The Journal of Membrane Biology

, Volume 247, Issue 9–10, pp 909–924 | Cite as

Synthesis, Characterization and Applications of a Perdeuterated Amphipol

  • Fabrice Giusti
  • Jutta Rieger
  • Laurent J. Catoire
  • Shuo Qian
  • Antonio N. Calabrese
  • Thomas G. Watkinson
  • Marina Casiraghi
  • Sheena E. Radford
  • Alison E. Ashcroft
  • Jean-Luc PopotEmail author


Amphipols are short amphipathic polymers that can substitute for detergents at the hydrophobic surface of membrane proteins (MPs), keeping them soluble in the absence of detergents while stabilizing them. The most widely used amphipol, known as A8-35, is comprised of a polyacrylic acid (PAA) main chain grafted with octylamine and isopropylamine. Among its many applications, A8-35 has proven particularly useful for solution-state NMR studies of MPs, for which it can be desirable to eliminate signals originating from the protons of the surfactant. In the present work, we describe the synthesis and properties of perdeuterated A8-35 (perDAPol). Perdeuterated PAA was obtained by radical polymerization of deuterated acrylic acid. It was subsequently grafted with deuterated amines, yielding perDAPol. The number-average molar mass of hydrogenated and perDAPol, ~4 and ~5 kDa, respectively, was deduced from that of their PAA precursors, determined by size exclusion chromatography in tetrahydrofuran following permethylation. Electrospray ionization–ion mobility spectrometry–mass spectrometry measurements show the molar mass and distribution of the two APols to be very similar. Upon neutron scattering, the contrast match point of perDAPol is found to be ~120 % D2O. In 1H-1H nuclear overhauser effect NMR spectra, its contribution is reduced to ~6 % of that of hydrogenated A8-35, making it suitable for extended uses in NMR spectroscopy. PerDAPol ought to also be of use for inelastic neutron scattering studies of the dynamics of APol-trapped MPs, as well as small-angle neutron scattering and analytical ultracentrifugation.


Amphipol A8-35 Deuteration Mass spectrometry NMR 


1D, 2D, 3D

One-, two- and three-dimensional, respectively


Sodium poly(acrylate)-based amphipol with a weight-average molar mass close to 8 kDa and containing 35 % of free carboxylate


Sodium poly(acrylate)-based amphipol with a weight-average molar mass close to 8 kDa and containing 75 % of free carboxylate


Acrylic acid


Acrylic acid-d4






Analytical ultracentrifugation


Neutron scattering contrast match point


Transfer constant


A8-35 with perdeuterated side chains





\(\overline{{DP_{n} }}\)

Average degree of polymerization in number


Differential viscometry


Molar mass dispersity


Electrospray ionization


Hydrogenated A8-35


Heteronuclear single quantum correlation




Ion mobility spectrometry


Inelastic neutron scattering


Leukotriene B4

\(\overline{{M_{n} }}\)

Number-average molar mass

mQ water

Water purified on a A10 advantage millipore system


Mass spectrometry

\(\overline{{M_{w} }}\)

Weight-average molar mass


Non-ionic amphipol




Nuclear Overhauser effect


NOE spectroscopy


Poly(acrylic acid)


Perdeuterated A8-35


Poly(ethylene oxide)




Refractive index


Stokes radius


Small-angle neutron scattering


Sulfonated amphipol derived from A8-75


Size-exclusion chromatography


Transfer agent


Thioglycolic acid






Width at half-height



Particular thanks are due to Alain Fradet (UPMC - CNRS, IPCM) for his support and his comments on the manuscript, to Gaëlle Pembouong and Marion Chenal (same laboratory) for assistance with SEC analyses, to Christophe Tribet (Ecole Normale Supérieure, Paris) for his kind help at interpreting the results of the SEC experiments and to the Biotechnology and Biological Sciences Research Council of the UK for funding for the Synapt HDMS mass spectrometer (BB/E012558/1), ANC (BB/K000659/1) and TGW (BB/K501827/1). This work was supported by the French Centre National de la Recherche Scientifique (CNRS), by Université Paris-7 Denis Diderot, by grant “DYNAMO,” ANR-11-LABX-0011-01 from the French “Initiative d’Excellence” program, by the Office of Biological and Environmental Research, US Department of Energy (Bio-SANS, operated by ORNL’s Center for Structural Molecular Biology) and the Scientific User Facilities Division, Office of Basic Energy Sciences, US Department of Energy (ORNL’s High Flux Isotope Reactor).


