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Figure 1. Identification of contaminants in selected samples of crude and final heparins by agarose gel electrophoresis. Aliquots (5 µL, 5–10 µg) of heparin samples were applied to 0.55% agarose gel and submitted to electrophoresis in 0.05M PDA or Ba/PDA discontinuous system as described in Methods. The arrow shows the presence of slow migrating bands which are not present in standard heparin. In the discontinuous Ba/PDA buffer this is most evident due to a clear separation of slow migrating bands. The figure also shows differences in the amounts of these contaminants among both the crude and final product heparin. C7, C8, C9, C10, C11 and C16: crude heparin samples; F1, F4, F8, F9, F10 and F11: final product heparin samples; HEP: standard porcine heparin; CS/DS/HS: standard mixture of chondroitin sulfate (CS), dermatan sulfate (DS), and heparan sulfate (HS); S: heparin slow moving component, I: heparin intermediate moving component and F: heparin fast moving component; Or: origin.
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Figure 2. Analysis of crude and final heparins by UV scanning spectroscopy (190–320 nm). UV spectra were performed on UV/VIS spectrophotometer using 1 mg/mL solution in water in the range of 190–320 nm, with a reading rate of 120 nm/min with a resolution of 1 nm at room temperature. Standard heparin UV spectra shows a narrow peak of 190–210 nm whereas, on the other hand, contaminated heparins have a broader peak around 200–220 nm, as well as an additional broad signal around 240–260 nm (nucleic acids, peptides). C7, C9 and C16: crude heparin samples; F1, F8 and F9: final heparin samples).
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Figure 3. Analysis of heparinase and heparitinase II products from crude and final heparin samples. Crude and final heparin samples were incubated with heparinase and heparitinase II and the degradation products analyzed by paper chromatography as described. The results show that in the presence of the contaminants there is a decrease in the degradation of heparin by heparinase, suggesting an inhibition of the enzyme. BHep: bovine standard heparin; PHep: porcine standard heparin; Crude heparins (C7 to C11); Final heparin samples (F8, F9 and F11); ΔTetra: unsaturated pentasulfated tetrasaccharide; (ΔU2S-GlcNS,6S): unsaturated trisulfated disaccharide; (ΔUGlcNS,6S): disulfated unsaturated disaccharide; (ΔU-GlcNS): unsaturated N-sulfated disaccharide; Or: origin.
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Figure 4. Purification of sulfated contaminants by ion exchange chromatography. (A) Elution profile of the crude heparin preparation C8 on Q-Sepharose ion exchange chromatography. I, II and III: the fractions shown in A were combined according to their elution profile and named peaks I to III. (B): Agarose gel electrophoresis in PDA buffer of Q-Sepharose fractions. Peak III corresponds to the contaminant OSC8, isolated from C8 crude heparin. CS/DS/HS: standard mixture of chondroitin sulfate (CS), dermatan sulfate (DS) and heparan sulfate (HS); Or: Origin.
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Figure 5. 1H-NMR spectra of standard OSCS and sulfated contaminants isolated from crude (OSC16) and final heparin samples (OSF4, OSF8 and OSF9). The isolated contaminants purified from crude and final heparin batches were structurally characterized by nuclear magnetic resonance (1H NMR). Solutions of 20 mg/mL of each sample in deuterium oxide (99.9%) were analyzed for the acquisition of free induction decay (FID) using 16 scans, 90° pulse and 20 s delay at 37 °C in a Bruker Spectrometer DRX-500. Sulfated contaminants purified from batches of crude (OSC16) and final heparin (OSF4, OSF8 and OSF9) display similar 1H NMR spectra of standard OSCS.
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Figure 6. 1H-NMR spectra of sulfated contaminants isolated from crude heparins OSC8 and OSC9 show distinct structure. The isolated contaminants purified were structurally characterized by nuclear magnetic resonance (1H NMR). Solutions of 20 mg/mL of each sample in deuterium oxide (99.9%) were analyzed for the acquisition of free induction decay (FID) using 16 scans, 90° pulse and 20 s delay at 37 °C in a Bruker Spectrometer DRX-500. Sulfated contaminants purified from crude batches (OSC8 and OSC9) in the 1H NMR spectra show signals in the 4.55 ppm region, marked with asterisks (*), that may correspond to non-fully substituted glucuronic acid residues and are not detected in OSCS.
