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. NMR Spectroscopy The sample (70 mg) was deuterium-exchanged by lyophilization from D2O, dissolved in 700 uL D2O (99.996 % D). A gradient enhanced heteronuclear single quantum coherence (gHSQC) spectrum was acquired on a Varian Inova-500 MHz spectrometer at 313 K (40 ?C). Proton chemical shifts were measured relative to DSS (delta=0.00 ppm). The spectra were recorded with a spectral width in the proton dimension of 3259 Hz and in the carbon dimension of 12569 Hz. The acquisition time was 0.20 s, and 128 increments were collected with 64 scans each. The one-bond C-H coupling constant was set at 140 Hz. Heparinase digestion A 20 uL aliquot of a 20 g/L solution of low molecular weight heparin (AVT and HSP) in water was diluted with 80 uL 100 mM NaOAc buffer, pH 7, containing 2 mM calcium acetate and 1 g/L BSA. The mixture was then treated with 20 uL 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 uL portion of the heparinase-digested sample was treated with 20 uL 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 x 250 mm Waters Spherisorb analytical column with 5um particle size at 23 ?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 NaClO4. After 5 min at 98 % A, a linear gradient was applied to reach 70 % 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 (Tables 1-4). (eq. 1) A = peak area from the SAX chromatogram MW = mass average molecular weight of enoxaparin = 4400 g/mol (from Opocrin) MMi = molecular weight of each individual di- or tetrasaccharide
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