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

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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)


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

  1. Microcentrifuge tubes (1.5 ml) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3456 )
  2. 5 mm NMR tubes (Norell, catalog number: XR-55TM-7 )
  3. Chondroitin sulfate A (from bovine trachea) (Sigma-Aldrich, catalog number: C8529 )
  4. Dermatan sulfate (Sigma-Aldrich, catalog number: C3788 )
  5. Chondroitin sulfate C (from shark cartilage, contains ~10% chondroitin sulfate A) (Sigma-Aldrich, catalog number: C4384 )
  6. Heparan sulfate (from porcine mucosa) (Celsus Laboratories, catalog number: HO-03103 )
  7. Heparin (from porcine mucosa) (New Zealand Pharmaceuticals, batch number: 5108356 )
  8. Heparanoid (from porcine mucosa) (New Zealand Pharmaceuticals, batch number: BX-090-010 )
  9. Deuterium oxide (99.9 atom%) (Cambridge Isotope Laboratories, catalog number: DLM-4-100 )
  10. Tert-butanol (t-BuOH, ACS reagent) (Sigma-Aldrich, catalog number: 360538 )


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


  1. Software Topspin 2.6


  1. Sample preparation
    Exchangable protons in the sample are treated to a deuterium exchange regime before NMR analysis: 
    1. Add D2O (1 ml) to a sample (~12 mg) of the glucosaminoglycan in an Eppendorf tube and agitate the sample to complete dissolution.
    2. The sample is freeze dried in the ‘loosely capped’ tube and this process repeated twice more.
    3. Finally the sample is made up with D2O (0.7 ml) containing 0.2 mg/ml t-BuOH as an internal reference (δ 1H 1.24 ppm) and transferred to the NMR tube for analysis.
  2. Sample concentration
    The concentration of 17 mg/ml has been found to be the minimum necessary for highly reproducible 2-dimensional NMR. The 1H NMR spectra of this class of compound is significantly affected by pH, cross-linking capable species (e.g., Ca2+) and the salt form of the sulfated moieties (Liu et al., 2009; Guerrini et al., 2008). If such contamination is suspected, the sample should be converted to the sodium salt and dialysed (3,500 MW cutoff) against water exhaustively, before freeze-drying and deuterium exchange.
  3. Data collection
    1. Spectra were collected at 30 °C on a three-channel Bruker Avance III 500 (installed August 2007). Proton frequency was 499.843 MHz and the probe was a Bruker two channel 5-mm broadband observe nuclei probe (31P-109Ag) equipped with actively shielded 50-G/cm Z-axis Pulsed Field Gradients (PFG).
    2. Data acquisition used a spectral width of 10,302 Hz, 65,536 data points, 30-degree excitation pulse, 32 transients, each with a 2-sec delay time and an FID acquisition time of 1.59 sec.
    3. Spectra with solvent-suppression were recorded: low-power continuous-wave pre-saturation was followed by a composite-observe pulse utilizing the ‘zgcppr’ Bruker sequence (software Topspin 2.6).

Data analysis

  1. The individual GAG’s are readily identified by their distinctive 1H NMR spectra (Figure 1). The presence of contaminating chondroitin or dermatan sulfate in a sample of heparan sulfate or heparin is apparent by examination of the glycosaminoglycan acetate resonances (Carnachan, 2016). For both heparin and heparan sulfate this acetate resonance occurs at δ 2.034 ppm and any additional acetate resonance downfield indicates the presence of other GAG’s (see Figure 2). In addition, heparin and heparan sulfate have resonances at δ 3.25, 3.37 and > 5.0 ppm that distinguish them from chondroitins or dermatan.

    Figure 1. 1H NMR spectra (D2O, 25 °C, with HDO suppression, referenced to t-BuOH) of heparin, heparan sulfate, chondroitin sulfates and dermatan sulfate. Chondroitin-A is a mixture of chondroitins A&C.

    Figure 2. 1H NMR spectra of the acetate methyl resonances (D2O, 25 °C, with HDO suppression, referenced to t-BuOH) of heparin, heparan sulfate, chondroitin sulfates and dermatan sulfate. Chondroitin-A is a mixture of chondroitins A&C.

  2. By way of demonstration, 1H NMR spectra that are typical of those recorded during a purification process from Heparanoid (see Note 6) are presented in Figure 3.

    Figure 3. 1H NMR spectra of Heparanoid and partially purified fractions obtained from DEAE-Sepharose chromatographic separation. A. Very crude Heparanoid with significant protein contribution; B. Heparanoid - different batch to A; C and D. GAG mixtures that have a significant proportion of heparin-like materials; E. Fraction that is > 85% HS (D2O, 25 °C, with HDO suppression [discontinuous peak at δ 4.6 ppm], referenced to t-BuOH).