  1. Banères J-L, Popot J-L, Mouillac B (2011) New advances in production and functional folding of G protein-coupled receptors. Trends Biotechnol 29:314–322CrossRefGoogle Scholar
  2. Bazzacco P, Billon-Denis E, Sharma KS, Catoire LJ, Mary S, Le Bon C, Point E, Banères J-L, Durand G, Zito F, Pucci B, Popot J-L (2012) Non-ionic homopolymeric amphipols: application to membrane protein folding, cell-free synthesis, and solution NMR. Biochemistry 51:1416–1430CrossRefGoogle Scholar
  3. Berry KD, Bailey KM, Beal J, Diawara Y, Funk L, Steve Hicks J, Jones AB, Littrell KC, Pingali SV, Summers PR, Urban VS, Vandergriff DH, Johnson NH, Bradley BJ (2012) Characterization of the neutron detector upgrade to the GP-SANS and Bio-SANS instruments at HFIR. Nucl Instr Meth Phys Res A 693:179–185CrossRefGoogle Scholar
  4. Catoire LJ, Zoonens M, van Heijenoort C, Giusti F, Popot J-L, Guittet E (2009) Inter- and intramolecular contacts in a membrane protein/surfactant complex observed by heteronuclear dipole-to-dipole cross-relaxation. J Magn Res 197:91–95CrossRefGoogle Scholar
  5. Catoire LJ, Damian M, Giusti F, Martin A, van Heijenoort C, Popot J-L, Guittet E, Banères J-L (2010a) Structure of a GPCR ligand in its receptor-bound state: leukotriene B4 adopts a highly constrained conformation when associated to human BLT2. J Am Chem Soc 132:9049–9057CrossRefGoogle Scholar
  6. Catoire LJ, Zoonens M, van Heijenoort C, Giusti F, Guittet E, Popot J-L (2010b) Solution NMR mapping of water-accessible residues in the transmembrane β-barrel of OmpX. Eur Biophys J 39:623–630CrossRefGoogle Scholar
  7. Catoire LJ, Damian M, Baaden M, Guittet E, Banères J-L (2011) Electrostatically-driven fast association and perdeuteration allow detection of transferred cross-relaxation for G protein-coupled receptor ligands with equilibrium dissociation constants in the high-to-low nanomolar range. J Biomol NMR 50:191–195CrossRefGoogle Scholar
  8. Catoire LJ, Warnet XL, Warschawski DE (2014) Micelles, bicelles, amphipols, nanodiscs, liposomes or intact cells: The hitch-hiker guide to membrane protein study by NMR. In: Mus-Veteau I (ed) Membrane protein production for structural analysis. Springer, Berlin (in press)Google Scholar
  9. Charvolin D, Perez J-B, Rouvière F, Giusti F, Bazzacco P, Abdine A, Rappaport F, Martinez KL, Popot J-L (2009) The use of amphipols as universal molecular adapters to immobilize membrane proteins onto solid supports. Proc Natl Acad Sci USA 106:405–410CrossRefGoogle Scholar
  10. Couvreur L, Lefay C, Belleney J, Charleux B, Guerret O, Magnet S (2003) First nitroxide-mediated controlled free-radical polymerization of acrylic acid. Macromolecules 36:8260–8267CrossRefGoogle Scholar
  11. Czerski L, Sanders CR (2000) Functionality of a membrane protein in bicelles. Anal Biochem 284:327–333CrossRefGoogle Scholar
  12. Dahmane T, Damian M, Mary S, Popot J-L, Banères J-L (2009) Amphipol-assisted in vitro folding of G protein-coupled receptors. Biochemistry 48:6516–6521CrossRefGoogle Scholar
  13. Dahmane T, Giusti F, Catoire LJ, Popot J-L (2011) Sulfonated amphipols: synthesis, properties and applications. Biopolymers 95:811–823CrossRefGoogle Scholar
  14. Diab C, Tribet C, Gohon Y, Popot J-L, Winnik FM (2007a) Complexation of integral membrane proteins by phosphorylcholine-based amphipols. Biochim Biophys Acta 1768:2737–2747CrossRefGoogle Scholar
