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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   

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参见作者原研究论文

本实验方案简略版
Carbohydrate Polymers
Nov 2016

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

  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

  1. Digest procedure
    1. Enzyme solutions (50 µl) as received (-80 °C) are permitted to warm to RT then diluted to 500 µl with the appropriate storage buffer giving, nominally, 0.5 IU per 500 µl. Aliquots (5 µl) are stored frozen (-80 °C) until needed. These 5 µl aliquots contain 5 mIU of activity according to the supplier's specification. However, when the enzyme activities were assayed using Celsus heparan sulfate and the digestion conditions described below, they were found to be 2.8, 5.0 and 13.8 mIU for Heparin lyases (HL) I, II and III, respectively. HL activities should be independently verified prior to starting the depolymerisation procedure.
    2. Heparin or HS (1 mg, in duplicate) is dissolved in a microcentrifuge tube (1.5 ml) with the digestion media (470 µl).
    3. Sequential enzyme addition is completed.
      1. HL I (one 5 µl aliquot) is added and the solution incubated (37 °C, 2 h, inversion at 9 rpm).
      2. HL III (5 µl) is added and incubation continued (1 h).
      3. HL II (5 µl) is added and incubation continued (18 h).
      4. All three enzymes (an additional 5 µl of each) are added at the same time and the solution incubated (gentle inversion, 9 rpm) for a further 24 h.
    4. Digests are terminated by heating (100 °C, 5 min).
    5. Samples are centrifuged (14,000 x g, 10 min) and the supernatant recovered.
    6. The supernatants (2 mg/ml) are then accurately diluted with water to give 100 µg/ml solutions, by taking 50 µl of supernatant and adding 950 µl of water. The 100 µg/ml solutions are then filtered (0.22 µm) and analysed.

  2. Analysis procedure
    1. HPLC is completed using a binary solvent system and a flow rate of 1 ml/min at 40 °C. Following long-term storage and prior to injecting the first sample, the column is conditioned by flushing thoroughly with water, then with 100% B for 10 min before equilibrating the column with the starting conditions (0% B) for 2 min. Digest and standard solutions (see below) are injected by autosampler (50 µl injection volume) and the disaccharides separated using a gradient system, 0% B (1 min), 0% to 35% B (over 31 min), 35% to 65% B (over 15 min), 100% B (10 min). Post analysis column re-equilibration 0% B (3 min).
    2. Disaccharides are detected by absorbance at their absorbance maxima of 232 nm.
    3. Standard curves for each disaccharide standard are prepared by dissolution of each of the twelve heparin-derived disaccharide standards (supplied as 1.0 mg samples) to give 1 mg/ml stock solutions in water. These stock solutions are in turn used to prepare a twelve disaccharide standard mix containing 20 µg/ml of each standard which was subsequently serially diluted to prepare solutions containing 10, 5, 2.5, 1.25, 0.625 and 0.3125 µg/ml of each disaccharide. These standard solutions are analysed with each batch of digests (see Figure 1). Linear calibration curves (concentration vs peak area) with R2 values of > 0.998 can be generated.
    4. For the enzyme digests, data reported are the average of two digests. Each digest is analysed by HPLC in duplicate (Figure 1). Disaccharides in the digest can be identified by elution times relative to the twelve heparin-derived disaccharide standards and quantified using the calibration curves.


      Figure 1. HPLC chromatogram showing resolution of the twelve heparin-derived disaccharides present in the standard mix. Retention times and species: 2.158 min, Δ-UA-GlcN; 5.092 min, Δ-UA-GlcNAc; 6.567 min, Δ-UA-GlcN(6S); 8.425 min, Δ-UA(2S)-GlcN; 12.175 min, Δ-UA-GlcNS; 14.817 min, Δ-UA-GlcNAc(6S); 16.942 min, Δ-UA(2S)-GlcNAc; 24.092 min, Δ-UA(2S)-GlcN(6S); 26.233 min, Δ-UA-GlcNS(6S); 28.950 min, Δ-UA(2S)-GlcNS; 36.208 min, Δ-UA(2S)-GlcNAc(6S); 44.775 min, Δ-UA(2S)-GlcNS(6S). The elution order of the twelve disaccharide standards was initially determined by running each species separately under the chromatographic conditions described above. 