  3. The co-occurrence of the heparin and heparan sulfate N-acetate methyl resonances at δ 2.034 ppm make using this peak for assessing the relative proportion of these species in a sample problematic. However, the intensity of this peak relative to the remainder of the spectrum can be indicative of whether a sample is more heparan sulfate-like, or, more heparin-like. The peak height for the acetate resonance of heparin is equivalent to the peak height of the methine resonances observed at δ ~3.8 ppm, while for heparan sulfate the acetate resonance is ~2.5 fold greater than the methine resonances (see Figure 2). Low levels (even up to < 15%) of contaminating heparin in a sample of heparan sulfate are very difficult to confirm or quantitate by 1H NMR (Keire et al., 2010), and are more readily assessed using 2-D NMR techniques (Guerrini et al., 2005).
  4. A semi-quantitative purity assessment can be undertaken for samples containing primarily one GAG (> 90%). Using a known concentration of t-BuOH in D2O and a pure sample of a particular GAG a standard curve may be generated. GAG was weighed out accurately into ~10, 5, 2 and 0.5 mg aliquots and made up with exactly 1.0 ml of t-BuOH standard solution for NMR analysis. Using integrated areas, the ratio of the acetate peak of the GAG divided by the t-BuOH resonance plotted against the known amount of GAG in solution generates a straight-line relation (Figure 4). A sample of unknown purity made up in the same concentration and volume of t-BuOH solution can therefore be assessed for purity compared to the reference material. Such a standard curve is particularly useful in assessing possible contributions from salt and water concentrations that are effectively invisible to this NMR analysis and not trivial to measure in a non-destructive manner on small samples (< 5 mg).

    Figure 4. Standard curve plotting the ratio of acetate and internal-standard resonances (integrated areas from 1H NMR) against the known mass of HS (> 95% w/w) in the sample (t-BuOH standard solution 0.20 mg/ml)


  1. GAG’s, either as a dry lyophilized solid or as a dilute solution (D2O, 5 °C) are stable indefinitely (> 5 yr). Dry solids appear to undergo no degradation over time either at room temperature (RT) or freezer conditions (to -70 °C).
  2. Completing the D2O exchange process has only a subtle effect on the NMR spectra, and so for routine analysis or during purification-monitoring this step is often omitted.
  3. Concentrated samples (> 60 mg/ml) (prepared for example for 13C analysis or rapid 2-D NMR techniques) exhibit significant line broadening and chemical shift changes in the 1H NMR spectrum, thus reference 1H NMR must be recorded as dilute solutions only.
  4. We have demonstrated in the laboratory that freshly lyophilized HS will adsorb 13% w/w of water on standing. This is a reproducible mass gain on standing and readily-reversible process by lyophilization. Samples are routinely stored at -70 °C and so are allowed to warm to RT in air-tight containers before exposure to the atmosphere to minimize water uptake during handling.
  5. The presence of salts can be subjectively noted by the physical appearance of the GAG. Heparin and HS are fluffy white ‘cotton-wool’ like solids. The presence of salt at ~> 10% w/w generates a more rigid, tough off-white solid which adsorbs atmospheric water at an accelerated rate compared to the de-salted material. This is particularly important to note for weighing small amounts of HS that are subsequently entering a dose-response bioassay.
  6. Heparanoid is a complex mixture of GAG’s recovered from the side-streams produced in the production of Heparin from porcine mucosa. The composition of material described as Heparanoid varies significantly (see Figures 3i and 3ii), dependant on which side-streams of the Heparin process are combined. Thus the levels of protein, individual GAG’s (including Heparin), salts and other biological materials can vary wildly in individually sourced materials and discrete batches. One method (Shworak, 2001) of purifying HS is partitioning on a diethylaminoethyl-Sepharose (DEAE-Sepharose) column. Column elution may be achieved by step-wise increments in ionic strength (e.g., sodium chloride in phosphate buffer). For analysis by NMR fractions are desalted (dialysis), lyophilized and the resultant amorphous white solid exchanged with D2O before 1H NMR spectra recorded (unpublished work).


This research was supported in part by the New Zealand Ministry of Business, Innovation and Employment, and the Kiwi Innovation Network (KiwiNet, VL001298). The collaborative research completed with Drs Simon M. Cool, Victor Nurcombe and R. Alex A. Smith (Institute of Medical Biology, Agency for Science, Technology and Research, Immunos, Singapore) is acknowledged.


