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Studying Glycosaminoglycan–Protein Interactions Using Capillary Electrophoresis

  • Aiye LiangEmail author
  • Umesh R. Desai
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1229)

Abstract

Methods for studying interactions between glycosaminoglycans (GAGs) and proteins have assumed considerable significance as their biological importance increases. Capillary electrophoresis (CE) is a powerful method to study these interactions due to its speed, high efficiency, and low sample/reagent consumption. In addition, CE works effectively under a wide range of physiologically relevant conditions. This chapter presents state-of-the-art on CE methods for studying GAG–protein interactions including affinity capillary electrophoresis (ACE), capillary zone electrophoresis (CZE), frontal analysis (FA)/frontal analysis continuous capillary electrophoresis (FACCE), and capillary electrokinetic chromatography (CEC) with detailed experimental protocols for ACE and CZE methods.

Key words

Affinity capillary electrophoresis Biophysical technique Capillary electrophoresis Capillary zone electrophoresis GAG–protein interactions Glycosaminoglycans Heparin 

Notes

Acknowledgments

This work was supported by grants SC EPSCoR/IDeA and SCICU to AL and grants HL090586 and HL107152 from the National Institutes of Health to URD. We thank Ms. Yingzi Jin of VCU for helping with the preparation of this chapter.

References

  1. 1.
    Mulloy B, Linhardt RJ (2001) Order out of complexity—protein structures that interact with heparin. Curr Opin Struct Biol 11: 623–628PubMedCrossRefGoogle Scholar
  2. 2.
    Yamada S, Sakamoto K, Tsuda H, Yoshida K, Sugiura M, Sugahara K (1999) Structural studies of octasaccharides derived from the low-sulfated repeating disaccharide region and octasaccharide serines derived from the protein linkage region of porcine intestinal heparin. Biochemistry 38:838–847PubMedCrossRefGoogle Scholar
  3. 3.
    Jiao QC, Liu Q, Sun C, He H (1999) Investigation on the binding site in heparin by spectrophotometry. Talanta 48:1095–1101PubMedCrossRefGoogle Scholar
  4. 4.
    Gallagher JT, Lyon M (2000) Heparan sulfate (molecular structure and interactions with growth factors and morphogens). In: Lozzo RV (ed) Proteoglycans (structure, biology, and molecular interactions). Marcel Dekker, Inc., New York, NY, pp 27–60Google Scholar
  5. 5.
    Whitelock JM, Iozzo RV (2005) Heparan sulfate: a complex polymer charged with biological activity. Chem Rev 105:2745–2764PubMedCrossRefGoogle Scholar
  6. 6.
    Dong J, Peters-Libeu CA, Weisgraber KH, Segelke BW, Rupp B, Capila I, Hernaiz MJ, LeBrun LA, Linhardt RJ (2001) Interaction of the N-terminal domain of apolipoprotein E4 with heparin. Biochemistry 40:2826–2834PubMedCrossRefGoogle Scholar
  7. 7.
    Fromm JR, Hileman RE, Caldwell EEO, Weiler JM, Linhardt RJ (1995) Differences in the interaction of heparin with arginine and lysine and the importance of these basic-amino-acids in the binding of heparin to acidic fibroblast growth-factor. Arch Biochem Biophys 323: 279–287PubMedCrossRefGoogle Scholar
  8. 8.
    Heegaard NHH, De Lorenzi E (2005) Interactions of charged ligands with beta (2)-microglobulin conformers in affinity capillary electrophoresis. Biochim Biophys Acta 1753:131–140PubMedCrossRefGoogle Scholar
  9. 9.
    Heegaard NHH (1998) Capillary electrophoresis for the study of affinity interactions. J Mol Recognit 11:141–148PubMedCrossRefGoogle Scholar
  10. 10.
    Heegaard NHH, Mortensen HD, Roepstorff P (1995) Demonstration of a heparin-binding site in serum amyloid-P component using affinity capillary electrophoresis as an adjunct technique. J Chromatogr A 717:83–90PubMedCrossRefGoogle Scholar
  11. 11.
    Heegaard NHH (1999) Microscale characterization of the structure-activity relationship of a heparin-binding glycopeptide using affinity capillary electrophoresis and immobilized enzymes. J Chromatogr A 853:189–195PubMedCrossRefGoogle Scholar
  12. 12.
    Heegaard NHH, Heegaard PMH, Roepstorff P, Robey FA (1996) Ligand-binding sites in human serum amyloid P component. Eur J Biochem 239:850–856PubMedCrossRefGoogle Scholar
