GAG ISOLATION AND SAX-HPLC OF 2 SAMPLES

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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: GAG Isolation The frozen tissue was ground in liquid nitrogen and extracted with 10 ml acetone (twice for 24 h) and once with diethyl ether (5 ml, 30 min) and dried under vacuum. The tissue was resuspended in 2 mL pronase digestion buffer (0.1 M Tris-HCl, pH 8.0, 2 mM CaCl2, 1% Triton X-100), 1.6 mg pronase was added, and the tissue was digested with shaking at 55 ¿C (1). After 24 h, a second 1.6 mg aliquot of pronase was added and digestion continued for 24 h. The enzyme was inactivated by heating to 100 ¿C for 15 min. The buffer was adjusted to 2 mM MgCl2, benzonase (100 mU) was added, and the sample was incubated for 2 h at 37 ¿C. After inactivation of the enzyme (15 min 100 ¿C) the undigested tissue was precipitated by centrifugation for 15 min at 12000 g. The supernatant was applied to a DEAE-Sephacel column (2 mL), washed with 20 mL equilibration buffer (20 mM Tris-HCl, pH 7.5, 0.1 M NaCl), and eluted with 6 mL elution buffer (20 mM Tris-HCl pH 7.5, 2 M NaCl). The sample was treated with 1 mL 10 % (w/v) NaBH4 in 2N NaOH, and incubated overnight at 4 ¿C (2). The reaction was stopped by adding glacial acetic acid until no bubbles were formed and the pH was neutral. The sample was freeze-dried, desalted using a PD10 column (GE Healthcare), again freeze-dried, and dissolved in 50 ¿L water. GAG lyase digestion A 10-¿L aliquot of the sample solution was dissolved 50 mM ammonium acetate, pH 7, and treated with 10 ¿L of the appropriate GAG lyase solution (1 U/mL). The enzyme solutions used were a) chondroitinases ABC and AC, b) chondroitinase ABC, and c) heparinases I, II, and III. The samples were incubated at 37 ¿C overnight and heated to 100 ¿C 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 gradients: 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 (3). 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.