  1. Carnachan, S. M., Bell, T. J., Sims, I. M., Smith, R. A., Nurcombe, V., Cool, S. M. and Hinkley, S. F. (2016). Determining the extent of heparan sulfate depolymerisation following heparin lyase treatment. Carbohydr Polym 152: 592-597.
  2. Carnachan, S. C. and Hinkley, S. F. R. (2017). Heparan sulfate identification and characterisation: Method II. Enzymatic depolymerisation and SAX-HPLC analysis to determine disaccharide composition. Bio-protocl 7(07): e2197.
  3. Guerrini, M., Beccati, D., Shriver, Z., Naggi, A., Viswanathan, K., Bisio, A., Capila, I., Lansing, J. C., Guglieri, S., Fraser, B., Al-Hakim, A., Gunay, N. S., Zhang, Z., Robinson, L., Buhse, L., Nasr, M., Woodcock, J., Langer, R., Venkataraman, G., Linhardt, R. J., Casu, B., Torri, G. and Sasisekharan, R. (2008). Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events. Nat Biotechnol 26 (6): 669-675.
  4. Guerrini, M., Naggi, A., Guglieri, S., Santarsiero, R. and Torri, G. (2005). Complex glycosaminoglycans: profiling substitution patterns by two-dimensional nuclear magnetic resonance spectroscopy. Anal Biochem 337 (1): 3547.
  5. Keire, D. A., Mans, D. J., Ye, H., Kolinski, R. E. and Buhse, L. F. (2010). Assay of possible economically motivated additives or native impurities levels in heparin by 1H NMR, SAX-HPLC, and anticoagulation time approaches. J Pharm Biomed Anal 52(5): 656-664.
  6. Liu, H., Zhang, Z. and Linhardt, R. J. (2009). Lessons learned from the contamination of heparin. Nat Prod Rep 26 (3): 313-321.
  7. Shworak, N. W. (2001). High-specific-activity 35S-labeled heparan sulfate prepared from cultured cells. In: Iozzo, R. V. (Ed.). Proteoglycan Protocols. Methods in Molecular Biology pp: 79-89.


肝素和硫酸乙酰肝素(HS)可以从复杂的生物学基质中纯化出来,通常以亚毫克数量分离,但不能通过光谱学方法明确鉴定。 该方案详细介绍了一种方法,通过H核磁共振快速评估HS的总体组成特征和近似纯度。 Carnachan和Hinkley(2017)可以找到一种免费的硫酸乙酰肝素鉴定和表征方法。

存在一些用于肝素和HS分析的方法。 该方案旨在提供一种可重现和广泛应用的通过核磁共振(NMR)快速鉴定肝素/ HS的方法。 可以以非破坏性的方式容易地评估小样品(〜0.3mg),以确定纯化肝素样分子时生物样品中发现的其他常见污染物的近似纯度。 本文描述的方法旨在提供适用于没有经验的糖胺聚糖(GAG)分析的实验室的逐步方案。

关键字:硫酸乙醯肝素, 糖胺聚糖, 化学表征, NMR


  1. 微量离心管(1.5ml)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:3456)
  2. 5毫米NMR管(Norell,目录号:XR-55 -7)
  3. 硫酸软骨素A(来自牛气管)(Sigma-Aldrich,目录号:C8529)
  4. 硫酸皮肤素(Sigma-Aldrich,目录号:C3788)
  5. 硫酸软骨素C(来自鲨鱼软骨,含有〜10%硫酸软骨素A)(Sigma-Aldrich,目录号:C4384)
  6. 硫酸肝素(来自猪粘膜)(Celsus Laboratories,目录号:HO-03103)
  7. 肝素(来自猪粘膜)(新西兰制药,批号:5108356)
  8. 肝素(来自猪粘膜)(新西兰制药,批号:BX-090-010)
  9. 氧化氘(99.9原子%)(剑桥同位素实验室,目录号:DLM-4-100)
  10. 叔丁醇(叔丁醇,ACS试剂)(Sigma-Aldrich,目录号:360538)


