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

Susan M. Carnachan; Simon F.R. Hinkley

doi:10.21769/BioProtoc.2197

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
CN
- EN - English
- CN - 中文

Peer-reviewed

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 email

发布: 2017年04月05日第7卷第7期 DOI: 10.21769/BioProtoc.2197 浏览次数: 8992

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

下载 PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Carbohydrate Polymers, featuring study using the protocol.

Nov 2016

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

通过双外糖苷酶消化 α-肌聚糖来鉴定基质聚糖

Ishita Chandel and Kevin P. Campbell

2023年09月20日 786 阅读

利用表面等离子体共振技术检测莢膜多糖（CPS）与KpACE的相互作用

Zhe Wang [...] Chao Cai

2025年06月20日 1991 阅读

检测和分离拟南芥种子黏液中鼠李半乳糖醛酸Ⅱ的新方法

Dayan Sanhueza and Susana Saez-Aguayo

2025年09月05日 300 阅读

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)

Glycosaminoglycan’s (糖胺聚糖)

Chemical characterization (化学表征)

Enzymatic depolymerisation (酶解聚合)

HPLC (HPLC)

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

Microcentrifuge tubes (1.5 ml) (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 3456 )
Syringe filters (0.22 μm) (hydrophilic PTFE) (MicroScience Hydraflow, catalog number: MS SF13HY022 )
Glass HPLC vials (2 ml) (Thermo Fisher Scientific, catalog number: THC11090500 )
Septum lids (Thermo Fisher Scientific, catalog number: THC11090500 )
Heparan sulfate (from porcine mucosa) (Celsus Laboratories, catalog number: HO-03103 )
Heparin lyase I (heparinase I or heparitinase III, EC 4.2.2.7, [0.5 IU]) (IBEX Technologies, catalog number: 50-010 )
Heparin lyase II (heparinase II or heparitinase II, no EC number assigned, [0.5 IU]) (IBEX Technologies, catalog number: 50-011 )
Heparin lyase III (heparinase III or heparitinase I, EC 4.2.2.8, [0.5 IU]) (IBEX Technologies, catalog number: 50-012 )
Heparin disaccharide standards produced by the action of bacterial heparinase on high grade porcine heparin (Iduron, catalog numbers: HD001 - HD008 and HD010 - HD013 )
Water (distilled) (Sartorius arium^® pro UV ultrafiltered; > 18.5 MΩ)
Bovine serum albumin (BSA) (Thermo Fisher Scientific, Gibco^TM, catalog number: 30060727 )
Na₂HPO₄·H₂O (EMD Millipore, catalog number: 106586 )
NaOAc (ACS) (EMD Millipore, catalog number: 1062680250 )
CaOAc·xH₂O (Sigma-Aldrich, catalog number: 25011 )
Sodium chloride (NaCl) (99.99%) (EMD Millipore, Suprapur^®, catalog number: 1.06406 )
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
Phosphoric acid (85%) (Sigma-Aldrich, catalog number: 79606 )
Enzyme storage buffer (see Recipes)
Digestion media (see Recipes)
Mobile phases for HPLC analysis (see Recipes)

Equipment

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

Procedure

English

中文翻译

文章信息

版权信息

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.