STRUCTURAL ANALYSIS OF ONE LOW MOLECULAR WEIGHT HEPARIN SAMPLE

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。 肝素酶消化 将20 μ L等份的20 g/L低分子量肝素（Clexane，07004）水溶液用80 μ L 100 mM NaOAc缓冲液（pH 7）稀释，其中含有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。 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。 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70% 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

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. Heparinase digestion A 20 uL aliquot of a 20 g/L solution of low molecular weight heparin (Clexane, 07004) 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. 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 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 70 % 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