  13. 13.
    Heegaard NHH (1998) A heparin-binding peptide from human serum amyloid P component characterized by affinity capillary electrophoresis. Electrophoresis 19:442–447PubMedCrossRefGoogle Scholar
  14. 14.
    Bohlin ME, Kogutowska E, Blomberg LG, Heegaard NHH (2004) Capillary electrophoresis- based analysis of phospholipid and glycosaminoglycan binding by human beta(2)-glycoprotein. J Chromatogr A 1059:215–222PubMedCrossRefGoogle Scholar
  15. 15.
    Gunnarsson K, Valtcheva L, Hjerten S (1997) Capillary zone electrophoresis for the study of the binding of antithrombin to low-affinity heparin. Glycoconj J 14:859–862PubMedCrossRefGoogle Scholar
  16. 16.
    Heegaard NHH, Nilsson S, Guzman NA (1998) Affinity capillary electrophoresis: important application areas and some recent developments. J Chromatogr B 715:29–54CrossRefGoogle Scholar
  17. 17.
    Heegaard NHH, Nissen MH, Chen DDY (2002) Applications of on-line weak affinity interactions in free solution capillary electrophoresis. Electrophoresis 23:815–822PubMedCrossRefGoogle Scholar
  18. 18.
    McKeon J, Holland LA (2004) Determination of dissociation constants for a heparin-binding domain of amyloid precursor protein and heparins or heparan sulfate by affinity capillary electrophoresis. Electrophoresis 25:1243–1248PubMedCrossRefGoogle Scholar
  19. 19.
    Liu JP, Abid S, Hail ME, Lee MS, Hangeland J, Zein N (1998) Use of affinity capillary electrophoresis for the study of protein and drug interactions. Analyst 123:1455–1459PubMedCrossRefGoogle Scholar
  20. 20.
    Varenne A, Gareil P, Colliec-Jouault S, Daniel R (2003) Capillary electrophoresis determination of the binding affinity of bioactive sulfated polysaccharides to proteins: study of the binding properties of fucoidan to antithrombin. Anal Biochem 315:152–159PubMedCrossRefGoogle Scholar
  21. 21.
    Tissot B, Montdargent B, Chevolot L, Varenne A, Descroix S, Gareil P, Daniel R (2003) Interaction of fucoidan with the proteins of the complement classical pathway. Biochim Biophys Acta 1651:5–16PubMedCrossRefGoogle Scholar
  22. 22.
    Heegaard NHH, He X, Blomberg LG (2006) Binding of Ca2+, Mg2+, and heparin by human serum amyloid P component in affinity capillary electrophoresis. Electrophoresis 27:2609–2615PubMedCrossRefGoogle Scholar
  23. 23.
    Hamazaki H (1987) Ca2+ mediated association of human-serum amyloid-P component with heparan-sulfate and dermatan-sulfate. J Biol Chem 262:1456–1460PubMedGoogle Scholar
  24. 24.
    Li XA, Hatanaka K, Guo L, Kitamura Y, Yamamoto A (1994) Binding of serum amyloid-P component to heparin in human serum. Biochim Biophys Acta 1201:143–148PubMedCrossRefGoogle Scholar
  25. 25.
    Heegaard NHH, Hansen SI, Holm J (2006) A novel specific heparin-binding activity of bovine folate-binding protein characterized by capillary electrophoresis. Electrophoresis 27: 1122–1127PubMedCrossRefGoogle Scholar
  26. 26.
    Liang A, Raghuraman A, Desai UR (2009) Capillary electrophoretic study of small, highly sulfated, non-sugar molecules interacting with antithrombin. Electrophoresis 30:1544–1551PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Anderot M, Nilsson M, Vegvari A, Moeller EH, Weert M, Isaksson R (2009) Determination of dissociation constants between polyelectrolytes and proteins by affinity capillary electrophoresis. J Chromatogr B 877:892–896CrossRefGoogle Scholar
  28. 28.
    Kinoshita M, Kakehi K (2005) Analysis of the interaction between hyaluronan and hyaluronan-binding proteins by capillary affinity electrophoresis: significance of hyaluronan molecular size on binding reaction. J Chromatogr B 816: 289–295CrossRefGoogle Scholar
  29. 29.
    Gotti R, Parma B, Spelta F, Liverani L (2013) Affinity capillary electrophoresis in binding study of antithrombin to heparin from different sources. Talanta 105:366–371PubMedCrossRefGoogle Scholar
  30. 30.
    Heegaard NH, Roepstorff P, Melberg SG, Nissen MH (2002) Cleaved beta 2- microglobulin partially attains a conformation that has amyloidogenic features. J Biol Chem 277: 11184–11189PubMedCrossRefGoogle Scholar
  31. 31.
