SAX-HPLC OF 6 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: Heparinase digestion A 20 ¿L aliquot of a 20 g/L solution of low molecular weight heparin (AVT and HSP) 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. Reduction A 60 ¿L portion of the heparinase-digested sample was treated with 20 ¿L of a 30 g/L solution of NaBH4 in H2O for at least 24 h at 23 ¿C. 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. Analytes were detected by their UV absorbance at 232 nm using the following system. Solvent A: 2.5 mM Na-phosphate, pH 3.5; Solvent B: 2.5 mM Na-phosphate, pH 3.5, 1.2 M NaCl. After 5 min at 97 % A, a linear gradient was applied to reach 85 % B after 50 min. The flow rate was 1.4 mL/min. The percentage of chains terminating in anhydro forms was determined according to equation 1 using the integration values of the SAX-HPLC chromatograms .