  1. NMR光谱仪(Bruker,型号:Bruker Avance DPX-500)
  2. 冷冻干燥机(Eyela,型号:FD-1)
  3. 移液器(0.5-10μl,20-200μl和100-1,000μl)(Eppendorf)


  1. 软件Topspin 2.6


  1. 样品制备
    1. 将D 2 O(1ml)加入到在Eppendorf管中的葡糖胺聚糖的样品(〜12mg)中,并搅拌样品以完全溶解。
    2. 将样品在"松散封闭"管中冷冻干燥,此过程重复两次。
    3. 最后,将样品与含有0.2mg/ml-t-BuOH的D 2 O(0.7ml)作为内标(δ 1 H 1.24ppm),并转移到NMR管中进行分析
  2. 样品浓度
    已经发现17mg/ml的浓度是高度可重现的2维NMR所必需的最小值。这类化合物的<1/1H NMR谱显着受pH,可交联的物质(例如,Ca 2+)的影响,和硫酸盐部分的盐形式(Liu et al。,2009; Guerrini等人,2008)。如果怀疑这种污染物,则应在冷冻干燥和氘转化之前,将样品转化为钠盐并透析(3,500MW截止)以防止水分残留。
  3. 数据收集
    1. 在三通道Bruker Avance III 500(安装于2007年8月)的30℃下收集光谱。质子频率为499.843MHz,探针是配备有主动屏蔽的50-G/cm Z轴脉冲场梯度(PFG)的Bruker双通道5-mm宽度观察核探针(31P-109Ag)。
    2. 数据采集采用10,302Hz,65,536个数据点,30度激励脉冲,32个瞬变的频谱宽度,每个具有2秒延迟时间和1.59秒的FID采集时间。
    3. 记录具有溶剂抑制的光谱:低功率连续波预饱和后,使用"zgcppr"Bruker序列(软件Topspin 2.6)的复合观察脉冲。


  1. 单独的GAG可以通过其独特的1 H NMR谱(图1)来识别。硫酸乙酰肝素或肝素样品中污染的软骨素或硫酸皮肤素的存在通过检测糖胺聚糖乙酸盐共振是显而易见的(Carnachan,2016)。对于肝素和硫酸乙酰肝素,这种乙酸盐共振发生在δ2.034ppm,任何另外的乙酸盐共振下场表明存在其他GAG(见图2)。此外,肝素和硫酸乙酰肝素在δ3.25,3.37和> 5.0 ppm,将其与软骨素或皮肤病区分开来。

    图1. 1 H NMR谱(D 2 O,25°C,具有HDO抑制,参考-TuOH)肝素,硫酸乙酰肝素,硫酸软骨素和硫酸皮肤素。软骨素A是软骨素A和C的混合物。

    参考图1所示的乙酸甲酯共振(D 2 O,25℃,HDO抑制)的1 H NMR谱,参考<肝素,硫酸乙酰肝素,硫酸软骨素和硫酸皮肤素。

  2. 通过证明,从Heparanoid纯化过程中记录的那些(参见注释6)的典型的1 H NMR谱见图3。

    图3.从DEAE-Sepharose色谱分离得到的类肝素和部分纯化级分的1 H NMR谱。A.非常粗的具有显着蛋白质贡献的肝素类; B.肝素 - 不同批次A; C和D.GAG混合物具有相当大比例的肝素样物质; E.分数> 85%HS(D 2 O,25°C,具有HDO抑制[δ4.6ppm的不连续峰),参考于-BuOH)。

  3. 肝素和硫酸乙酰肝素N-乙酸甲酯共振在δ2.034ppm处的共同发生使用该峰来评估这些物质在样品中的相对比例是有问题的。然而,相对于光谱的其余部分,该峰的强度可以指示样品是否更多是硫酸肝素样,或更多的肝素样。肝素的乙酸盐共振的峰高相当于在δ〜3.8ppm处观察到的次甲基共振的峰高,而对于硫酸乙酰肝素,乙酸盐共振比次甲基共振大约2.5倍(参见图2)。硫酸乙酰肝素样品中的污染性肝素的低水平(甚至高达<15%)非常难以通过1 H NMR(Keire等人)进行确认或定量。 ,2010),并且使用二维核磁共振技术更容易评估(Guerrini等人,2005)。
  4. 可以对主要包含一个GAG(> 90%)的样品进行半定量纯度评估。在D 2 O中使用已知浓度的t-BuOH和特定GAG的纯样品可以产生标准曲线。将GAG精确称取至〜10,5,2和0.5mg等分试样,并用正好1.0ml的用于NMR分析的标准溶液制成。使用综合区域; GAG的乙酸酯峰除以相对于溶液中已知的GAG量的曲线的比率产生直线关系(图4)。因此,与参考材料相比,可以评估在相同浓度和体积的溶液中制备的未知纯度的样品的纯度。这种标准曲线在评估盐和水浓度的可能贡献方面特别有用,该浓度对于该NMR分析是有效的不可见的,在小样品(<5mg)上以非破坏性方式测量不是微不足道的。

    图4.标准曲线绘制了乙酸盐和内部标准共振的比例(来自 1的NMR的积分面积)与已知质量的HS(> 95%w/w)样品(标准溶液,0.20mg/ml)