    Dimitrellos V, Lamari FN, Militsopoulou M, Kanakis I, Karamanos NK (2003) Capillary electrophoresis and enzyme solid phase assay for examining the purity of a synthetic heparin proteoglycan-like conjugate and identifying binding to basic fibroblast growth factor. Biomed Chromatogr 17:42–47PubMedCrossRefGoogle Scholar
  32. 32.
    Heegaard NHH, Robey FA (1992) Use of capillary zone electrophoresis to evaluate the binding of anionic carbohydrates to synthetic peptides derived from human serum amyloid-P component. Anal Chem 64:2479–2482PubMedCrossRefGoogle Scholar
  33. 33.
    Hernaiz MJ, LeBrun LA, Wu Y, Sen JW, Linhardt RJ, Heegaard NHH (2002) Characterization of heparin binding by a peptide from amyloid P component using capillary electrophoresis, surface plasmon resonance and isothermal titration calorimetry. Eur J Biochem 269:2860–2867PubMedCrossRefGoogle Scholar
  34. 34.
    Guijt-van Duijn RM, Frank J, van Dedem GWK, Baltussen E (2000) Recent advances in affinity capillary electrophoresis. Electrophoresis 21:3905–3918PubMedCrossRefGoogle Scholar
  35. 35.
    Militsopoulou M, Lamari F, Karamanos NK (2003) Capillary electrophoresis: a tool for studying interactions of glycans/proteoglycans with growth factors. J Pharm Biomed Anal 32:823–828PubMedCrossRefGoogle Scholar
  36. 36.
    Ling X, Liu Y, Fan H, Zhong Y, Li D, Wang Y (2007) Studies on interactions f programmed cell death 5 (PDCD5) and its related peptides with heparin by capillary zone electrophoresis. Anal Bioanal Chem 387:909–916PubMedCrossRefGoogle Scholar
  37. 37.
    Liu Y, Zhang S, Ling X, Li Y, Zhang Y, Han W, Wang Y (2008) Analysis of the interactions between the peptides from secreted human CKLF1 and heparin using capillary zone electrophoresis. J Pept Sci 14:984–988PubMedCrossRefGoogle Scholar
  38. 38.
    Liang A, He X, Du Y, Wang K, Fung Y, Lin B (2004) Capillary zone electrophoresis investigation of the interaction between heparin and granulocyte-colony stimulating factor. Electrophoresis 25:870–875PubMedCrossRefGoogle Scholar
  39. 39.
    Liang A, He X, Du Y, Wang K, Fung Y, Lin B (2005) Capillary zone electrophoresis characterization of low molecular weight heparin binding to interleukin 2. J Pharm Biomed Anal 38:408–413PubMedCrossRefGoogle Scholar
  40. 40.
    Liang A, Chao Y, Liu X, Du Y, Wang K, Qian S, Lin B (2005) Separation, identification, and interaction of heparin oligosaccharides with granulocyte-colony stimulating factor using capillary electrophoresis and mass spectrometry. Electrophoresis 26:3460–3467PubMedCrossRefGoogle Scholar
  41. 41.
    Liang A, Du Y, Wang K, Lin B (2006) Quantitative investigation of interaction between granulocyte-macrophage colony-stimulating factor and heparin by capillary zone electrophoresis. J Sep Sci 29:1637–1641PubMedCrossRefGoogle Scholar
  42. 42.
    Liang A, Liu X, Du Y, Wang K, Lin B (2008) Further characterization of the binding of heparin to granulocyte colony-stimulating factor: importance of sulfate groups. Electrophoresis 29:1286–1290PubMedCrossRefGoogle Scholar
  43. 43.
    Liang A, Zhou X, Wang Q, Liu X, Qin J, Du Y, Wang K, Lin B (2006) Interactions of dextran sulfates with granulocyte colony-stimulating factor and their effects on leukemia cells. Electrophoresis 27:3195–3201PubMedCrossRefGoogle Scholar
  44. 44.
    Liang A, Zhou X, Wang Q, Liu X, Liu X, Du Y, Wang K, Lin B (2006) Structural features in carrageenan that interact with a heparin-binding hematopoietic growth factor and modulate its biological activity. J Chromatogr B 843:114–119CrossRefGoogle Scholar
  45. 45.
    Lipponen K, Liu Y, Patricia WS, Oorni K, Kovanen PT, Riekkola M (2012) Capillary electrochromatography and quartz crystal microbalance, valuable techniques in the study of heparin-lipoprotein interactions. Anal Biochem 424:71–78PubMedCrossRefGoogle Scholar
  46. 46.
    Wu XJ, Linhardt RJ (1998) Capillary affinity chromatography and affinity capillary electrophoresis of heparin binding proteins. Electrophoresis 19:2650–2653PubMedCrossRefGoogle Scholar
  47. 47.
    VanderNoot VA, Hileman RE, Dordick JS, Linhardt RJ (1998) Affinity capillary electrophoresis employing immobilized glycosaminoglycan to resolve heparin-binding peptides. Electrophoresis 19:437–441PubMedCrossRefGoogle Scholar
  48. 48.
