SAX-HPLC, GLYCOSYL COMPOSITION, AND NMR OF 7 SAMPLES

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 用肝素酶I、II和III完全解聚为二糖 将20 μ L等份的20 g/L低分子量肝素（AVT和HSP）水溶液用80 μ L 100 mM NaOAc缓冲液（pH 7）稀释，该缓冲液含有2 mM乙酸钙和1 g/L BSA。 然后用20 μ L肝素酶I、II和III（各0.5 U/mL）在10 mM磷酸钾缓冲液（pH 7，含2 g/L BSA）中的混合物处理混合物，并在23 ℃下孵育。 48小时后，通过将混合物煮沸2分钟来淬灭反应。 肝素酶I、肝素酶II或两者混合物部分解聚（仅批次93485、93689、12074、15057和DA 1194） 所有4份样本均单独用肝素酶I、肝素酶II以及肝素酶I和肝素酶II的混合物处理。 将150 uL等份的2 g/L低分子量肝素（AVT和HSP）溶液（溶于含有2 mM醋酸钙的20 mM NH 4 AAC缓冲液（pH 7）中）用20 mU肝素酶（I或II或I和II）处理，并在37 ° C下孵育。 24小时后，通过将混合物煮沸2分钟来淬灭反应。 减少 将60 uL肝素酶消化样品用20 uL 30 g/L NaBH 4水溶液在23 ℃下处理至少24 h。 二糖混合物的SAX-HPLC SAX-HPLC在Agilent系统上进行，使用4.6沃茨Spherisorb分析柱，粒径为5 μ m，温度为45 ℃。使用以下系统通过232 nm处的UV吸光度检测分析物。 溶剂A：2.5 mM磷酸钠，pH 3.5;溶剂B：2.5 mM磷酸钠，pH 3.5，1.2 M NaClO 4。在98% A下5分钟后，施加线性梯度以在50分钟后达到50% B。 使用SAX-HPLC色谱图的积分值，根据公式1测定以脱水形式终止的链的百分比（表1-4）。 （eq.第一章 A = SAX色谱图的峰面积 MW =依诺肝素的质均分子量= 4400 g/mol（来自Opocrin） MMi =各二糖或四糖的分子量 部分解聚产物的SAX-HPLC 使用与上述相同的条件，但修改了缓冲液梯度：在97% A下5 min后，应用线性梯度以在50 min时达到50% B，然后应用线性梯度以在82 min时达到100% B。 糖基组成 糖基组成分析进行了组合的气相色谱/质谱（GC/MS）的全-O-三甲基甲硅烷基（TMS）衍生物的单糖甲基糖苷的样品通过酸性甲醇分解。 通过重复（3 X）甲醇分解和再-N-乙酰化，将1 mg各样品完全分解和解聚。对于每次重复，将干燥样品与0.5 mL 3 M HCl的MeOH溶液一起加热至100 ℃ 2 h，蒸发，用200 uL MeOH、100 uL吡啶和100 uL乙酸酐处理，并在空气流下干燥。 在最后的N-乙酰化步骤之后，将样品与200 uL Tri-Sil在80 ° C下加热20 min。 TMS甲基糖苷的GC/MS分析在与5970 MSD连接的HP 5890 GC上进行，使用Supelco DB-1熔融石英毛细管柱（30 m ² 0.25 mm ID）。 NMR光谱 样品通过冻干从D2 O进行氘交换，并溶解在700 μ L D2 O（99.96%D）中。 在Varian Inova-500 MHz光谱仪上在313 K（40 ℃）下获得梯度异质结单量子相干（gHSQC）光谱。 相对于DSS（S=0.00 ppm）测量质子化学位移。 记录光谱，质子维度的光谱宽度为3 kHz，碳维度的光谱宽度为15-20 kHz（取决于样品量和可用的仪器时间）。 采集时间为0.20 s，采集200个增量，每个增量64-128次扫描。 单键C-H偶合常数设定为140 Hz。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Complete depolymerization to disaccharides with heparinases I, II, and III A 20 uL aliquot of a 20 g/L solution of low molecular weight heparin (AVT and HSP) in water was diluted with 80 uL 100 mM NaOAc buffer, pH 7, containing 2 mM calcium acetate and 1 g/L BSA. The mixture was then treated with 20 uL of a mixture of heparinases I, II, and III (0.5 U/mL each) in 10 mM potassium phosphate buffer, pH 7, containing 2 g/L BSA and incubated at 23 ¿C. After 48 h, the reaction was quenched by boiling the mixture for 2 min. Partial depolymerization with heparinase I, heparinase II, or a mixture of both (Lots 93485, 93689, 12074, 15057, and DA1194 only) All 4 samples were treated with heparinase I alone, heparinase II alone, and with a mixture of heparinases I and II. A 150 uL aliquot of a 2 g/L solution of low molecular weight heparin (AVT and HSP) in 20 mM NH4OAc buffer, pH 7, containing 2 mM calcium acetate was treated with 20 mU of heparinase (I or II or I and II) and incubated at 37 ¿C. After 24 h, the reaction was quenched by boiling the mixture for 2 