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.
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