Advertisement

Colloid and Polymer Science

, Volume 291, Issue 7, pp 1759–1769 | Cite as

Polyelectrolyte–protein interaction at low ionic strength: required chain flexibility depending on protein average charge

  • Florian CapitoEmail author
  • Romas Skudas
  • Bernd Stanislawski
  • Harald Kolmar
Original Contribution

Abstract

The effect of low ionic strength leading to reduced polyelectrolyte–protein interactions has been shown by in silico and in vitro experiments, suggesting polyelectrolyte rigidity increasing at low ionic strength, thus leading to reduced interactions with proteins. This contribution elucidates polyelectrolyte–protein precipitation in the 0–2.6-mS cm−1 ionic strength regime with polyelectrolyte rigidity determinations, using viscosimetry at these conditions, also considering protein charge distributions, using different proteins. Precipitation yields increased from 5 to 40 % at low ionic strength to up to 90 % at intermediate ionic strength, depending on protein and polyelectrolyte type, using lysozyme and three different monoclonal antibodies. Comparing precipitation behavior of the monoclonal antibodies, a qualitative correlation between required polyelectrolyte flexibility to enhance protein precipitation and protein average charge as well as hydrophobicity of the antibodies was discovered. Antibodies with lower average charge and less hydrophobicity required more flexible polyelectrolytes to enhance precipitation behavior by allowing interaction of the polyelectrolytes with proteins, attaching to positively charged protein patches while “circumnavigating” negatively charged protein areas. In contrast, antibodies with higher protein average charge showed increasing precipitation yields up to 90 % already at lower ionic strength, associated with then more rigid polyelectrolyte structures. Therefore, designing polyelectrolytes with specific chain flexibility could help to improve precipitation behavior toward specific target proteins in polyelectrolyte-driven purification techniques.

Keywords

Polyelectrolyte flexibility Proteins Structure–property relations Viscosity 

Notes

Acknowledgments

The authors are grateful to Merck KGaA for financial and technical support for this project. Thanks to Merck Millipore for antibody supply. Thanks to Mikhail Kozlov, Merck Millipore, and Johann Bauer, Merck KGaA, for helpful advice on this project, and Alexandra Hill and Simon Geissler, both Merck KGaA, for providing the rheometer.