min. Reduction A 60 uL portion of the heparinase-digested sample was treated with 20 uL of a 30 g/L solution of NaBH4 in H2O for at least 24 h at 23 ¿C. SAX-HPLC of disaccharide mixture SAX-HPLC was carried out on an Agilent system using a 4.6¿250 mm Waters Spherisorb analytical column with 5um particle size at 45 ¿C. Analytes were detected by their UV absorbance at 232 nm using the following system. Solvent A: 2.5 mM Na-phosphate, pH 3.5; Solvent B: 2.5 mM Na-phosphate, pH 3.5, 1.2 M NaClO4. After 5 min at 98 % A, a linear gradient was applied to reach 50 % B after 50 min. The flow rate was 1.4 mL/min. The percentage of chains terminating in anhydro forms was determined according to equation 1 using the integration values of the SAX-HPLC chromatograms (Tables 1-4). (eq. 1) A = peak area from the SAX chromatogram MW = mass average molecular weight of enoxaparin = 4400 g/mol (from Opocrin) MMi = molecular weight of each individual di- or tetrasaccharide SAX-HPLC of partially depolymerized product The same conditions as above were used with a modification of the buffer gradient: After 5 min at 97 % A, a linear gradient was applied to reach 50 % B at 50 min, and then a linear gradient was applied to reach 100 % B at 82 min. Glycosyl composition Glycosyl composition analysis was performed by combined gas chromatography/mass spectrometry (GC/MS) of the per-O-trimethylsilyl (TMS) derivatives of the monosaccharide methyl glycosides produced from the sample by acidic methanolysis. 1mg of each samples was completely desulfated and depolymerized by repeated (3 X) methanolysis and re-N-acetylation. For each repetition, the dry sample was heated to 100 ¿C with 0.5 mL 3 M HCl in MeOH for 2 h, evaporated, treated with 200 uL MeOH, 100 uL pyridine, and 100 uL acetic anhydride, and dried under a stream of air. After the last N-acetylation step, the sample was heated for 20 min at 80 ¿C with 200 uL Tri-Sil. GC/MS analysis of the TMS methyl glycosides was performed on an HP 5890 GC interfaced to a 5970 MSD, using a Supelco DB-1 fused silica capillary column (30m ¿ 0.25 mm ID). NMR Spectroscopy The sample was deuterium-exchanged by lyophilization from D2O and dissolved in 700 uL D2O (99.96 % D). Gradient heteronuclear single quantum coherence (gHSQC) spectra were acquired on a Varian Inova-500 MHz spectrometer at 313 K (40 ¿C). Proton chemical shifts were measured relative to DSS (S=0.00 ppm). The spectra were recorded with a spectral width in the proton dimension of 3 kHz and in the carbon dimension of 15-20 kHz (depending on sample amount and instrument time available). The acquisition time was 0.20 s, and 200 increments were collected with 64-128 scans each. The one-bond C-H coupling constant was set to 140 Hz.