STRUCTURAL ANALYSIS OF ONE LOW MOLECULAR WEIGHT HEPARIN SAMPLE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 肝素酶消化 用含2 mM醋酸钙和1g/L牛血清白蛋白的80ul 100 mm NaOAc缓冲液，pH 7，稀释20ul等份低分子肝素(来克索，07004)溶液。然后用20ul的肝素酶I、II和III的混合物(各0.5U/mL)在10 mM磷酸二氢钾缓冲液中处理，pH为7，含有2g/L牛血清白蛋白，在23℃孵育，48h后，将混合物煮沸2min，使反应熄灭。 减少 将60ul肝素酶消化的样品用20ul的30g/L的NaBH4水溶液在23℃下处理至少24小时。 SAX-高效液相色谱 采用Agilent系统，色谱柱为4.6 mm Waters Spherisorb，粒度为5微米，检测波长232 nm，检测波长232 nm。 溶剂A：2.5 mM磷酸二氢钠，pH 3.5；溶剂B：2.5 mM磷酸二钠，pH 3.5，1.2M NaClO4。在98%A下5min后，施加线性梯度以在50min后达到70%B。流速为1.4毫升/分钟。 根据公式1，使用SAX-HPLC色谱图的积分值来确定以脱水形式终止的链的百分比(表1-4)。 (情商。1) A=SAX色谱图的峰面积 Mw=依诺肝素的质量平均分子量=4400g/mol(来自Opocrin) Mmi=每个二糖或四糖的相对分子质量

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