Data analysis

  1. A typical HPLC chromatogram for an enzyme digest of heparan sulfate is displayed in Figure 2.


    Figure 2. HPLC chromatogram of a typical enzymatic digest of commercial porcine mucosal heparan sulfate showing separation of the Δ-disaccharide constituents. Peaks marked with an asterisk represent uncharacterized species that appear reproducibly in chromatographic runs.

  2. Using the standard curves and integrated areas for each of the disaccharide peaks it is possible to determine the proportion of each disaccharide present in the sample. This data can be used to calculate the normalised proportion of each disaccharide and the total mass of disaccharides produced by the digestion (Table 1).
  3. The compositions reported are the mean from the four HPLC runs; duplicate analysis of the two digests completed on each sample.

    Table 1. Disaccharide normalised composition and mass recovery for a commercial porcine mucosal heparan sulfate

Notes

  1. Enzyme activity is tested using the same conditions as above and using the reference HS specified in the materials and methods above.
  2. The split peaks observed for some disaccharides in the HPLC traces have been attributed to the two anomeric forms of the reducing sugar moiety. It is important to be consistent and either quantitate individual anomeric species (Beccati et al., 2016), or, as in this protocol, the peak areas are pooled for quantifying disaccharides from sample digests.
  3. The disaccharide with an N-unsubstituted amine group and no sulfation (ΔUA-GlcN) can be problematic to quantify on this ion-exchange column. Care should be taken to ensure that this species, which is poorly-retained on the column, is not co-eluting with the solvent front and providing erroneous values.
  4. Some additional peaks were reproducibly detected in the chromatograms of digests (see species labelled * on Figure 2). These are tentatively attributed to tetrasaccharides resulting from incomplete enzymatic depolymerisation but have not been characterised fully.
  5. The data generated using this SAX-HPLC method can be used to quantify the total mass of unsaturated disaccharides produced by heparin lyase depolymerisation and, in theory, should provide a guide as to the purity of the heparan sulfate. However, if heparin or specific linkages not susceptible to enzymatic depolymerisation are present, then the extent of depolymerisation determined using this method can be misleading in a purity calculation (Yamada et al., 1993; Carnachan et al., 2016; Mulloy et al., 2016).
  6. During the analysis of an unknown HS material, the reference HS described in this protocol (Celsus) is always digested and analysed in duplicate to ensure that the protocol is operating within expectations.

Recipes

  1. Enzyme storage buffer
    Heparin lyases (HL) are stored with BSA (0.1%, w/v) in 50 mM sodium phosphate buffer prepared by the addition of Na2HPO4·H2O to RO water with adjustment of the pH by the addition of concentrated phosphoric acid to give pH 7.1 for HL I and II and pH 7.6 for HL III. 100 mM NaCl is added to the storage buffer for HL I only
  2. Digestion media
    NaOAc (100 mM) with CaOAc (2 mM) and adjustment of the pH to 7.0 by the addition of 1 N NaOH or HCl
  3. Mobile phases for HPLC analysis
    Water (eluent A) and 2 M NaCl (aqueous, eluent B) are both prepared and adjusted to pH 3.5 using 1 N HCl immediately prior to analysis

Acknowledgments

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.

References

  1. Beccati, D., Lech, M., Ozug, J., Gunay, N. S., Wang, J., Sun, E. Y., Pradines, J. R., Farutin, V., Shriver, Z., Kaundinya, G. V. and Capila, I. (2016). An integrated approach using orthogonal analytical techniques to characterize heparan sulfate structure. Glycoconj J 34(1): 107-117.
  2. 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.
  3. Carnachan, S. M. and Hinkley, S. F. R. (2017). Heparan Sulfate Identification and Characterisation: Method I. Heparan Sulfate Identification by NMR analysis. Bio-protocl 7(07): e2196.
  4. Mulloy, B., Wu, N., Gyapon-Quast, F., Lin, L., Zhang, F., Pickering, M. C., Linhardt, R. J., Feizi, T. and Chai, W. (2016). Abnormally high content of free glucosamine residues identified in a preparation of commercially available porcine intestinal heparan sulfate. Anal Chem 88(13): 6648-6652.
  5. Skidmore, M. A., Guimond, S. E., Dumax-Vorzet, A. F., Atrih, A., Yates, E. A. and Turnbull, J. E. (2006). High sensitivity separation and detection of heparan sulfate disaccharides. J Chromatogr A 1135 (1): 52-56.
  6. Skidmore, M. A., Guimond, S. E., Dumax-Vorzet, A. F., Yates, E. A. and Turnbull, J. E. (2010). Disaccharide compositional analysis of heparan sulfate and heparin polysaccharides using UV or high-sensitivity fluorescence (BODIPY) detection. Nat Protoc 5 (12): 1983-1992.
  7. Yamada, S., Yoshida, K., Sugiura, M., Sugahara, K., Khoo, K. H., Morris, H. R. and Dell, A. (1993). Structural studies on the bacterial lyase-resistant tetrasaccharides derived from the antithrombin III-binding site of porcine intestinal heparin. J Biol Chem 268(7): 4780-4787.

