DISACCHARIDE ANALYSIS BY SAX-HPLC OF 3 SAMPLES

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 研究方法： 肝素酶消化 20磅用80 μ L 100 mM NaOAc缓冲液（pH 7）（含2 mM乙酸钙和1 g/L BSA）稀释20 g/L GAG样品水溶液的1 L等分试样。 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 μ C下孵育。 48小时后，通过将混合物煮沸2分钟来淬灭反应。 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，25 ℃，使用以下梯度： 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 NaCl。 如（1）所述，通过柱后衍生进行检测。简言之，从二元HPLC泵向来自柱的洗脱液中以0.5 mL/min加入0.25 M NaOH和1% 2-氰基乙酰胺的1：1混合物。然后在10-m反应盘管中将洗脱液加热至120 ℃，然后在30-cm冷却盘管中冷却，并导入Shimadzu荧光检测器。激发波长为346 nm，发射波长为410 nm。

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 the GAG sample 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. 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 using the following gradient: Solvent A: 2.5 mM Na-phosphate, pH 3.5 Solvent B: 2.5 mM Na-phosphate, pH 3.5, 1.2 M NaCl. Detection was performed by post-column derivatization as described (1). Briefly, to the eluent from the column was added, from a binary HPLC pump, a 1:1 mixture of 0.25 M NaOH and 1 % 2-cyanoacetamide at 0.5 mL/min. The eluent was then heated to 120 ¿C in a 10-m reaction coil, followed by cooling in a 30-cm cooling coil, and directed into a Shimadzu fluorescence detector. Excitation wavelength was 346 nm and emission wavelength was 410 nm.