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Heparan Sulfate Identification and Characterisation: Method II. Enzymatic Depolymerisation and SAX-HPLC Analysis to Determine Disaccharide Composition

硫酸乙酰肝素的鉴定和表征：方法II.酶解聚合和SAX-HPLC分析确定二糖组成

SC Susan M. Carnachan
SH Simon F.R. Hinkley

发布: 2017年04月05日第7卷第7期 DOI: 10.21769/BioProtoc.2197 浏览次数: 9753

评审: Vivien Jane Coulson-Thomas Masahiro MoritaAnonymous reviewer(s)

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Cover of Carbohydrate Polymers, featuring study using the protocol.

Nov 2016

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Abstract

Heparan sulfate (HS) is purified from complex matrices and often not fully characterised to validate its assignment. The characterisation of heparins and heparan sulfates through enzymatic depolymerisation and subsequent strong anion-exchange high performance liquid chromatography (SAX-HPLC) analysis and quantitation of the resulting disaccharides is a critical tool for assessing the structural composition of this class of compound. This protocol details a methodology to reproducibly determine the disaccharide composition of heparan sulfate by enzymatic depolymerisation and SAX-HPLC analysis. A complementary method for identification and characterisation of heparan sulfate can be found at Carnachan and Hinkley (2017).

Keywords: Heparan sulfate (硫酸乙醯肝素) search Glycosaminoglycan’s (糖胺聚糖) search Chemical characterization (化学表征) search Enzymatic depolymerisation (酶解聚合) search HPLC (HPLC) search

Background

A number of methods exist for the structural analysis of heparin and HS. This protocol aims to provide an optimised methodology for the enzymatic depolymerisation of heparin and HS and the analysis and quantification of the disaccharides produced therein. Very few published analyses consider all aspects of the gross composition including the extent of depolymerisation in conjunction with the disaccharide composition obtained (Skidmore et al., 2006 and 2010; Carnachan et al., 2016). This is particularly worrisome when the sample is subsequently utilised in a biological assay that is invariably dose-dependent. The enzymatic procedure described herein is the culmination of a detailed study investigating the conditions necessary for optimal enzyme activity and HS depolymerisation (Carnachan et al., 2016). This procedure is intended to provide a stepwise protocol suitable for a laboratory inexperienced in glycosaminoglycan (GAG) analysis.

Materials and Reagents

  1. Microcentrifuge tubes (1.5 ml) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3456 )
  2. Syringe filters (0.22 μm) (hydrophilic PTFE) (MicroScience Hydraflow, catalog number: MS SF13HY022 )
  3. Glass HPLC vials (2 ml) (Thermo Fisher Scientific, catalog number: THC11090500 )
  4. Septum lids (Thermo Fisher Scientific, catalog number: THC11090500 )
  5. Heparan sulfate (from porcine mucosa) (Celsus Laboratories, catalog number: HO-03103 )
  6. Heparin lyase I (heparinase I or heparitinase III, EC 4.2.2.7, [0.5 IU]) (IBEX Technologies, catalog number: 50-010 )
  7. Heparin lyase II (heparinase II or heparitinase II, no EC number assigned, [0.5 IU]) (IBEX Technologies, catalog number: 50-011 )
  8. Heparin lyase III (heparinase III or heparitinase I, EC 4.2.2.8, [0.5 IU]) (IBEX Technologies, catalog number: 50-012 )
  9. Heparin disaccharide standards produced by the action of bacterial heparinase on high grade porcine heparin (Iduron, catalog numbers: HD001 - HD008 and HD010 - HD013 )
  10. Water (distilled) (Sartorius arium® pro UV ultrafiltered; > 18.5 MΩ)
  11. Bovine serum albumin (BSA) (Thermo Fisher Scientific, GibcoTM, catalog number: 30060727 )
  12. Na2HPO4·H2O (EMD Millipore, catalog number: 106586 )
  13. NaOAc (ACS) (EMD Millipore, catalog number: 1062680250 )
  14. CaOAc·xH2O (Sigma-Aldrich, catalog number: 25011 )
  15. Sodium chloride (NaCl) (99.99%) (EMD Millipore, Suprapur®, catalog number: 1.06406 )
  16. Hydrochloric acid (HCl) (37%) (EMD Millipore, catalog number: 100317 ) and NaOH (Sigma-Aldrich, catalog number: S8045 ) made up to 1 N aqueous solutions for adjusting pH
  17. Phosphoric acid (85%) (Sigma-Aldrich, catalog number: 79606 )
  18. Enzyme storage buffer (see Recipes)
  19. Digestion media (see Recipes)
  20. Mobile phases for HPLC analysis (see Recipes)

Equipment

  1. High performance liquid chromatography (HPLC) machine (Infinity with multiple wavelength detector) (Agilent Technologies, model: 1260 )
  2. Strong anion-exchange HPLC column (4 x 250 mm) (ProPacTM PA1, Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 039658 ) with guard column (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 039657 )
  3. Pipettors (0.5-10 μl, 20-200 μl and 100-1,000 μl) (Eppendorf)
  4. Incubator (BINDER, model: KBF-115 )
  5. Rotator (Cole-Parmer, Stuart, model: SB3 )
  6. Centrifuge (Eppendorf, model: Minispin® plus , catalog number: 5453000011)

Procedure

English
中文翻译

文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Carnachan, S. M. and Hinkley, S. F. R. (2017). Heparan Sulfate Identification and Characterisation: Method II. Enzymatic Depolymerisation and SAX-HPLC Analysis to Determine Disaccharide Composition. Bio-protocol 7(7): e2197. DOI: 10.21769/BioProtoc.2197.

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分类

生物化学 > 糖类 > 二糖

生物化学 > 糖类 > 多糖

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