简介

从复杂基质中纯化硫酸肝素(HS),并且通常没有完全表征以验证其分配。 通过酶促解聚和随后的强阴离子交换高效液相色谱(SAX-HPLC)分析和定量所得二糖来描述肝素和硫酸乙酰肝素是评估该类化合物的结构组成的关键工具。 该方案详述了通过酶促解聚和SAX-HPLC分析可重复地确定硫酸乙酰肝素的二糖组成的方法。 Carnachan和Hinkley(2017)可以找到一种用于硫酸乙酰肝素鉴定和表征的补充方法。

肝素和HS的结构分析存在一些方法。 该方案旨在为肝素和HS的酶解解提供优化的方法,并分析和定量其中产生的二糖。 非常少的公开分析考虑了总组合物的所有方面,包括解聚程度与获得的二糖组合的结合(Skidmore等人,2006和2010; Carnachan等人。,2016)。 当样品随后用于总是剂量依赖性的生物测定中时,这尤其令人担忧。 本文描述的酶程序是研究调查最佳酶活性和HS解聚所需条件的详细研究的最终结果(Carnachan等人,2016)。 该程序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, 酶解聚合, HPLC

材料和试剂

  1. 微量离心管(1.5ml)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:3456)
  2. 注射器过滤器(0.22μm)(亲水性PTFE)(MicroScience Hydraflow,目录号:MS SF13HY022)
  3. 玻璃HPLC小瓶(2ml)(Thermo Fisher Scientific,目录号:THC11090500)
  4. 隔垫盖(Thermo Fisher Scientific,目录号:THC11090500)
  5. 硫酸肝素(来自猪粘膜)(Celsus Laboratories,目录号:HO-03103)
  6. 肝素裂解酶I(肝素酶I或肝素酶III,EC 4.2.2.7,[0.5IU])(IBEX Technologies,目录号:50-010)
  7. 肝素裂解酶II(肝素酶II或肝素酶II,无EC编号,[0.5IU])(IBEX Technologies,目录号:50-011)
  8. 肝素裂解酶III(肝素酶III或肝素酶I,EC 4.2.2.8,[0.5IU])(IBEX Technologies,目录号:50-012)
  9. 通过细菌肝素酶对高级猪肝素(Iduron,目录号:HD001-HD008和HD010-HD013)的作用产生的肝素二糖标准
  10. 水(蒸馏)(Sartorius arium ®超UV过滤;>18.5MΩ)
  11. 牛血清白蛋白(BSA)(Thermo Fisher Scientific,Gibco TM,目录号:30060727)
  12. Na 2 HPO 4 H 2 O(EMD Millipore,目录号:106586)
  13. NaOAc(ACS)(EMD Millipore,目录号:1062680250)
  14. CaOAc·xH 2 O(Sigma-Aldrich,目录号:25011)
  15. 氯化钠(NaCl)(99.99%)(EMD Millipore,Suprapur ,目录号:1.06406)
  16. 用于调节pH值的1N盐水(HCl)(37%)(EMD Millipore,目录号:100317)和NaOH(Sigma-Aldrich,目录号:S8045)
  17. 磷酸(85%)(Sigma-Aldrich,目录号:79606)
  18. 酶储存缓冲液(参见食谱)
  19. 消化媒体(见食谱)
  20. HPLC分析的流动相(见配方)

