This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Heparinase digestionA 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.ReductionA 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-HPLCSAX-HPLC was carried out on an Agilent system using a 4.6x250 mm Waters Spherisorb analytical column with 5um particle size at 45 C. Analytes were detected by their UV absorbance at 232 nm using the following system.Glycosyl compositionGlycosyl composition analysis was performed by combined gas chromatography/mass spectrometry (GC/MS) of the per-O-trimethylsilyl (TMS) derivatives of the monosaccharide methyl glycosides produced from the sample by acidic methanolysis.1mg of each samples was completely desulfated and depolymerized by repeated (3 X) methanolysis and re-N-acetylation. For each repetition, the dry sample was heated to 100 C with 0.5 mL 3 M HCl in MeOH for 2 h, evaporated, treated with 200 uL MeOH, 100 uL pyridine, and 100 uL acetic anhydride, and dried under a stream of air. After the last N-acetylation step, the sample was heated for 20 min at 80 C with 200 uL Tri-Sil. GC/MS analysis of the TMS methyl glycosides was performed on an HP 5890 GC interfaced to a 5970 MSD, using a Supelco DB-1 fused silica capillary column (30m x 0.25 mm ID).NMR SpectroscopyThe sample was deuterium-exchanged by lyophilization from D2O and dissolved in 700 uL D2O (99.996 % D). Gradient heteronuclear single quantum coherence (gHSQC) spectra were 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 3 kHz and in the carbon dimension of 15-20 kHz (depending on sample amount and instrument time available). The acquisition time was 0.20 s, and 200 increments were collected with 64-128 scans each. The one-bond C-H coupling constant was set to 140 Hz.
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