Chondroitin ac lyase

Solutions containing CS from the pharmaceutical preparations and CS standards (5 μg of each) were applied in an agarose-gel (0.5%). The preparations submitted to enzymatic digestion were incubated with 0.01 units of chondroitin AC lyase from Flavobacterium heparinum, recombinant expressed in E. coli (Sigma-Aldrich) in 100 μL 0.05 M Tris:HCl (pH 8.0), supplemented with 5 mM EDTA and 15 mM sodium acetate at 37 °C for 12 h. After incubation, the samples were heated at dried-bath at 80 °C for 15 min to stop the reaction through denaturation of the enzyme, cooled in ice cold and applied in the agarose-gel. Electrophoresis run for 1 h at 110 V in 0.05 M 1,3-diaminopropane acetate (pH 9.0). CS in the gel were fixed with 0.1% N-cetyl-N,N,N-trimethylammonium bromide solution for 12 h, dried and stained with 0.1% toluidine blue in 0.1:5:5 (v/v) acetic acid:ethanol:water.

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.

Ascidian (Halocynthia roretzi) tunics were purchased from a fresh market in Tongyeong, Korea. The tunics were washed with tap water and kept at −40 °C. Chondroitin AC lyase (EC 4.2.2.5) from Arthrobacter aurenses, chondroitin ABC lyase (EC 4.2.2.4) from Proteus vulgaris, monosaccharide standards, 1,9-dimethylmethylene blue, glucuronolactone and toluidine blue-O were obtained from Sigma-Aldrich (St. Louis, MO, USA); DEAE-Sepharose was obtained from GE Healthcare Bio-Sciences AB (Uppsala, Sweden). Human follicle dermal papilla (HFDP) cells (C-12071) were purchased from PromoCell GmbH (Heidelberg, Germany). Cells were cultured in follicle dermal papilla cell growth medium (C-26501, PromoCell GmbH, Heidelberg, Germany) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA, USA), 50 units/mL of penicillin and 50 mg/mL of streptomycin. All the other reagents were of analytical grade.