DISACCHARIDE ANALYSIS OF 3 GAG SAMPLES BY SAX-HPLC (HS AND CS)

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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 0.09 mg/mL GAG, 50 mM NH4OAc buffer, pH 7, and 0.1 mU/mL chondroitinase (F. heparinum, Sigma) or heparinases I, II, and III (F. heparinum, Grampian) was incubated at 37 ¿C for 24 h. The enzyme was inactivated by heating to 100 ¿C for 2 min and the samples were centrifuged prior to HPLC analysis. 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.