硫酸乙酰肝素的鉴定和表征：方法I.通过NMR分析鉴定硫酸乙酰肝素

Susan M. Carnachan; Simon F.R. Hinkley

doi:10.21769/BioProtoc.2196

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Heparan Sulfate Identification and Characterisation: Method I. Heparan Sulfate Identification by NMR Analysis

硫酸乙酰肝素的鉴定和表征：方法I.通过NMR分析鉴定硫酸乙酰肝素

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.2196 浏览次数: 8164

评审: Vivien Jane Coulson-ThomasMasahiro MoritaAnonymous reviewer(s)

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Abstract

Heparin and heparan sulfate (HS) may be purified from complex biological matrices and are often isolated in sub-milligram quantities but not unequivocally identified by spectroscopic means. This protocol details a methodology to rapidly assess the gross compositional features and approximate purity of HS by ¹H nuclear magnetic resonance. A complimentary 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)

NMR (NMR)

Background

A number of methods exist for the analysis of heparin and HS. This protocol aims to provide a reproducible and widely applicable method for the rapid identification of heparin/HS by nuclear magnetic resonance (NMR). Small samples (~0.3 mg) can be readily assessed in a non-destructive manner to ascertain an approximate purity that identifies other common contaminants found in biological samples when purifying heparin-like molecules. The procedures described herein are 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 )
5 mm NMR tubes (Norell, catalog number: XR-55^TM-7 )
Chondroitin sulfate A (from bovine trachea) (Sigma-Aldrich, catalog number: C8529 )
Dermatan sulfate (Sigma-Aldrich, catalog number: C3788 )
Chondroitin sulfate C (from shark cartilage, contains ~10% chondroitin sulfate A) (Sigma-Aldrich, catalog number: C4384 )
Heparan sulfate (from porcine mucosa) (Celsus Laboratories, catalog number: HO-03103 )
Heparin (from porcine mucosa) (New Zealand Pharmaceuticals, batch number: 5108356 )
Heparanoid (from porcine mucosa) (New Zealand Pharmaceuticals, batch number: BX-090-010 )
Deuterium oxide (99.9 atom%) (Cambridge Isotope Laboratories, catalog number: DLM-4-100 )
Tert-butanol (t-BuOH, ACS reagent) (Sigma-Aldrich, catalog number: 360538 )

Equipment

NMR spectrometer (Bruker, model: Bruker Avance DPX-500 )
Freeze drier (Eyela, model: FD-1 )
Pipettors (0.5-10 μl, 20-200 μl and 100-1,000 μl) (Eppendorf)

Software

Software Topspin 2.6

Procedure

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Carnachan, S. M. and Hinkley, S. F. R. (2017). Heparan Sulfate Identification and Characterisation: Method I. Heparan Sulfate Identification by NMR Analysis. Bio-protocol 7(7): e2196. DOI: 10.21769/BioProtoc.2196.