GAG PROFILE OF 2 SAMPLES BY SAX-HPLC

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： 软骨素酶消化 将含有0.1 mg/mLGAG、50mMNH4OAc缓冲液、pH 6(葛根素酶和透明质酸酶)或pH 7(软骨素酶和肝素酶)、0.1mU/mL或20mU/mL(仅透明质酸酶)的L总体积的溶液在37℃下孵育24 h，在透明质酸酶消化的情况下，在55℃下再孵育2 h，加热到100℃灭活2分钟，样品进行高效液相色谱分析。 使用了以下酶： 软骨素酶ABC(肝素F. 软骨素酶AC(A.auresens，Sigma) 肝素酶I、II和III(F·肝素，格兰宾) 透明质酸酶(S.hyalurolyticus，Northstar) 角聚糖酶II(芽孢杆菌属，北极星属) SAX-高效液相色谱 SAX-HPLC在Agilent系统上进行，使用4.6？250 mm Waters Spherisorb分析柱，粒度为5？m，25℃。 溶剂A：2.5 mM磷酸二氢钠，pH 3.5 溶剂B：2.5 mM磷酸二氢钠，pH 3.5，1.2M氯化钠。 流速为1.0mL/min。 进样量为10？L。 采用柱后衍生化方法进行检测。简单地说，将0.25M氢氧化钠和1%2-氰基乙酰胺的1：1混合物加入到柱上的洗脱液中，以0.5mL/min的速度从二元高效液相色谱泵中加入。洗脱液在10米的反应线圈中加热到120℃，然后在50厘米的冷却线圈中冷却，并直接进入岛津荧光检测器。激发波长为346 nm，发射波长为410 nm。 商业标准双糖(DExtra实验室)用于校准肝素酶和软骨素酶消化混合物。对于其他混合物，以酶消化的正品硫酸角蛋白(AMS Biotech)和透明质酸(Sigma)样品为标准。

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: Chondroitinase digestion A solution (100 ¿L total volume) containing 0.1 mg/mL GAG, 50 mM NH4OAc buffer, pH 6 (kratanase and hyaluronidase) or pH 7 (chondroitinase and heparinase), and 0.1 mU/mL or 20 mU/mL (hyaluronidase only) GAG lyase was incubated at 37 ¿C for 24 h. In the case of hyaluronidase digestion, the samples were incubated for an additional 2 h at 55 ¿C. The enzyme was inactivated by heating to 100 ¿C for 2 min and the samples were centrifuged prior to HPLC analysis. The following enzymes were used: Chondroitinase ABC (F. heparinum, Sigma) Chondroitinase AC (A. aurescens, Sigma) Heparinases I, II, and III (F. heparinum, Grampian) Hyaluronidase (S. hyalurolyticus, Northstar) Keratanase II (Bacillus sp., Northstar) 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. Solvent A: 2.5 mM Na-phosphate, pH 3.5 Solvent B: 2.5 mM Na-phosphate, pH 3.5, 1.2 M NaCl. The flow rate was 1.0 mL/min. Injection volume was 10 ¿L. Detection was performed by post-column derivatization. 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 50-cm cooling coil, and directed into a Shimadzu fluorescence detector. Excitation wavelength was 346 nm and emission wavelength was 410 nm. Commercial standard disaccharides (Dextra Laboratories) were used for calibration for heparinase and chondroitinase digestion mixtures. For the other mixtures enzyme digested authentic keratan sulfate (AMS Biotechnology) and hyaluronic acid (Sigma) samples were used as standards.