References

  1. 1.
    Shahidi F, Synowiecki J (1991) Isolation and characterization of nutrients and value-added products from snow crab (Chionoecetes opilio) and shrimp (Pandalus borealis) processing discards. J Agric Food Chem 39:1527–1532. doi: 10.1021/jf00008a032 CrossRefGoogle Scholar
  2. 2.
    Sudharshan NR, Hoover DG, Knorr D (1992) Antibacterial action of chitosan. Food Biotechnol 6:257–272. doi: 10.1080/08905439209549838 CrossRefGoogle Scholar
  3. 3.
    Papineau AM, Hoover DG, Knorr D, Farkas DF (1991) Antimicrobial effect of water-soluble chitosans with high hydrostatic pressure. Food Biotechnol 5:45–57. doi: 10.1080/08905439109549790 CrossRefGoogle Scholar
  4. 4.
    Gross RA, Kalra B (2002) Biodegradable polymers for the environment. Science 297:803–807. doi: 10.1126/science.297.5582.803 CrossRefGoogle Scholar
  5. 5.
    Muzzarelli RAA, Weckx M, Fillipini O, Lough C (1989) Characteristic properties of N-carboxybutyl chitosan. Carbohydr Poly 11:293–296. doi: 10.1016/0144-8617(89)90005-2 CrossRefGoogle Scholar
  6. 6.
    Bolto B, Gregory J (2007) Organic polyelectrolytes in water treatment. Water Research 41:2301–2324. doi: 10.1016/j.watres.2007.03.012 CrossRefGoogle Scholar
  7. 7.
    Allen TM, Cullis PR (2004) Drug delivery systems: entering the mainstream. Science 303:1818–1822. doi: 10.1126/science.1095833 CrossRefGoogle Scholar
  8. 8.
    Jeong B, Bae YH, Lee DS, Kim SW (1997) Biodegradable block copolymers as injectable drug-delivery systems. Nature 388:860–862. doi: 10.1038/42218 CrossRefGoogle Scholar
  9. 9.
    Schmaljohann D (2006) Thermo- and pH-responsive polymers in drug delivery. Adv Drug Delivery Rev 58:1655–1670. doi: 10.1016/j.addr.2006.09.020 CrossRefGoogle Scholar
  10. 10.
    Gillies ER, Fréchet JM (2005) Dendrimers and dendritic polymers in drug delivery. J Drug Discovery Today 10:35–43. doi: 10.1016/S1359-6446(04)03276-3 CrossRefGoogle Scholar
  11. 11.
    Svec F, Fréchet JM (1992) Continuous rods of macroporous polymer as high-performance liquid chromatography separation media. J Anal Chem 64:820–822. doi: 10.1021/ac00031a022 CrossRefGoogle Scholar
  12. 12.
    Šmigol V, Švec F, Hosoya K, Wang Q, Fréchet JM (1992) Monodisperse polymer beads as packing material for high-performance liquid chromatography. Synthesis and properties of monodisperse polystyrene and poly(methacrylate) latex seeds. Die Angewandte Makromolekulare Chemie 195:151–164. doi: 10.1002/apmc.1992.051950112 CrossRefGoogle Scholar
  13. 13.
    Barrande M, Beurroies I, Denoyel R, Tatárová I, Gramblička M, Polakovič M, Joehnck M, Schulte M (2009) Characterisation of porous materials for bioseparation. J Chromatogr A 1216:6906–6916. doi: 10.1016/j.chroma.2009.07.075 CrossRefGoogle Scholar
  14. 14.
    Kamath N, D'Souza SF (1991) Immobilization of ureolytic cells through flocculation and adhesion on cotton cloth using polyethylenimine. Enzyme Microb Technol 13:935–938. doi: 10.1016/0141-0229(91)90112-N CrossRefGoogle Scholar
  15. 15.
    Ovenden C, Xiao H (2002) Flocculation behaviour and mechanisms of cationic inorganic microparticle/polymer systems. Colloids and Surfaces A 197:225–234. doi: 10.1016/S0927-7757(01)00903-7 CrossRefGoogle Scholar
  16. 16.
    Hönig W, Kula RM (1976) Selectivity of protein precipitation with polyethylene glycol fractions of various molecular weights. Anal Biochem 72:502–512. doi: 10.1016/0003-2697(76)90560-1 CrossRefGoogle Scholar
  17. 17.
    Gervais DP, Pfeiffer KA (2010) (Pfizer Limited) US patent 20100204455, August 12Google Scholar
  18. 18.
    Fahrner R, Franklin J, McDonald P, Peram T, Sisodiya V, Victa C (2008) (Genentech, Inc.) International patent WO/2008/091,740, January 10Google Scholar
  19. 19.
    Gronke RS, Jaquez OA (2009) (Biogen Idec MA Inc.) US Patent 12/425,328, April 16Google Scholar
  20. 20.
    Ramanan S, Stenson R (2008) (Amgen Inc.) International patent WO/2008/100,578, August 21Google Scholar
  21. 21.
    McDonald P, Victa C, Carter-Franklin JN, Fahrner R (2009) Selective antibody precipitation using polyelectrolytes: a novel approach to the purification of monoclonal antibodies. Biotechnol Bioeng 102:1141–1151. doi: 10.1002/bit.22127 CrossRefGoogle Scholar
  22. 22.
    Peram T, McDonald P, Carter-Franklin J, Fahrner R (2010) Monoclonal antibody purification using cationic polyelectrolytes: an alternative to column chromatography. Biotechnol Prog 26:1322–1331. doi: 10.1002/btpr.437 CrossRefGoogle Scholar
  23. 23.
