SAX-HPLC AND NMR ANALYSIS OF EIGHT SAMPLES

八个样品的 SAX-HPLC 和 NMR 分析

• 批准号: 7957555
• 负责人: Parastoo Azadi
• 金额: $ 0.22万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7957555
• 关键词: Aliquot; Area; Buffers; Computer Retrieval of Information on Scientific Projects Database; Digestion; Disaccharides; Enoxaparin; Equation; Funding; Furuncles; Grant; Heparin Lyase; Incubated; Individual; Institution; Low-Molecular-Weight Heparin; Molecular Weight; Particle Size; Phosphate Buffer; Potassium Phosphate; Reaction; Research; Research Personnel; Resources; Sampling; Solutions; Solvents; Source; System; United States National Institutes of Health; Water; calcium acetate; inorganic phosphate

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 (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. 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 4.6x250 mm Waters Spherisorb analytical column with 5 um 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 NaCl. After 5 min at 95 % A, a linear gradient was applied to reach 85 % 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 1-8). (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

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 肝素酶消化 将20 g/l的低分子量肝素（AVT和HSP）溶液的20 uL等分试样用80 ul 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，将混合物用20 ul的肝素I，II和III和III（每分0.5 u/ml）的混合在10 mM磷酸钾缓冲液中，pH 7，含有2 g/L BSA，并在23 h中孵育，并在48 h时孵育。在48 H时，将反应煮沸2分钟，将反应淬灭。 减少 将60 ul的肝素酶消化样品用20 uL在H2O中的30 g/l溶液在23€c时至少24小时处理24小时。 SAX-HPLC SAX-HPLC使用4.6x250 mM Waters Spherisorb分析柱在安捷伦系统上进行，其粒径为45€�C。使用以下系统在232 nm处通过其UV吸光度检测到分析物。 溶剂A：2.5 mm Na磷酸盐，pH 3.5;溶剂B：2.5 mm Na-磷酸盐，pH 3.5，1.2 m NaCl。在95％A处5分钟后，将线性梯度应用于50分钟后达到85％B。流速为1.4 mL/min。 使用公式1使用SAX-HPLC色谱图（表1-8）根据公式1确定止血形式中终止的链的百分比。 （等式1） A =萨克斯色谱图的峰面积 MW =依诺肝素的质量平均分子量= 4400 g/mol（来自Opocrin） MMI =每个单独的二糖的分子量