  15. Diab C, Winnik FM, Tribet C (2007b) Enthalpy of interaction and binding isotherms of non-ionic surfactants onto micellar amphiphilic polymers (amphipols). Langmuir 23:3025–3035CrossRefGoogle Scholar
  16. Elter, S, Raschle, T, Arens, S, Gelev, V, Etzkorn, M, Wagner, G (2014). The use of amphipols for NMR structural characterization of 7-TM proteins (submitted for publication)CrossRefGoogle Scholar
  17. Etzkorn M, Raschle T, Hagn F, Gelev V, Rice AJ, Walz T, Wagner G (2013) Cell-free expressed bacteriorhodopsin in different soluble membrane mimetics: biophysical properties and NMR accessibility. Structure 21:394–401CrossRefGoogle Scholar
  18. Etzkorn, M, Zoonens, M, Catoire, LJ, Popot, J-L, Hiller, S (2014). How amphipols embed membrane proteins: Global solvent accessibility and interaction with a flexible protein terminus. J Membr Biol (in press)Google Scholar
  19. Fernandez, A, Le Bon, C, Baumlin, N, Giusti, F, Crémel, G, Popot, J-L, Bagnard, D (2014) In vivo characterization of the biodistribution profile of amphipols (submitted for publication)Google Scholar
  20. Giusti F, Popot J-L, Tribet C (2012) Well-defined critical association concentration and rapid adsorption at the air/water interface of a short amphiphilic polymer, amphipol A8-35: a study by Förster resonance energy transfer and dynamic surface tension measurements. Langmuir 28:10372–10380CrossRefGoogle Scholar
  21. Giusti, F, Kessler, P, Westh Hansen, R, Della Pia, EA, Le Bon, C, Mourier, G, Popot, J-L, Martinez, KL, Zoonens, M (2014). Synthesis of polyhistidine- or imidazole-bearing amphipols and their use for immobilized metal affinity chromatography and surface plasmon resonance studies of membrane proteins (in preparation)Google Scholar
  22. Glück JM, Wittlich M, Feuerstein S, Hoffmann S, Willbold D, Koenig BW (2009) Integral membrane proteins in nanodiscs can be studied by solution NMR spectroscopy. J Am Chem Soc 131:12060–12061CrossRefGoogle Scholar
  23. Gohon Y, Pavlov G, Timmins P, Tribet C, Popot J-L, Ebel C (2004) Partial specific volume and solvent interactions of amphipol A8-35. Anal Biochem 334:318–334CrossRefGoogle Scholar
  24. Gohon Y, Giusti F, Prata C, Charvolin D, Timmins P, Ebel C, Tribet C, Popot J-L (2006) Well-defined nanoparticles formed by hydrophobic assembly of a short and polydisperse random terpolymer, amphipol A8-35. Langmuir 22:1281–1290CrossRefGoogle Scholar
  25. Gohon Y, Dahmane T, Ruigrok R, Schuck P, Charvolin D, Rappaport F, Timmins P, Engelman DM, Tribet C, Popot J-L, Ebel C (2008) Bacteriorhodopsin/amphipol complexes: structural and functional properties. Biophys J 94:3523–3537CrossRefGoogle Scholar
  26. Guinier A, Fournet G (1955) Small-angle scattering of X-rays. John Wiley and sons, New YorkGoogle Scholar
  27. Hagn F, Etzkorn M, Raschle T, Wagner G (2013) Optimized phospholipid bilayer nanodiscs facilitate high-resolution structure determination of membrane proteins. J Am Chem Soc 135:1919–1925CrossRefGoogle Scholar
  28. Hamberg M, Svensson J, Samuelsson B (1974) Prostaglandin endoperoxides. A new concept concerning the mode of action and release of prostaglandins. Proc Natl Acad Sci USA 71:3824–3828CrossRefGoogle Scholar
  29. Hernandez H, Robinson CV (2007) Determining the stoichiometry and interactions of macromolecular assemblies from mass spectrometry. Nat Protocols 2:715–726CrossRefGoogle Scholar
  30. Huynh, KW, Cohen, MR, Moiseenkova-Bell, VY (2014). Application of amphipols for structure-functional analysis of TRP channels (submitted for publication)CrossRefGoogle Scholar
