This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Fragment Mapping Heparinase digestion [unreadable]A buffer solution (20 mM ammonium acetate, pH 7;2 mM calcium acetate) containing 300 [unreadable]g heparin and 20 mU heparinase I was incubated at 37 [unreadable]C. After 24 h, the reaction was quenched by boiling the mixture for 2 min. SAX-HPLC - SAX-HPLC was carried out on an Agilent system using a 4.6[unreadable]250 mm Waters Spherisorb analytical column with 5 [unreadable]m particle size at 25 [unreadable]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. The following gradient was used at 1.4 mL/min flow rate. Heparinase digestion A 1 [unreadable]L aliquot of a 20 g/L solution of heparin in water was diluted with 60.5 [unreadable]L water and treated with 7.5 [unreadable]L 200 mM NH4OAc buffer, pH 7, containing 20 mM calcium acetate. The mixture was then treated with 2 [unreadable]L each of heparinases I, II, and III (5 U/mL each) in 10 mM potassium phosphate buffer, pH 7, containing 2 g/L BSA and incubated at 37 [unreadable]C for 24 h. The enzymes were inactivated by heating the samples to boiling for 2 min. ESI-MS The samples were diluted with 425 [unreadable]L 50 % MeOH and infused into the MS instrument at 5 [unreadable]L/min. Mass spectrometry was performed on a Thermo Scientific LTQ Orbitrap Discovery instrument in ESI mode under direct infusion. Tuning was performed using a 3 [unreadable]M solution of heparin disaccharide IV-A in 50 % MeOH. Capillary temperature was 300 [unreadable]C, capillary voltage -13 V, spray voltage -3.2 kV, and sheath gas flow rate was 18. Data were acquired for 3 min for each sample. Heparinase digestion A 20 [unreadable]L aliquot of a 20 g/L solution of heparin in water was diluted with 80 [unreadable]L 100 mM NaOAc buffer, pH 7, containing 2 mM calcium acetate and 1 g/L BSA. The mixture was then treated with 20 [unreadable]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 [unreadable]C for 48 h. The enzymes were inactivated by heating the samples to boiling for 2 min. Reduction A 30 [unreadable]L portion of the heparinase-digested sample was treated with 10 [unreadable]L of a 30 g/L solution of NaBH4 in H2O for 72 h at 23 [unreadable]C. A mixture of 12 heparin disaccharides was also reduced in the same manner. SAX-HPLC SAX-HPLC was carried out on an Agilent system using a 4.6[unreadable]250 mm Waters Spherisorb analytical column with 5[unreadable]m particle size at 23 [unreadable]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. Heparinase digestion A 20 [unreadable]L aliquot of a 20 g/L solution of heparin in water was diluted with 80 [unreadable]L 100 mM NaOAc buffer, pH 7, containing 2 mM calcium acetate and 1 g/L BSA. The mixture was then treated with 20 [unreadable]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 [unreadable]C for 48 h. The enzymes were inactivated by heating the samples to boiling for 2 min. NMR The samples (30-35 mg) were dissolved in D2O (99.9 % D, Aldrich) and lypholized to remove exchangeable protons. The samples were then dissolved in 0.3 mL D2O (99.96 % D, Cambridge Isotope Laboratories), 20 [unreadable]L 50 mg/mL DSS was added, and the solutions were placed in 3-mm NMR tubes. Proton 1-D NMR spectra were recorded on a Varian Inova 600 NMR spectrometer at 60 [unreadable]C. For Lot 2421, 2-D gCOSY, gHSQC, and TOCSY spectra were recorded in addition to the 1-D experiments.
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