Skip to main content
SpringerLink
Log in

Nuclear Magnetic Resonance Methods in Structural Characterization of Glycosaminoglycans

  • Protocol
  • First Online:
Glycosaminoglycans

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2303))

Abstract

Glycosaminoglycans (GAGs) are sulfated glycans of complex structure and multiple biological actions. They are composed of disaccharide repeating units of alternating uronic acid/galactose and hexosamine. Sulfation patterns are an additional structural variation of these polymers. Nuclear magnetic resonance (NMR) spectroscopy is one of the most powerful analytical techniques employed in structural analysis of GAGs. 1D and 2D NMR spectra, both homonuclear 1H and heteronuclear 1H-13C, are the commonest NMR methods used. This chapter describes the overall experimental methods and materials necessary for adequate preparation of GAG samples for NMR investigations aimed to unveil the main structural characteristics of these biomacromolecules. The NMR methods discussed here cover all three isotopes (1H, 13C, and 15N) that can be exploited in structural analysis of GAGs. These NMR methods are described from an overall standpoint, to be applied to any GAG family, extracted from either natural or synthetic sources and destined to either basic research or pharmaceutical applications.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 149.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 199.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 329.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Pomin VH (2015) NMR structural determination of unique invertebrate glycosaminoglycans endowed with medical properties. Carbohydr Res 413:41–50. https://doi.org/10.1016/j.carres.2015.05.004

    Article  CAS  PubMed  Google Scholar 

  2. Rudd TR, Yates EA, Hricovini M (2009) Spectroscopic and theoretical approaches for the determination of heparin saccharide structure and the study of protein-glycosaminoglycan complexes in solution. Curr Med Chem 16(35):4750–4766. https://doi.org/10.2174/092986709789878193

    Article  CAS  PubMed  Google Scholar 

  3. Pomin VH (2014) NMR-based dynamics of free glycosaminoglycans in solution. Analyst 139(15):3656–3665. https://doi.org/10.1039/C4AN00531G

    Article  CAS  PubMed  Google Scholar 

  4. Pomin VH (2014) Solution NMR conformation of glycosaminoglycans. Prog Biophys Mol Biol 114(2):61–68. https://doi.org/10.1016/j.pbiomolbio.2014.01.001

    Article  CAS  PubMed  Google Scholar 

  5. Soares PA, Queiroz IN, Pomin VH (2017) NMR structural biology of sulfated glycans. J Biomol Struct Dyn 35(5):1069–1084. https://doi.org/10.1080/07391102.2016.1171165

    Article  CAS  PubMed  Google Scholar 

  6. Pomin VH, Wang X (2018) Glycosaminoglycan-protein interactions by nuclear magnetic resonance (NMR) spectroscopy. Molecules 23(9):2314. https://doi.org/10.3390/molecules23092314

    Article  CAS  PubMed Central  Google Scholar 

  7. Beecher CN, Larive CK (2015) Methods for measuring exchangeable protons in glycosaminoglycans. In: Balagurunathan K, Nakato H, Desai UR (eds) Glycosaminoglycans: chemistry and biology. Springer, New York, NY, pp 173–187. https://doi.org/10.1007/978-1-4939-1714-3_16

    Chapter  Google Scholar 

  8. Joseph PRB, Poluri KM, Sepuru KM, Rajarathnam K (2015) Characterizing protein–glycosaminoglycan interactions using solution NMR spectroscopy. In: Balagurunathan K, Nakato H, Desai UR (eds) Glycosaminoglycans: chemistry and biology. Springer, New York, NY, pp 325–333. https://doi.org/10.1007/978-1-4939-1714-3_26

    Chapter  Google Scholar 

  9. Victor XV, Tran VM, Kuberan B, Nguyen TK (2015) Preparation of isotope-enriched heparan sulfate precursors for structural biology studies. Methods Mol Biol 1229:43–48. https://doi.org/10.1007/978-1-4939-1714-3_6

    Article  CAS  PubMed  Google Scholar 

  10. Jones C, Mulloy B (1993) The application of nuclear magnetic resonance to structural studies of polysaccharides. Methods Mol Biol 17:149–167. https://doi.org/10.1385/0-89603-215-9:149

    Article  CAS  PubMed  Google Scholar 

  11. Pomin VH, Mulloy B (2018) Glycosaminoglycans and proteoglycans. Pharmaceuticals 11(1):27

    Article  Google Scholar 

  12. Lindahl UCJ, Kimata K et al (2017) Proteoglycans and sulfated glycosaminoglycans. In: Varki ACR, Esko JD et al (eds) Essentials of glycobiology, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 207–221. https://doi.org/10.1101/glycobiology.3e.017

    Chapter  Google Scholar 

  13. Pavão MSG, Mourão PAS, Mulloy B, Tollefsen DM (1995) A unique dermatan sulfate-like glycosaminoglycan from ascidian:: its structure and the effect of its unusual sulfation pattern on anticoagulant activity. J Biol Chem 270(52):31027–31036. https://doi.org/10.1074/jbc.270.52.31027