  31. Kang CB, Li Q (2011) Solution NMR study of integral membrane proteins. Curr Opin Struct Biol 15:560–569CrossRefGoogle Scholar
  32. Kanu AB, Dwivedi P, Tam M, Matz L, Hill HH Jr (2008) Ion mobility: mass spectrometry. J Mass Spectrom 43:1–22CrossRefGoogle Scholar
  33. Kay LE, Ikura M, Tschudin R, Bax A (1990) Three-dimensional triple resonance NMR spectroscopy of isotopically enriched proteins. J Magn Reson 89:496–514Google Scholar
  34. Kline SR (2006) Reduction and analysis of SANS and USANS data using IGOR Pro. J Appl Crystall 39:895–900CrossRefGoogle Scholar
  35. Le Bon, C, Della Pia, EA, Giusti, F, Lloret, N, Zoonens, M, Martinez, KL, Popot, J-L (2014a). Synthesis of an oligonucleotide-derivatized amphipol and its use to trap and immobilize membrane proteins. Nucleic Acids Res (in press)Google Scholar
  36. Le Bon, C, Popot, J-L, Giusti, F (2014b). Labeling and functionalizing amphipols for biological applications. J Membr Biol (in press)Google Scholar
  37. Leney AC, McMorran LM, Radford SE, Ashcroft AE (2012) Amphipathic polymers enable the study of functional membrane proteins in the gas phase. Anal Chem 84:9841–9847CrossRefGoogle Scholar
  38. Loubat C, Javidan A, Boutevin B (2000) Etude de la télomérisation de l’acide acrylique par les mercaptans. 1. Télomérisation de l’acide acrylique par l’acide thioglycolique. Influence de la nature du solvant sur la valeur de la constante de transfert et de k p/√k te. Macromol Chem Phys 201:2845–2852CrossRefGoogle Scholar
  39. Lynn GW, Heller W, Urban V, Wignall GD, Weiss K, Myles DAA (2006) A dedicated facility for neutron structural biology at Oak Ridge National Laboratory. Physica B 385–386:880–882CrossRefGoogle Scholar
  40. Mazhab-Jafari MT, Marshall CB, Stathopulos PB, Kobashigawa Y, Stambolic V, Kay LE, Inagaki F, Ikura M (2013) Membrane-dependent modulation of the mTOR activator Rheb: NMR observations of a GTPase tethered to a lipid-bilayer nanodisc. J Am Chem Soc 135:3367–3370CrossRefGoogle Scholar
  41. McGregor C-L, Chen L, Pomroy NC, Hwang P, Go S, Chakrabartty A, Privé GG (2003) Lipopeptide detergents designed for the structural study of membrane proteins. Nat Biotechnol 21:171–176CrossRefGoogle Scholar
  42. Opačić, M, Giusti, F, Broos, J, Popot, J-L (2014). Isolation of Escherichia coli mannitol permease, EIImtl, trapped in amphipol A8-35 (submitted for publication)Google Scholar
  43. Pavia AA, Pucci B, Riess JG, Zarrif L (1992) New perfluoroalkyl telomeric non-ionic surfactants: synthesis, physicochemical and biological properties. Makromol Chem 193:2505–2517CrossRefGoogle Scholar
  44. Perlmutter JD, Drasler WJ, Xie W, Gao J, Popot J-L, Sachs JN (2011) All-atom and coarse-grained molecular dynamics simulations of a membrane protein stabilizing polymer. Langmuir 27:10523–10537CrossRefGoogle Scholar
  45. Perlmutter, JD, Popot, J-L, Sachs, JN (2014). Molecular dynamics simulations of a membrane protein/amphipol complex (submitted for publication)Google Scholar
  46. Picard M, Dahmane T, Garrigos M, Gauron C, Giusti F, le Maire M, Popot J-L, Champeil P (2006) Protective and inhibitory effects of various types of amphipols on the Ca2+-ATPase from sarcoplasmic reticulum: a comparative study. Biochemistry 45:1861–1869CrossRefGoogle Scholar