    Netz RR, Joanny JF (1999) Complexation between a semiflexible polyelectrolyte and an oppositely charged sphere. Macromolecules 32:9026–9040. doi: 10.1021/ma990264+ CrossRefGoogle Scholar
  24. 24.
    Carlsson F, Linse P, Malmsten M (2001) Monte Carlo simulations of polyelectrolyte–protein complexation. J Phys Chem B 105:9040–9049. doi: 10.1021/jp010360o CrossRefGoogle Scholar
  25. 25.
    Seyrek E, Dubin PL, Tribet C, Gamble EA (2003) Ionic strength dependence of protein–polyelectrolyte interactions. Biomacromolecules 4:273–282. doi: 10.1021/bm025664a CrossRefGoogle Scholar
  26. 26.
    Hattori T, Hallberg R, Dubin PL (2000) Roles of electrostatic interaction and polymer structure in the binding of β-lactoglobulin to anionic polyelectrolytes: measurement of binding constants by frontal analysis continuous capillary electrophoresis. Langmuir 16:9738–9743. doi: 10.1021/la000648p CrossRefGoogle Scholar
  27. 27.
    Dobrynin AV, Rubinstein M (2003) Effect of short-range interactions on polyelectrolyte adsorption at charged surfaces. J Phys Chem B 107:8260–8269. doi: 10.1021/jp0225323 CrossRefGoogle Scholar
  28. 28.
    Moss JM, VanDamme MP, Murphy WH, Preston BN (1997) Dependence of salt concentration on glycosaminoglycan–lysozyme interactions in cartilage. Arch Biochem Biophys 348:49–55. doi: 10.1006/abbi.1997.0365 CrossRefGoogle Scholar
  29. 29.
    Marky NL, Manning GS (2000) An interpretation of small-ion effects on the electrostatics of the λ repressor DNA complex. J Am Chem Soc 122:6057–6066. doi: 10.1021/ja9942437 CrossRefGoogle Scholar
  30. 30.
    Antonov M, Mazzawi M, Dubin PL (2009) Entering and exiting the protein–polyelectrolyte coacervate phase via nonmonotonic salt dependence of critical conditions. Biomacromolecules 11:51–59. doi: 10.1021/bm900886k CrossRefGoogle Scholar
  31. 31.
    Schiessel H, Pincus P (1998) Counterion-condensation-induced collapse of highly charged polyelectrolytes. Macromolecules 31:7953–7959. doi: 10.1021/ma980823x CrossRefGoogle Scholar
  32. 32.
    Stoll S, Chodanowski P (2002) Polyelectrolyte adsorption on an oppositely charged spherical particle Chain Rigidity Effects Macromolecules 35:9556–9562. doi: 10.1021/ma020272h CrossRefGoogle Scholar
  33. 33.
    Cooper CL, Dubin PL, Kayitmazer AB, Turksen S (2005) Polyelectrolyte–protein complexes. Curr Opin Colloid Interface Sci 10:52–78. doi: 10.1016/j.cocis.2005.05.007 CrossRefGoogle Scholar
  34. 34.
    Liao Q, Dobrynin AV, Rubinstein M (2003) Molecular dynamics simulations of polyelectrolyte solutions: nonuniform stretching of chains and scaling behavior. Macromolecules 36:3386–3398. doi: 10.1021/ma025995f CrossRefGoogle Scholar
  35. 35.
    Baeurle SA, Nogovitsin EA (2007) Challenging scaling laws of flexible polyelectrolyte solutions with effective renormalization concepts. Polymer 48:4883–4899. doi: 10.1016/j.polymer.2007.05.080 CrossRefGoogle Scholar
  36. 36.
    Carlsson F, Malmsten M, Linse P (2003) Protein–polyelectrolyte cluster formation and redissolution: a Monte Carlo study. J Am Chem Soc 125:3140–3149. doi: 10.1021/ja020935a CrossRefGoogle Scholar
  37. 37.
    de Gennes PG, Pincus P, Velasco RM, Brochard F (1976) Remarks on polyelectrolyte conformation. J Physique 37:1461–1473. doi: 10.1051/jphys:0197600370120146100 CrossRefGoogle Scholar
  38. 38.
    Jonsson M, Linse P (2001) Polyelectrolyte–macroion complexation. II. Effect of chain flexibility. J Chem Phys 115:10975–10985. doi: 10.1063/1.1417508 CrossRefGoogle Scholar
  39. 39.
    Akinchina A, Linse P (2002) Monte Carlo simulations of polyion–macroion complexes. 1. Equal absolute polyion and macroion charges. Macromolecules 35:5183–5193. doi: 10.1021/ma012052u CrossRefGoogle Scholar
  40. 40.
    Skepö M, Linse P (2003) Complexation, phase separation, and redissolution in polyelectrolyte–macroion solutions. Macromolecules 36:508–519. doi: 10.1021/ma020634l CrossRefGoogle Scholar
  41. 41.
    Kayitmazer AB, Seyrek E, Dubin PL, Staggemeier BA (2003) Influence of chain stiffness on the interaction of polyelectrolytes with oppositely charged micelles and proteins. J Phys Chem B 107:8158–8165. doi: 10.1021/jp034065a CrossRefGoogle Scholar
  42. 42.
    Barrat JL, Joanny JF (1993) Persistence length of polyelectrolyte chains Europhys Lett 24:333–338. doi: 10.1209/0295-5075/24/5/003 CrossRefGoogle Scholar
  43. 43.
    Drifford M, Delsanti M (2001) Polyelectrolyte solutions with multivalent added salts: stability, structure, and dynamics. In: Radeva T (ed) Physical chemistry of polyelectrolytes, surfactant science series. Marcel Dekker, New York, pp 149–161Google Scholar