设备

  1. 高效液相色谱(HPLC)机器(Infinity与多波长检测器)(Agilent Technologies,型号:1260)
  2. 使用保护柱(Thermo Fisher,强力阴离子交换HPLC柱(4×250mm)(ProPac TM PA1,Thermo Fisher Scientific,Thermo Scientific TM,目录号:039658) Scientific,Thermo Scientific TM ,目录号:039657)
  3. 移液器(0.5-10μl,20-200μl和100-1,000μl)(Eppendorf)
  4. 孵化器(BINDER,型号:KBF-115)
  5. 旋转器(Cole-Parmer,Stuart,型号:SB3)
  6. 离心机(Eppendorf,型号:Minispin ® plus,目录号:5453000011)

程序

  1. 摘要程序
    1. 允许接受的酶溶液(50μl)(-80℃)温热至室温,然后用合适的储存缓冲液稀释至500μl,标称每500μl为0.5IU。将等分试样(5μl)冷冻保存(-80℃)直至需要。然而,这5μl等分试样根据供应商的规格含有5mIU的活性;当使用硫酸肝素硫酸酶和下述消化条件测定酶活性时,分别发现肝素裂解酶(HL)I,II和III分别为2.8,5.0和13.8mIU。在开始解聚过程之前,HL活性应独立验证。
    2. 用消化培养基(470μl)将肝素或HS(1mg,一式两份)溶于微量离心管(1.5ml)中。
    3. 顺序酶添加完成。
      1. 加入HL I(5μl等分试样),将溶液温育(37℃,2h,以9rpm倒置)。
      2. 加入HL III(5μl)并继续培养(1小时)
      3. 加入HL II(5μl),继续培养(18 h)
      4. 同时加入所有三种酶(每种另外5μl),将溶液孵育(轻轻倒置,9rpm)另外24小时。
    4. 消化通过加热终止(100℃,5分钟)。
    5. 将样品离心(14,000 x g,10分钟),上清液回收。
    6. 然后将上清液(2mg/ml)用水精确稀释,得到100μg/ml溶液,通过取50μl上清液并加入950μl水。然后将100μg/ml溶液过滤(0.22μm)并分析。

  2. 分析程序
    1. 使用二元溶剂系统和在40℃下1ml/min的流速完成HPLC。在长期储存后,在注入第一个样品之前,将柱用水充分冲洗,然后用100%B冲洗10分钟,然后用起始条件(0%B)平衡柱2分钟。采用自动进样器(50μl注射体积)注射摘要和标准溶液(参见下文),使用0%B(1分钟),0%至35%B(超过31分钟),35%梯度系统分离二糖,35% 65%B(超过15分钟),100%B(10分钟)。后分析柱重新平衡0%B(3分钟)
    2. 通过吸光度在232nm的最大吸光度下检测二糖
    3. 通过溶解12种肝素衍生的二糖标准品(作为1.0mg样品提供)中的每一种制备每种二糖标准物的标准曲线,得到1mg/ml储备液在水中。这些储备溶液又用于制备含有20μg/ml每种标准物的十二个二糖标准混合物,随后连续稀释以制备每种二糖含有10,5,25,225,0.625和0.3125μg/ml的溶液。这些标准解决方案用每批消化分析(见图1)进行分析。线性校准曲线(浓度对峰面积)R< 2>值>可以生成0.998。
    4. 对于酶消化,报告的数据是两个消化的平均值。每份摘要通过HPLC分析一式两份(图1)。可以通过相对于十二肝素衍生的二糖标准品的洗脱时间来鉴定消化中的二糖并使用校准曲线进行定量。


      图1.显示标准混合物中存在的十二个肝素衍生的二糖的分辨率的HPLC色谱图。保留时间和种类:2.158分钟,Δ-UA-GlcN; 5.092分钟,Δ-UA-GlcNAc; 6.567分钟,Δ-UA-GlcN(6S); 8.425分钟,Δ-UA(2S)-GlcN; 12.175分钟,Δ-UA-GlcNS; 14.817分钟,Δ-UA-GlcNAc(6S); 16.942分钟,Δ-UA(2S)-GlcNAc; 24.092分钟,Δ-UA(2S)-GlcN(6S); 26.233分钟,Δ-UA-GlcNS(6S); 28.950分钟,Δ-UA(2S)-GlcNS; 36.208分钟,Δ-UA(2S)-GlcNAc(6S); 44.775分钟,Δ-UA(2S)-GlcNS(6S)。最初通过在上述色谱条件下分别运行各物种来确定十二二糖标准品的洗脱顺序。 