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Figure 7. Two-dimensional NMR COSY spectra for the standard OSCS and the isolated OSC9. 1H NMR spectroscopy and 1H1H two-dimensional COSY homology was performed according to the USP monograph for sodium heparin. In the OSCS COSY spectra (red), the U2-U3 correlation signal is shifted, as expected, downfield since position 3 is replaced by a sulfate (electronegative) group. On the other hand, the OSC9 COSY spectra (blue) shows a downfield unlinked U2-U3 correlation signal which suggests the complete non-sulfation at the 3-position of the glucuronic acid residues.
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Figure 8. Analysis by Fluorophore Assisted Carbohydrate Electrophoresis (FACE) of the products formed from OSC8 by the exhaustive degradation with Chondroitinases AC and ABC. The structural characteristics of the OSC8 were investigated by degradation with chondroitinases AC and ABC under exhaustive degradation conditions (72 h and 0.3 enzyme units). The products formed were derivatized with AMAC and analyzed by polyacrylamide gel electrophoresis in Tris Borate pH 8.3 buffer, and displayed by UV light absorption, as described in Methods. OSC8 is not a substrate for the enzymes, since no products could be detected by this methodology that enhances the detection of products. ΔDi2S: unsaturated 2-sulfated disaccharide; ΔDi4S: unsaturated 4-sulfated disaccharide; ΔDi6S: unsaturated 6-sulfated disaccharide; ΔDi4,6S: unsaturated 4,6-disulfated disaccharide; ΔDi2,6S: unsaturated 2,6-disulfated disaccharide; ΔDi2,4S: unsaturated 2,4-disulfated disaccharide; ΔDi2,4,6S: unsaturated 2,4,6-trisulfated disaccharide; ΔDi0S: non-sulfated unsaturated disaccharide; Standard: mixture of standard unsaturated disaccharides; Tetra: tetrasaccharides; Oligo: oligosaccharides; OSC8: sulfated contaminant purified from C8 crude heparin; CS: standard chondroitin sulfate; DS: standard dermatan sulfate; AC: chondroitinase AC; ABC: chondroitinase ABC; Or: Origin.
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Figure 9. Kinetics of heparin degradation by heparinase in the presence of standard OSCS and sulfated contaminants isolated from crude and final heparin. The assay was performed using an UV spectrophotometer at 232 nm with temperature regulation. Incubation was performed using both recombinant heparinase and purified heparinase from F. heparinum in 50 mM Hepes buffer, 150 mM NaCl, pH 7.0. The enzyme was preincubated with 10 mM calcium acetate at 30 °C, and then standard heparin added. The formation of the unsaturated products was monitored at 232 nm for 10 min. Afterwards, different amounts of the isolated contaminants were added, and the formation of products monitored for 10 min at 232 nm. The sulfated contaminants isolated from crude heparins (OSC8 and OSC9) and final product (OSF4 and OSF8) as well as standard OSCS inhibit the action of heparinase upon heparin in a dose and time dependent manner. OSCS: standard oversulfated chondroitin sulfate; OSC8: sulfated contaminant purified from crude heparin C8; OSC9: sulfated contaminant purified from crude heparin C9; OSF4: sulfated contaminant purified from final heparin F4; OSF8: sulfated contaminant isolated from final heparin product F8.
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Figure 10. Isolated sulfated contaminants bind to endothelial cells. Endothelial cells were cultured in 96-well plates in F12 medium containing 10% FBS. The cells were exposed to different concentrations of biotinylated heparin plus molar excess of standard heparin or sulfated contaminants. Binding of biotinylated heparin to cells was detected by incubation with Europium-conjugated streptavidin and fluorescence analyzed on a fluorescence reader. Biotinylated heparin binds to EC in a dose dependent manner. This binding is decreased in the presence of excess molar amounts of all the tested contaminants as also observed for standard heparin. Heparin: standard heparin; OSCS: standard oversulfated chondroitin sulfate; OSC8: sulfated contaminant purified isolated from crude heparin preparation C8; OSF8: sulfated contaminant purified from final heparin product F8.
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