    Hattori T, Kimura K, Seyrek E, Dubin PL (2001) Binding of bovine serum albumin to heparin determined by turbidimetric titration and frontal analysis continuous capillary electrophoresis. Anal Biochem 295:158–167PubMedCrossRefGoogle Scholar
  49. 49.
    Hattori T, Kimura K, Seyrek E, Dubin PL (2001) The use of frontal analysis continuous capillary electrophoresis to compare protein binding by natural and synthetic polyelectrolyte. Anal Sci 17:93–95CrossRefGoogle Scholar
  50. 50.
    Saux TL, Varenne V, Perreau F, Siret L, Duteil S, Duhau L, Gareil P (2006) Determination of the binding parameters for antithrombin-heparin fragment systems by affinity and frontal analysis continuous capillary electrophoresis. J Chromatogr A 1132:289–296PubMedCrossRefGoogle Scholar
  51. 51.
    Seyrek E, Dubin PL, Henriksen J (2007) Nonspecific electrostatic binding characteristics of the heparin-antithrombin interaction. Biopolymers 86:249–259PubMedCrossRefGoogle Scholar
  52. 52.
    Fermas S, Gonnet F, Varenne A, Gareil P, Daniel R (2007) Frontal analysis capillary electrophoresis hyphenated to electrospray ionization mass spectrometry for the characterization of the antithrombin/heparin pentasaccharide complex. Anal Chem 79: 4987–4993PubMedCrossRefGoogle Scholar
  53. 53.
    He X, Ding Y, Li D, Lin B (2004) Recent advances in the study of biomolecular interactions by capillary electrophoresis. Electrophoresis 25:697–711PubMedCrossRefGoogle Scholar
  54. 54.
    Scatchard G (1949) The attraction of proteins for small molecules and ions. Ann N Y Acad Sci 51:660–672CrossRefGoogle Scholar
  55. 55.
    Keyes RS, Bobst AM (1993) A comparative study of Scatchard-type and linear lattice models for the analysis of EPR competition experiments with spin-labeled nucleic acids and sin. Biophys Chem 45:281–303PubMedCrossRefGoogle Scholar
  56. 56.
    Klotz IM, Hunston DL (1971) Properties of graphical representations of multiple classes of binding sites. Biochemistry 10:3065–3069PubMedCrossRefGoogle Scholar
  57. 57.
    Colton IJ, Carbeck JD, Rao J, Whitesides GM (1998) Affinity capillary electrophoresis: a physical-organic tool for studying interactions in biomolecular recognition. Electrophoresis 19:367–382PubMedCrossRefGoogle Scholar
  58. 58.
    Olson ST, Björk I, Sheffer R, Craig PA, Shore JD, Choay J (1992) Role of the antithrombin-binding pentasaccharide in heparin acceleration of antithrombin-proteinase reactions. Resolution of the antithrombin conformational change contribution to heparin rate enhancement. J Biol Chem 267:12528–12538PubMedGoogle Scholar
  59. 59.
    Desai UR, Petitou M, Björk I, Olson ST (1998) Mechanism of heparin activation of antithrombin. Role of individual residues of the pentasaccharide activating sequence in the recognition of native and activated states of antithrombin. J Biol Chem 273: 7478–7487PubMedCrossRefGoogle Scholar
  60. 60.
    Gunnarsson GT, Desai UR (2002) Interaction of designed sulfated flavanoids with antithrombin: lessons on the design of organic activators. J Med Chem 45:4460–4470PubMedCrossRefGoogle Scholar
  61. 61.
    Gunnarsson GT, Desai UR (2002) Designing small, nonsugar activators of antithrombin using hydropathic interaction analyses. J Med Chem 45:1233–1243PubMedCrossRefGoogle Scholar
  62. 62.
    Gunnarsson GT, Riaz M, Adams J, Desai UR (2005) Synthesis of per-sulfated flavonoids using 2,2,2-trichloro ethyl protecting group and their factor Xa inhibition potential. Bioorg Med Chem 13:1783–1789PubMedCrossRefGoogle Scholar
  63. 63.
    Gunnarsson GT, Desai UR (2003) Exploring new non-sugar sulfated molecules as activators of antithrombin. Bioorg Med Chem Lett 13: 579–583CrossRefGoogle Scholar
  64. 64.
    Raghuraman A, Riaz M, Hindle M, Desai UR (2007) Rapid and efficient microwave-assisted synthesis of highly sulfated organic scaffolds. Tetrahedron Lett 48: 6754–6758PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Physical SciencesCharleston Southern UniversityNorth CharlestonUSA
  2. 2.Department of Medicinal Chemistry and Institute for Structural Biology and Drug DiscoveryVirginia Commonwealth UniversityRichmondUSA

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