  47. Planchard, N, Point, E, Dahmane, T, Giusti, F, Renault, M, Le Bon, C, Durand, G, Milon, A, Guittet, E, Zoonens, M, Popot, J-L, Catoire, LJ (2014). The use of amphipols for solution NMR studies of membrane proteins: advantages and limitations as compared to other solubilizing media. J Membr Biol (in press)Google Scholar
  48. Plevin, MJ, Boisbouvier J (2012). Isotope-labelling of methyl groups for NMR studies of large proteins. In M Clore and J Potts (eds) Recent developments in biomolecular NMR, Royal Society of Chemistry pp 1–24Google Scholar
  49. Pocanschi C, Popot J-L, Kleinschmidt JH (2013) Folding and stability of outer membrane protein A (OmpA) from Escherichia coli in an amphipathic polymer, amphipol A8-35. Eur Biophys J 42:103–118CrossRefGoogle Scholar
  50. Popot J-L (2010) Amphipols, nanodiscs, and fluorinated surfactants: three non-conventional approaches to studying membrane proteins in aqueous solutions. Annu Rev Biochem 79:737–775CrossRefGoogle Scholar
  51. Popot J-L, Althoff T, Bagnard D, Banères J-L, Bazzacco P, Billon-Denis E, Catoire LJ, Champeil P, Charvolin D, Cocco MJ, Crémel G, Dahmane T, de la Maza LM, Ebel C, Gabel F, Giusti F, Gohon Y, Goormaghtigh E, Guittet E, Kleinschmidt JH, Kühlbrandt W, Le Bon C, Martinez KL, Picard M, Pucci B, Rappaport F, Sachs JN, Tribet C, van Heijenoort C, Wien F, Zito F, Zoonens M (2011) Amphipols from A to Z. Annu Rev Biophys 40:379–408CrossRefGoogle Scholar
  52. Presser A, Hüfner A (2004) Trimethylsilyldiazomethane. A mild and efficient reagent for the methylation of carboxylic acids and alcohols in natural products. Monatsch Chem 135:1015–1022CrossRefGoogle Scholar
  53. Privé G (2009) Lipopeptide detergents for membrane protein studies. Curr Opin Struct Biol 19:1–7CrossRefGoogle Scholar
  54. Raschle T, Hiller S, Yu TY, Rice AJ, Walz T, Wagner G (2009) Structural and functional characterization of the integral membrane protein VDAC-1 in lipid bilayer nanodiscs. J Am Chem Soc 131:17777–17779CrossRefGoogle Scholar
  55. Raschle T, Hiller S, Etzkorn M, Wagner G (2010) Nonmicellar systems for solution NMR spectroscopy of membrane proteins. Curr Opin Struct Biol 20:471–479CrossRefGoogle Scholar
  56. Renault M (2008) Etudes structurales et dynamiques de la protéine membranaire KpOmpA par RMN en phase liquide et solide, Ph. D. Thesis, Université Paul Sabatier, ToulouseGoogle Scholar
  57. Salzmann M, Wider G, Pervushin K, Wüthrich K (1999) Improved sensitivity and coherence selection for [15N,1H]-TROSY elements in triple resonance experiments. J Biomol NMR 15:181–184CrossRefGoogle Scholar
  58. Sanders CR II, Landis GC (1995) Reconstitution of membrane proteins into lipid-rich bilayered mixed micelles for NMR studies. Biochemistry 34:4030–4040CrossRefGoogle Scholar
  59. Sanders CR, Prosser RS (1998) Bicelles: a model membrane system for all seasons? Structure 6:1227–1234CrossRefGoogle Scholar
  60. Shenkarev ZO, Lyukmanova EN, Paramonov AS, Shingarova LN, Chupin VV, Kirpich-ni-kov MP, Blommers MJ, Arseniev AS (2010) Lipid-protein nanodiscs as reference medium in detergent screening for high-resolution NMR studies of integral membrane proteins. J Am Chem Soc 132:5628–5629CrossRefGoogle Scholar
  61. Sprangers R, Velyvis A, Kay LE (2007) Solution NMR of supramolecular complexes: providing new insights into function. Nat Meth 4:697–703CrossRefGoogle Scholar
  62. Sverzhinsky, A, Qian, S, Yang, L, Allaire, M, Moraes, I, Ma, D, Chung, JW, Zoonens, M, Popot, J-L, Coulton, JW (2014). Amphipol-trapped ExbB—ExbD membrane protein complex from Escherichia coli: A biochemical and structural case study (submitted for publication)Google Scholar
