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Amino Acids

, Volume 51, Issue 4, pp 705–715 | Cite as

Isolation and characterization of collagen type I crosslink from skin: high-resolution NMR reveals diastereomers of hydroxylysinonorleucine crosslink

  • Rafea NaffaEmail author
  • Patrick J. B. Edwards
  • Gillian Norris
Original Article
  • 174 Downloads

Abstract

Skin is made up of mainly collagen type I and its structure is stabilised by the formation of covalent immature and mature crosslinks. In this study, collagen immature crosslink hydroxylysinonorleucine (HLNL) was isolated from bovine skin in high purity using two sequential purification steps. These consisted of preparative fibrous cellulose and size exclusion chromatography. The purified crosslink was then analysed using tandem mass spectrometry and high-resolution nuclear magnetic resonance (NMR) spectroscopy. The mass of singly and doubly charged ions of HLNL was 292.1865 and 146.5970 m/z and their optimised fragmentation energy was 17 keV and 5 keV, respectively. The 13C NMR of HLNL showed a doubled-up peak at 67.84 and 67.91 ppm which corroborated a diastereomeric form of collagen immature crosslink HLNL and both are chiroptically indistinguishable. The chemical structure was fully resolved using 1H, 13C and DEPT-135 high-resolution NMR spectroscopy and compared with other previous studies. We also obtained for the first time the 2D NMR spectra COSY and HSQC of HLNL. We therefore suggested that collagen organization into specific fibrils’ orientation may be affected by the different configuration of these diastereomers of HLNL.

Keywords

Skin Collagen type I Unusual amino acids Preparative chromatography NMR Hydroxylysinonorleucine crosslink 

Abbreviations

CF

Fibrous cellulose

COSY

Correlation spectroscopy

DEPT-135

Distortionless enhancement by polarization transfer-135

HLNL

Hydroxylysinonorleucine

HSQC

Heteronuclear single quantum coherence

NMR

Nuclear magnetic resonance

TIC

Total ion chromatogram

TLC

Thin layer chromatography

Notes

Acknowledgements

This work was supported by NZ Leather and Shoe Research Association (LASRA®), Palmerston North, New Zealand through the Ministry of Business, Innovation and Employment (MBIE) grant number LSRX1301 and LSRX1801.

Author contributions

The authors’ contributions to the manuscript were as follows: RN designed the research; RN, GN and PE conducted the research; and RN and GN wrote the paper. All authors have read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest financial or non-financial.

Research involving human participants and/or animals

No human participants or animal model were involved with this project, thus exempting this study from animal/human approval process.

Informed consent

No patient samples or bio-bank samples were used for this study, thus exempting informed consent requirement. We confirm that this work is new and original and that the manuscript has not been published elsewhere, nor is it under consideration by any other journal. Furthermore, all the authors listed contributed in some way to the work.

Supplementary material

726_2019_2708_MOESM1_ESM.docx (717 kb)
Supplementary material 1 (DOCX 717 kb)

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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  1. 1.NZ Leather and Shoe Research Association (LASRA®)Palmerston NorthNew Zealand
  2. 2.Institute of Fundamental SciencesMassey UniversityPalmerston NorthNew Zealand

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