    Article  PubMed  Google Scholar 

  14. Beni S, Limtiaco JFK, Larive CK (2011) Analysis and characterization of heparin impurities. Anal Bioanal Chem 399(2):527–539. https://doi.org/10.1007/s00216-010-4121-x

    Article  CAS  PubMed  Google Scholar 

  15. Tang Y, Cui Y, De Agostini A, Zhang L (2019) Biological mechanisms of glycan- and glycosaminoglycan-based nutraceuticals. In: Zhang L (ed) Progress in molecular biology and translational science, vol 163. Academic, New York, NY, pp 445–469. https://doi.org/10.1016/bs.pmbts.2019.02.012

    Chapter  Google Scholar 

  16. Pomin VH, Piquet AA, Pereira MS, Mourão PAS (2012) Residual keratan sulfate in chondroitin sulfate formulations for oral administration. Carbohydr Polym 90(2):839–846. https://doi.org/10.1016/j.carbpol.2012.06.009

    Article  CAS  PubMed  Google Scholar 

  17. Sitkowski J, Bednarek E, Bocian W, Kozerski L (2008) Assessment of oversulfated chondroitin sulfate in low molecular weight and unfractioned heparins diffusion ordered nuclear magnetic resonance spectroscopy method. J Med Chem 51(24):7663–7665. https://doi.org/10.1021/jm801198b

    Article  CAS  PubMed  Google Scholar 

  18. Pomin VH (2014) NMR chemical shifts in structural biology of glycosaminoglycans. Anal Chem 86(1):65–94. https://doi.org/10.1021/ac401791h

    Article  CAS  PubMed  Google Scholar 

  19. Pomin VH (2014) Holothurian fucosylated chondroitin sulfate. Mar Drugs 12(1):232–254. https://doi.org/10.3390/md12010232

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Santos GR, Porto AC, Soares PA, Vilanova E, Mourão PA (2017) Exploring the structure of fucosylated chondroitin sulfate through bottom-up nuclear magnetic resonance and electrospray ionization-high-resolution mass spectrometry approaches. Glycobiology 27(7):625–634. https://doi.org/10.1093/glycob/cwx031

    Article  CAS  PubMed  Google Scholar 

  21. York WS, Hantus S, Albersheim P, Darvill AG (1997) Determination of the absolute configuration of monosaccharides by 1H NMR spectroscopy of their per-O-(S)-2-methylbutyrate derivatives. Carbohydr Res 300(3):199–206. https://doi.org/10.1016/S0008-6215(97)00050-5

    Article  CAS  Google Scholar 

  22. Pomin VH, Sharp JS, Li X, Wang L, Prestegard JH (2010) Characterization of glycosaminoglycans by 15N NMR spectroscopy and in vivo isotopic labeling. Anal Chem 82(10):4078–4088. https://doi.org/10.1021/ac1001383

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Pomin VH (2013) Advances in glycosaminoglycanomics by 15N-NMR spectroscopy. Anal Bioanal Chem 405(10):3035–3048. https://doi.org/10.1007/s00216-013-6803-7

    Article  CAS  PubMed  Google Scholar 

  24. Spelta F, Liverani L, Peluso A, Marinozzi M, Urso E, Guerrini M, Naggi A (2019) SAX-HPLC and HSQC NMR spectroscopy: orthogonal methods for characterizing heparin batches composition. Front Med (Lausanne) 6:78–78. https://doi.org/10.3389/fmed.2019.00078

    Article  Google Scholar 

  25. Mauri L, Marinozzi M, Phatak N, Karfunkle M, St Ange K, Guerrini M, Keire DA, Linhardt RJ (2019) 1D and 2D-HSQC NMR: two methods to distinguish and characterize heparin from different animal and tissue sources. Front Med (Lausanne) 6:142–142. https://doi.org/10.3389/fmed.2019.00142

    Article  Google Scholar 

  26. Langeslay DJ, Beecher CN, Naggi A, Guerrini M, Torri G, Larive CK (2013) Characterizing the microstructure of heparin and heparan sulfate using N-sulfoglucosamine 1H and 15N NMR chemical shift analysis. Anal Chem 85(2):1247–1255. https://doi.org/10.1021/ac3032788

    Article  CAS  PubMed  Google Scholar 

  27. Mauri L, Boccardi G, Torri G, Karfunkle M, Macchi E, Muzi L, Keire D, Guerrini M (2017) Qualification of HSQC methods for quantitative composition of heparin and low molecular weight heparins. J Pharm Biomed Anal 136:92–105. https://doi.org/10.1016/j.jpba.2016.12.031

    Article  CAS  PubMed  Google Scholar 

  28. Pomin VH (2017) CHAPTER 10 analysis of glycosaminoglycans by 15N-NMR spectroscopy. In: NMR in glycoscience and glycotechnology. The Royal Society of Chemistry, London, pp 228–249. https://doi.org/10.1039/9781782623946-00228