  63. Tehei, M, Giusti, F, Popot, J-L, Zaccai, G (2014). Thermal fluctuations in amphipol A8-35 measured by neutron scattering (submitted for publication)Google Scholar
  64. Tribet C, Audebert R, Popot J-L (1996) Amphipols: polymers that keep membrane proteins soluble in aqueous solutions. Proc Natl Acad Sci USA 93:15047–15050CrossRefGoogle Scholar
  65. Tribet C, Diab C, Dahmane T, Zoonens M, Popot J-L, Winnik FM (2009) Thermodynamic characterization of the exchange of detergents and amphipols at the surfaces of integral membrane proteins. Langmuir 25:12623–12634CrossRefGoogle Scholar
  66. Tzitzilonis C, Eichmann C, Maslennikov I, Choe S, Riek R (2013) Detergent/nanodisc screening for high-resolution NMR studies of an integral membrane protein containing a cytoplasmic domain. PLoS ONE 8:e54378CrossRefGoogle Scholar
  67. Velasquez E, Pembouong G, Rieger J, Stoffelbach F, Boyron O, Charleux B, D’Agosto F, Lansalot M, Dufils P-E, Vinas J (2013) Poly(vinylidene chloride)-based amphiphilic block copolymers. Macromolecules 46:664–673CrossRefGoogle Scholar
  68. Warschawski DE, Arnold AA, Beaugrand M, Gravel A, Chartrand E, Marcotte I (2011) Choosing membrane mimetics for NMR structural studies of transmembrane proteins. Biochim Biophys Acta 1808:1957–1974CrossRefGoogle Scholar
  69. Weidner SM, Trimpin S (2010) Mass spectrometry of synthetic polymers. Anal Chem 82:4811–4829CrossRefGoogle Scholar
  70. Yokomizo T, Kato K, Terawaki K, Izumi T, Shimizu T (2000) A second leukotriene B(4) receptor, BLT2. A new therapeutic target in inflammation and immunological disorders. J Exp Med 192:421–432CrossRefGoogle Scholar
  71. Zaccai G, Jacrot B (1983) Small-angle neutron scattering. Annu Rev Biophys Bioeng 12:139–157CrossRefGoogle Scholar
  72. Zoonens, M, Popot, J-L (2014). Amphipols for each season (submitted for publication)Google Scholar
  73. Zoonens M, Catoire LJ, Giusti F, Popot J-L (2005) NMR study of a membrane protein in detergent-free aqueous solution. Proc Natl Acad Sci USA 102:8893–8898CrossRefGoogle Scholar
  74. Zoonens M, Giusti F, Zito F, Popot J-L (2007) Dynamics of membrane protein/amphipol association studied by Förster resonance energy transfer. Implications for in vitro studies of amphipol-stabilized membrane proteins. Biochemistry 46:10392–10404CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Fabrice Giusti
    • 1
  • Jutta Rieger
    • 2
    • 3
  • Laurent J. Catoire
    • 1
  • Shuo Qian
    • 4
  • Antonio N. Calabrese
    • 5
  • Thomas G. Watkinson
    • 5
  • Marina Casiraghi
    • 1
  • Sheena E. Radford
    • 5
  • Alison E. Ashcroft
    • 5
  • Jean-Luc Popot
    • 1
    Email author
  1. 1.Laboratoire de Physico-Chimie Moléculaire des Membranes Biologiques, UMR 7099, Institut de Biologie Physico-Chimique (FRC 550)Centre National de la Recherche Scientifique and Université Paris-7ParisFrance
  2. 2.UMR 8232, Institut Parisien de Chimie Moléculaire (IPCM), Equipe Chimie des PolymèresSorbonne Universités, UPMC Univ Paris 06ParisFrance
  3. 3.UMR 8232, Institut Parisien de Chimie Moléculaire (IPCM), Equipe Chimie des PolymèresCNRSParisFrance
  4. 4.Center for Structural Molecular Biology and Biology and Soft Matter DivisionOak Ridge National LaboratoryOak RidgeUSA
  5. 5.Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular BiologyUniversity of LeedsLeedsUK

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