  44. 44.
    Chodanowski P, Stoll S (2001) Polyelectrolyte adsorption on charged particles in the Debye–Hückel approximation. A Monte Carlo approach. Macromolecules 34:2320–2328. doi: 10.1021/ma000482z CrossRefGoogle Scholar
  45. 45.
    von Goeler F, Muthukumar M (1995) Polyelectrolyte brush density profiles. Macromolecules 28:6608–6617. doi: 10.1021/ma00123a031 CrossRefGoogle Scholar
  46. 46.
    Kong CY, Muthukumar M (1998) Monte Carlo study of adsorption of a polyelectrolyte onto charged surfaces. J Chem Phys 109:1522–1527. doi: 10.1063/1.476703 CrossRefGoogle Scholar
  47. 47.
    Minakata A, Takahashi H, Nishio T, Nagaya J, Tanioka A (2002) Effect of salts on the conductance of polylectrolyte solutions. Colloids Surf A 209:213–218CrossRefGoogle Scholar
  48. 48.
    Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-Pdb Viewer: an environment for comparative protein modeling. Electrophoresis 18:2714–2723. doi: 10.1002/elps.1150181505 CrossRefGoogle Scholar
  49. 49.
    Kiefer F, Arnold K, Künzli M, Bordoli L, Schwede T (2009) The SWISS-MODEL repository and associated resources. Nucleic Acids Res 37:D387–D392CrossRefGoogle Scholar
  50. 50.
    Bordoli L, Kiefer F, Arnold K, Benkert P, Battey J, Schwede T (2008) Protein structure homology modeling using SWISS-MODEL workspace. Nat Protocols 4:1–13. doi: 10.1038/nprot.2008.197 CrossRefGoogle Scholar
  51. 51.
    Daura X, Mark AE, van Gunsteren WF (1998) Parametrization of aliphatic CHn united atoms of GROMOS96 force field. J Comput Chem 19:535–547. doi: 10.1002/(SICI)1096-987X(19980415 CrossRefGoogle Scholar
  52. 52.
    Scott WRP, Hünenberger PH, Tironi IG, Mark AE, Billeter SR, Fennen J, Torda AE, Huber T, Krüger P, van Gunsteren WF (1999) The GROMOS biomolecular simulation program package. J Phys Chem A 103:3596–3607. doi: 10.1021/jp984217f CrossRefGoogle Scholar
  53. 53.
    Tanford C, Kirkwood JG (1957) Theory of protein titration curves. I. General equations for impenetrable spheres. J Am Chem Soc 79:5333–5347. doi: 10.1021/ja01577a001 CrossRefGoogle Scholar
  54. 54.
    Kogej K, Skerjanc J (2001) Surfactant binding to polyelectrolytes. In: Radeva T (ed) Physical chemistry of polyelectrolytes, surfactant science series. Marcel Dekker, New York, pp 793–828Google Scholar
  55. 55.
    Matsunami H, Kikuchi R, Ogawa K, Kokufuta E (2007) Light scattering study of complex formation between protein and polyelectrolyte at various ionic strengths Colloids Surf B 56:142–148. doi: 10.1016/j.colsurfb.2006.10.003 CrossRefGoogle Scholar
  56. 56.
    Tribet C (2001) Complexation between amphiphilic polyelectrolytes and proteins: from necklaces to gels. In: Radeva T (ed) Physical chemistry of polyelectrolytes, surfactant science series. Marcel Dekker, New York, pp 687–742Google Scholar
  57. 57.
    Petit F, Audebert R, Iliopoulos I (1995) Interactions of hydrophobically modified poly(sodium acrylate) with globular proteins. Colloid Polym Sci 273:777–781. doi: 10.1007/BF00658756 CrossRefGoogle Scholar
  58. 58.
    Tribet C, Porcar I, Bonnefont PA, Audebert R (1998) Association between hydrophobically modified polyanions and negatively charged bovine serum albumin. J Phys Chem B 102:1327–1333. doi: 10.1021/jp973022p CrossRefGoogle Scholar
  59. 59.
    Tricot M (1984) Comparison of experimental and theoretical persistence length of some polyelectrolytes at various ionic strengths. Macromolecules 17:1698–1704. doi: 10.1021/ma00139a010 CrossRefGoogle Scholar
  60. 60.
    Le Bret M (1982) Electrostatic contribution to the persistence length of a polyelectrolyte. J Chem Phys 76:6243–6255. doi: 10.1063/1.443027 CrossRefGoogle Scholar
  61. 61.
    Cooper CL, Goulding A, Kayitmazer AB, Ulrich S, Stoll S, Turksen S, Yusa S, Kumar A, Dubin PL (2006) Effects of polyelectrolyte chain stiffness, charge mobility, and charge sequences on binding to proteins and micelles. Biomacromolecules 7:1025–1035. doi: 10.1021/bm050592j CrossRefGoogle Scholar
  62. 62.
    Förster S, Schmidt M (1995) Polyelectrolytes in solution. Adv Polym Sci 120:52–128. doi: 10.1007/3-540-58704-7_2 Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Florian Capito
    • 1
    Email author
  • Romas Skudas
    • 2
  • Bernd Stanislawski
    • 2
  • Harald Kolmar
    • 1
  1. 1.Institute for Organic Chemistry and BiochemistryTechnische Universität DarmstadtDarmstadtGermany
  2. 2.Merck KGaADarmstadtGermany

Personalised recommendations