinman AO, Goñi FM, Gómez-Fernández JC, Moscarello MA, et al. Protein-lipid interactions. A nuclear magnetic resonance study of sarcoplasmic reticulum Ca2,Mg2+-ATPase, lipophilin, and proteolipid apoprotein-lecithin systems and a comparison with the effects of cholesterol. Biochemistry. 1979;18: 5893–5903. 160247

33. Paddy MR, Dahlquist FW, Davis JH, Bloom M. Dynamical and temperature-dependent effects of lipid-protein interactions. Application of deuterium nuclear magnetic resonance and electron paramagnetic resonance spectroscopy to the same reconstitutions of cytochrome c oxidase. Biochemistry. 1981;20: 3152–3162. doi: 10.1021/bi00514a026 6264951

34. Orlowski S, Champeil P. Kinetics of calcium dissociation from its high-affinity transport sites on sarcoplasmic reticulum ATPase. Biochemistry. Jan 15 1991a;30: 352–61.

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