数据分析

  1. 硫酸乙酰肝素酶消化物的典型HPLC色谱图如图2所示

    图2.商业猪粘膜硫酸乙酰肝素的典型酶消化物的HPLC色谱图,显示了Δ-二糖成分的分离。标有星号的峰代表在色谱运行中可重复出现的未表征的物质。 >
  2. 使用每个二糖峰的标准曲线和积分区域,可以确定样品中存在的每种二糖的比例。该数据可用于计算每种二糖的归一化比例和通过消化产生的二糖的总质量(表1)。
  3. 报道的组合物是四个HPLC运行的平均值;对每个样本完成的两个摘要的重复分析。

    表1.商业猪粘膜硫酸乙酰肝素的二糖标准化组成和质量回收

笔记

  1. 使用与上述相同的条件并使用上述材料和方法中指定的参考HS测试酶活性。
  2. 在HPLC痕迹中观察到的一些二糖的分裂峰归因于还原糖部分的两个端基异构形式。重要的是要保持一致并定量个别的异头物种(Beccati等人,2016),或者如本协议中一样,将峰面积合并用于从样品消化物定量二糖。 >
  3. 具有N-未取代的胺基并且没有硫酸化的二糖(ΔUA-GlcN)在该离子交换柱上定量是有问题的。应注意确保在柱上不良保留的物种不与溶剂前体共洗脱并提供错误的值。
  4. 在消化物的色谱图中再现性地检测到一些额外的峰(参见图2上标记的物质)。这些暂时归因于由不完全的酶解聚而产生的四糖,但尚未被充分表征
  5. 使用该SAX-HPLC方法产生的数据可用于量化由肝素裂解酶解聚产生的不饱和二糖的总质量,理论上应提供关于硫酸乙酰肝素的纯度的指导。然而,如果存在肝素或不易于酶促解聚的特异性连接,则使用该方法测定的解聚程度在纯度计算中可能是误导的(Yamada等人,1993; Carnachan et al。 ,2016; Mulloy等人,2016)。
  6. 在分析未知HS材料期间,本协议(Celsus)中描述的参考HS总是被一次消化和分析,以确保该协议在预期内运行。

食谱

  1. 酶储存缓冲液
    肝素裂解酶(HL)与BSA(0.1%,w/v)一起储存在通过加入Na 2 HPO 4 H·H 2 O 3制备的50mM磷酸钠缓冲液中> 2 O到RO水,通过加入浓磷酸调节pH值,给出HL I和II的pH 7.1,HL III的pH 7.6。将100mM NaCl加入仅用于HL I的存储缓冲液中
  2. 消化媒体
    NaOAc(100mM)与CaOAc(2mM),通过加入1N NaOH或HCl调节pH至7.0
  3. 用于HPLC分析的流动相
    制备水(洗脱剂A)和2M NaCl(水溶液,洗脱剂B),并在分析前立即用1N HCl调节至pH 3.5。

致谢

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。

参考文献

  1. Beccati,D.,Lech,M.,Ozug,J.,Gunay,NS,Wang,J.,Sun,EY,Pradines,JR,Farutin,V.,Shriver,Z.,Kaundinya,GV和Capila,I. (2016)。使用正交分析技术的综合方法表征硫酸乙酰肝素结构。糖酵母J 34(1):107-117。
  2. 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 。
  3. Carnachan,SM和Hinkley,SFR(2017)。硫酸肝素鉴定和表征:方法I.通过NMR分析的硫酸肝素鉴定。生物样品7(07):e2196。
  4. Mulloy,B.,Wu,N.,Gyapon-Quast,F.,Lin,L.,Zhang,F.,Pickering,MC,Linhardt,RJ,Feizi,T.and Chai,W。(2016) 在市售的制剂中鉴定的游离氨基葡萄糖残留异常高猪肝素硫酸乙酰肝素。分析化学 88(13):6648-6652。
  5. Skidmore,MA,Guimond,SE,Dumax-Vorzet,AF,Atrih,A.,Yates,EA和Turnbull,JE(2006)。  硫酸肝素二糖的高灵敏度分离和检测。 Chromatogr A 1135(1):52-56 。
  6. Skidmore,MA,Guimond,SE,Dumax-Vorzet,AF,Yates,EA和Turnbull,JE(2010)。  源自猪肠肝素抗凝血酶III结合位点的细菌裂解酶四糖的结构研究。 J Biol Chem 268(7):4780-4787。
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引用:Carnachan, S. M. and Hinkley, S. F. (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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