    Chapter  Google Scholar 

  29. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6(3):277–293. https://doi.org/10.1007/BF00197809

    Article  CAS  PubMed  Google Scholar 

  30. Pomin VH (2012) Unravelling glycobiology by NMR spectroscopy G, Stefana Petrescu. IntechOpen, London. https://doi.org/10.5772/48136. https://www.intechopen.com/books/glycosylation/unravelling-glycobiology-by-nmr-spectroscopy

    Book  Google Scholar 

  31. 0333 Heparin Sodium (2018) European Pharmacopoeia 2018:4913–4915

    Google Scholar 

  32. Heparin Sodium (2018) United States Pharmacopeia USP42-NF37 pp 2148–2153

    Google Scholar 

  33. Szajek AY, Chess E, Johansen K, Gratzl G, Gray E, Keire D, Linhardt RJ, Liu J, Morris T, Mulloy B, Nasr M, Shriver Z, Torralba P, Viskov C, Williams R, Woodcock J, Workman W, Al-Hakim A (2016) The US regulatory and pharmacopeia response to the global heparin contamination crisis. Nat Biotechnol 34(6):625–630. https://doi.org/10.1038/nbt.3606

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Lever R, Smailbegovic A, Riffo-Vasquez Y, Gray E, Hogwood J, Francis SM, Richardson NV, Page CP, Mulloy B (2016) Biochemical and functional characterization of glycosaminoglycans released from degranulating rat peritoneal mast cells: insights into the physiological role of endogenous heparin. Pulm Pharmacol Ther 41:96–102. https://doi.org/10.1016/j.pupt.2016.11.002

    Article  CAS  PubMed  Google Scholar 

  35. Guerrini M, Naggi A, Guglieri S, Santarsiero R, Torri G (2005) Complex glycosaminoglycans: profiling substitution patterns by two-dimensional nuclear magnetic resonance spectroscopy. Anal Biochem 337(1):35–47. https://doi.org/10.1016/j.ab.2004.10.012

    Article  CAS  PubMed  Google Scholar 

  36. Mulloy B, Wu N, Gyapon-Quast F, Lin L, Zhang F, Pickering MC, Linhardt RJ, Feizi T, Chai W (2016) Abnormally high content of free glucosamine residues identified in a preparation of commercially available porcine intestinal heparan sulfate. Anal Chem 88(13):6648–6652. https://doi.org/10.1021/acs.analchem.6b01662

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. 0828:Heparins, Low-Molecular-Mass (2019) European Pharmacopoeia 98:6923–6925

    Google Scholar 

Download references

Acknowledgments

This publication was supported by funds from the startup package provided from the University of Mississippi and the American Association of Colleges of Pharmacy 2019 New Investigator Award, all nominated to V.H.P. We acknowledge the invitation of Dr. Kuberan Balagurunathan, editor of the “Glycosaminoglycans—Chemistry & Biology” for the kind invitation to contribute a chapter to the book. NMR spectra shown in the figures were recorded at the National Institute for Biological Standards and Control, UK or at the Francis Crick Institute through provision of access to the MRC Biomedical NMR Centre. The Francis Crick Institute receives its core funding from Cancer Research UK (FC001029), the UK Medical Research Council (FC001029), and the Wellcome Trust (FC001029). This work was supported, in part, by Wellcome Trust Grant WT108430.

Author information

Authors and Affiliations

  1. Department of Biomolecular Sciences, Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, Oxford, MS, USA

    Vitor H. Pomin

  2. Glycosciences Laboratory, Department of Medicine, Imperial College, London, UK

    Barbara Mulloy

  3. Institute of Pharmaceutical Science, King’s College London, London, UK

    Barbara Mulloy

Authors
  1. Vitor H. Pomin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Barbara Mulloy
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Departments of Biology, Bioengineering, and Medicinal Chemistry, University of Utah, Salt Lake City, UT, USA

    Kuberan Balagurunathan

  2. Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA

    Hiroshi Nakato

  3. Department of Medicinal Chemistry and Institute for Structural Biology, Drug Discovery and Development, Virginia Commonwealth University, Richmond, VA, USA

    Umesh Desai

  4. Department of Neurobiology & Department of Nutrition & Integrative Physiology, University of Utah, Salt Lake City, UT, USA

    Yukio Saijoh

Rights and permissions

Reprints and permissions

Copyright information

© 2022 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Pomin, V.H., Mulloy, B. (2022). Nuclear Magnetic Resonance Methods in Structural Characterization of Glycosaminoglycans. In: Balagurunathan, K., Nakato, H., Desai, U., Saijoh, Y. (eds) Glycosaminoglycans. Methods in Molecular Biology, vol 2303. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1398-6_16

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1398-6_16

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1397-9

  • Online ISBN: 978-1-0716-1398-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation