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.Complete depolymerization to disaccharides with heparinases I, II, and III A 20 uL aliquot of a 20 g/L solution of low molecular weight heparin 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.Partial depolymerization with heparinase I, heparinase II, or a mixture of both (Lots 93485, 93689, 12074, 15057, and DA1194 only)All 4 samples were treated with heparinase I alone, heparinase II alone, and with a mixture of heparinases I and II.A 150 uL aliquot of a 2 g/L solution of low molecular weight heparin (AVT and HSP) in 20 mM NH4OAc buffer, pH 7, containing 2 mM calcium acetate was treated with 20 mU of heparinase (I or II or I and II) and incubated at 37 C. After 24 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-HPLC of disaccharide mixtureSAX-HPLC was carried out on an Agilent system using a 4.6 250 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.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 50 % 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 1-7). Equation 1A = peak area from the SAX chromatogramMW = mass average molecular weight of enoxaparin = 4400 g/mol (from Opocrin)MMi = molecular weight of each individual di- or tetrasaccharideSAX-HPLC of partially depolymerized productThe same conditions as above were used with a modification of the buffer gradient: After 5 min at 97 % A, a linear gradient was applied to reach 50 % B at 50 min, and then a linear gradient was applied to reach 100 % B at 82 min. Glycosyl composition analysis Glycosyl 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 Spectroscopy for determination of gluco/manno ratioThe sample was deuterium-exchanged by lyophilization from D2O and dissolved in 700 uL D2O (99.96 % 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 (s=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. Size-Exclusion ChromatographyOne TSK Gel column (SEC 1)One hundred mg enoxaparin (Hospira Lot # 93689) was dissolved in 1 mL water and filtered through a 0.22 um membrane filter. Fourteen injections of 25 uL each were made. Seven fractions were collected during each run.Separation was carried out on a TSKGel G2000SWxl column (7.8 mm ID X 30 cm, 5 um), equipped with a TSKGel guard column (6.0 mm ID X 4.0 cm, 7 um), using 0.25 M ammonium acetate, pH 6.0 as eluent at a flow rate of 0.5 mL/min. Detection was by UV at 232 nm.Two TSK Gel columns in series (SEC 2)To obtain more and better resolved fractions, the size-exclusion chromatography was repeated with two TSK Gel G2000SWxl columns in series, injecting 10 mL at a time. Twenty-six injections were made.Preparative Gel Filtration with Sephacryl S-100 (SEC 3)One hundred mg enoxaparin (lot Nos. 15057 and 93689) was dissolved in 1 mL water, and 500 uL of this solution were injected onto a 3.0 X 100 cm Sephacryl S-100 column with a flow rate of 1 mL/min. The eluate was monitored by UV detection at 254 nm and 3-mL fractions were collected. The fractions belonging to the same peaks were pooled and freeze-dried, then water was added and the samples were again freeze-dried to remove residual ammonium acetate. Hexa-, octa, deca, and dodecasaccharide fractions were analyzed according to SEC 2 to determine purity.Preparative SAX-HPLCThe lyophilized fraction 3 from SEC 1 was dissolved in 100 uL water, filtered, and injected onto the SAX column.The column used was a Waters Spherisorb SAX analytical column (4.6 mm ID X 25 cm), equipped with a SAX guard column. The eluents used were 25 mM sodium phosphate, pH 3.5 (Eluent A) and 2 M NaCl in 25 mM sodium phosphate, pH 3.5 (Eluent B). The following gradient was used:t/min 0 10 20 40 60 90 95 100 102% B 0 28 38 43 46 48 95 95 0Five fractions, A, B, C, D, and E were collected.NMR Spectroscopy for sequencingFractions E from both lots 15057 and 93689 were deuterium-exchanged by lyophilization from D2O, dissolved in 0.26 mL D2O, and placed in an NMR tube with magnetic susceptibility plugs (Shigemi). Proton-detected NMR spectra were acquired on a Varian Inova-800 MHz spectrometer at 298 K (25 C) using standard Varian pulse sequences. Proton chemical shifts were measured relative to internal acetone (SH = 2.225 ppm, SC = 31.07 ppm). TOCSY and NOESY mixing times were 0.06 and 0.30 s, respectively.Mass SpectrometrySamples were analyzed using a Bruker 9.4T QeFTMS. All samples were ionized in negative ion mode on an Apollo II Dual source. Samples were dissolved in 50:50:0.1 MeOH:H2O:NH3 to a concentration of 5 uM and introduced into the ESI source at a flow rate of 2 uL/min. For each acquisition, ions were accumulated in the external hexapole for 2 seconds. For each mass spectrum, 512k data points were acquired, 24 spectra were averaged, padded with one zero fill, and apodized with a sinebell window.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR005351-19
Application #
7721592
Study Section
Special Emphasis Panel (ZRG1-BNP (40))
Project Start
2008-02-01
Project End
2009-01-31
Budget Start
2008-02-01
Budget End
2009-01-31
Support Year
19
Fiscal Year
2008
Total Cost
$1,404
Indirect Cost
Name
University of Georgia
Department
Type
Organized Research Units
DUNS #
004315578
City
Athens
State
GA
Country
United States
Zip Code
30602
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