  1. 作为干燥冻干固体或稀释溶液(D 2 O 5,5℃),GAG无限期(> 5年)是稳定的。在室温(RT)或冷冻条件(至-70℃)下,干固体似乎不会随时间降解。
  2. 完成D 2 O交换过程对NMR光谱只有微妙的影响,因此对于常规分析或在纯化监测期间,通常省略此步骤。
  3. 浓缩样品(> 60mg/ml)(例如用于13℃或快速二维核磁共振技术制备)显示出 1 中的显着的线展宽和化学位移变化1 H NMR光谱,因此参考文献1必须将1H NMR记录为稀释溶液
  4. 我们已经在实验室证明,新鲜冻干的HS在静置时会吸附13%w/w的水。这是通过冻干固定和容易逆转的方法的可重复的质量增益。样品通常储存在-70°C,因此在暴露于大气中之前允许在气密容器中温热至室温,以尽量减少处理过程中的吸水量。
  5. 盐的存在可以通过GAG的物理外观来主观地注意。肝素和HS是蓬松的白色"棉毛"固体。盐的存在〜 10%w/w产生更硬,坚韧的灰白色固体,其与脱盐材料相比以更快的速度吸附大气水。对于随后进入剂量反应生物测定的少量HS进行称重尤为重要。
  6. 肝素是从生产肝素从猪粘膜中产生的侧流回收的GAG的复杂混合物。描述为肝素类的材料组成变化很大(参见图3i和3ii),取决于肝素过程的哪些侧流组合。因此,蛋白质,单个GAG(包括肝素),盐和其他生物材料的水平可以在单独来源的材料和离散批次中变化很大。纯化HS的一种方法(Shworak,2001)是在二乙基氨基乙基琼脂糖(DEAE-Sepharose)柱上分配。柱洗脱可以通过离子强度(例如,磷酸盐缓冲液中的氯化钠)逐步增加来实现。对于通过NMR分析进行分析,将其脱盐(透析),冷冻干燥,得到的无定形白色固体与D 2 O 2交换,然后记录(未发表的工作)。 />


这项研究部分得到了新西兰商业,创新和就业部以及新西兰创新网络(KiwiNet,VL001298)的支持。 Simon M. Cool博士,Victor Nurcombe博士和R. Alex A. Smith博士(新加坡免疫科学与技术研究所医学生物研究所)的合作研究得到了认可。


  1. Carnachan,SM,Bell,TJ,Sims,IM,Smith,RA,Nurcombe,V.,Cool,SM and Hinkley,SF(2016)。  确定肝素裂解酶治疗后硫酸乙酰肝素解聚的程度。 Carbohydr Polym 152:592-597 。
  2. Carnachan,SC和Hinkley,SFR(2017)。硫酸肝素鉴定和表征方法二酶解解和SAX-HPLC分析以确定二糖组成。生物样品7(07):e2197。
  3. Guerrini,M.,Beccati,D.,Shriver,Z.,Naggi,A.,Viswanathan,K.,Bisio,A.,Capila,I.,Lansing,JC,Guglieri,S.,Fraser,B.,Al -Hakim,A.,Gunay,NS,Zhang,Z.,Robinson,L.,Buhse,L.,Nasr,M.,Woodcock,J.,Langer,R.,Venkataraman,G.,Linhardt,RJ,Casu ,B.,Torri,G.和Sasisekharan,R。(2008)。硫酸化硫酸软骨素是与不良临床事件相关的肝素中的污染物。 Nat Biotechnol 26(6):669-675。
  4. Guerrini,M.,Naggi,A.,Guglieri,S.,Santarsiero,R。和Torri,G。(2005)。< a class ="ke-insertfile"href ="https://www.ncbi。 nlm.nih.gov/pubmed/15649373"target ="_ blank">复合糖胺聚糖:通过二维核磁共振光谱分析取代模式。分析生化 337(1):3547 。
  5. Keire,DA,Mans,DJ,Ye,H.,Kolinski,RE和Buhse,LF(2010)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih。通过1 H NMR,SAX-HPLC和抗凝时间方法测定肝素中可能的经济动力的添加剂或天然杂质水平。"药物生物学分析" em> 52(5):656-664。
  6. Liu,H.,Zhang,Z.和Linhardt,RJ(2009)。从肝素污染中获得的经验教训。Nat Prod Rep 26(3):313-321。
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引用:Carnachan, S. M. and Hinkley, S. F. (2017). Heparan Sulfate Identification and Characterisation: Method I. Heparan Sulfate Identification by NMR Analysis. Bio-protocol 7(7): e2196. DOI: 10.21769/BioProtoc.2196.

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