SAX-HPLC OF 6 SAMPLES

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 方法： 肝素酶消化 用含2 mM醋酸钙和1g/L牛血清白蛋白的80°L 100 mM NaOAc缓冲液，pH 7，稀释成20g/L低分子肝素水溶液。然后用肝素酶I、II和III的混合物20°L(各0.5U/mL)在10 mM磷酸二氢钾缓冲液中处理，pH 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60°L肝素酶消化的样品用20°L 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，粒径为5 mm，柱温为25℃，检测波长为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氯化钠。在97%A下5min后，施加线性梯度以在50min后达到85%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。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Methods: Heparinase digestion A 20 ¿L aliquot of a 20 g/L solution of low molecular weight heparin (AVT and HSP) in water was diluted with 80 ¿L 100 mM NaOAc buffer, pH 7, containing 2 mM calcium acetate and 1 g/L BSA. The mixture was then treated with 20 ¿L 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 ¿L portion of the heparinase-digested sample was treated with 20 ¿L 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 5¿m particle size at 25 ¿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 97 % 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 .