Chondroitin abc lyase

Seven danaparoid API batches (CAT271-277), one heparin sodium USP and one Nadroparin calcium samples were provided by Aspen Oss B.V., Oss, Netherlands. Heparin lyases I (EC 4.2.2.7), II and III (EC 4.2.2.8) were purchased from Grampian Enzymes, Aberdeen, UK. Chondroitin ABC lyase from Proteus vulgaris (EC 4.2.2.4), ammonium acetate (≥98%), sodium azide (≥99.0%), sodium nitrate (≥99.0%), sodium dihydrogen phosphate monohydrate (>98%), sodium hydrogen phosphate dihydrate (≥99.0%), trimethylsilyl-3-propionic acid (TSP 98% D), dibutylamine (≥99.5%), acetic acid (glacial, 99.9%), acetonitrile (LC-MS grade), methanol (LC-MS grade), ammonium chloride (≥99.5%), sodium nitrite (>95%), sodium tetraborate (≥98%), hydrochloric acid (≥37%) were purchased from Sigma Aldrich (Milan, Italy); calcium acetate (≥97%) from BDH; sodium acetate (≥99%) and NaOH (≥99%) from Merck (Kenilworth, NJ, USA); Amberlite IR 120 H⁺ and 0.1 M NaOH from Fluka Analytical (Milan, Italy).
Ethanol (96%) was purchased from Girelli Alcool (Milan, Italy); ethylenediaminetetraacetic acid (EDTA D16, 98%) from Cambridge Isotope Laboratories (Tewksbury, MA, USA) and deuterium oxide (≥99.9%) from Euriso-top (Saint-Aubin, France). Deionized water (conductivity less than 0.15 µS) was prepared with an osmosis inverse system (Culligan, Milan, Italy).

We treated 10 µL of the GAG samples with 5 mU of chondroitin ABC lyase (Sigma-Aldrich) and incubated them in 50 µL of 100 mM Tris / 150 mM sodium acetate buffer (pH 8.0) at 37 °C for 16 h. The reaction was blocked by boiling the solution for 1 min. We analysed the unsaturated disaccharides generated from hyaluronic acid (HA) and chondroitin sulphate (CS) after enzymatic treatment of the purified GAGs by strong anion exchange HPLC separation at 232 nm (column: Sphere-Image 80-5 SAX, Knauer, Berlin, Germany; equipment: Shimazdu, Duisburg, Germany, consisting of degasifier DGU-20A3, two pumps LC-20AT, autosampler SIL-20A, controller CBM-20A, detector SPD-20A; software: LCSolution). Isocratic separation was performed for 15 min with 10 mM NaH₂PO₄, pH 4.0 and afterwards linear gradient separation for another 20 min with 10 mM NaH₂PO₄, pH 4.0 to 33% 750 mM NaH₂PO₄, pH 4.0. Flow rate was 1.2 mL/min. Charge density was calculated by dividing the area under the curve (AUC) for CS by the AUC for (HA+CS).

GAGs (60 μg) extracted from archaeological human bone samples were suspended in MilliQ water (60 μL) and the sample divided in three, boiled for 15 min to inactivate any residual maxatase, and lyophilized. The first sample was suspended in 20 μL chondroitin AC lyase (1 U/mL, Sigma-Aldrich, St. Louis, MO), the second sample in 20 μL chondroitin ABC lyase (1 U/mL, Sigma-Aldrich) and the third sample in 20 μL MilliQ water. The samples were incubated overnight at 37°C and 5 μL of each sample analyzed by agarose gel electrophoresis as mentioned above. Digestion control samples (C4S and a mixture of CS, DS and HS extracted from shark cartilage; all 1 mg/mL) were also incubated in chondroitin AC lyase, chondroitin ABC lyase or MilliQ water overnight at 37°C and analyzed by agarose gel electrophoresis.