Digestion buffer (50 mM sodium acetate, 5 mM EDTA, 10 mM cysteine, PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/22026160 pH 5.0), containing 10 papain (w/w), and incubated at 60 , overnight. Soluble glycans were precipitated with two-volumes of ethanol and fractionated in an anion exchange chromatography on a Mono-Q-FPLC column (HR 5/5) (Amersham Pharmacia Biotech), equilibrated in 20 mM Tris/HCl (pH 8.8), containing 4 mM EDTA. The column was developed with a linear gradient of 0-2.0 M NaCl in the same buffer, at a flow rate of 1 mL/min. The fractions from the column were monitored by metachromatic property with dimethylmethylene blue, at 525 nm [25]. The metachromatic fractions were pooled, dialyzed, lyophilized and analyzed by agarose gel electrophoresis.Agarose gel electrophoresisascidians may also be related to an angiogenesis event. The alteration between a 2,4 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/21715270 disaccharide from Stolidobranchia to a 2,6 disaccharide in Phlebobranchia might be involved in the branchial vessel organization. One of the main differences between these two ascidian orders is the pharyngeal structure. Phlebobranch ascidians possess a simpler pharynx, without folds and usually withThe ascidian glycans were analyzed by agarose gel electrophoresis, as described [5]. Briefly, GAGs ( 15 mg) were applied to a 0.5 agarose gel and ran for 1 h at 100 V in 0.05 M 1,3-diaminopropane/acetate (pH 9.0). GAGs were fixed in the gel with 3-(2,2,2-Trifluoroethoxy)aniline hydrochloride 0.1 N-cetyl-N,N,Ntrimethylammonium bromide solution. After 12 h theKozlowski et al. BMC Biochemistry 2011, 12:29 http://www.biomedcentral.com/1471-2091/12/Page 8 ofgel was dried and stained with 0.1 toluidine blue in acetic acid/ethanol/water (0.1:5:5, v/v). Polyacrylamide gel electrophoresis-The molecular weight of the ascidian DSs was estimated by polyacrylamide gel electrophoresis. DS samples ( 10 mg) were applied to a 7.5 1-mm-thick polyacrylamide gel slab in 0.02 M sodium barbital (pH 8.6). After electrophoresis (100 V for 30 min), the gel was stained with 0.1 toluidine blue in 1 acetic acid and then washed for about 4 h in 1 acetic acid. The molecular weight markers were the same as those used previously [26].Enzymatic treatments with chondroitin lyasesincubated with 10 L of DS at several dilutions and 100 L of cephalin. After three minutes at 37 , 100 L of 0.25 M CaCl2 were added to the mixtures and the clotting time was recorded. The activity was expressed as units/mg using a standard unfractionated heparin (180 U/mg) curve.Heparin cofactor II-mediated inhibition of thrombin by ascidian DSsThe glycans extracted from ascidians ( 20 mg) were incubated with 0.005 U of chondroitin AC or ABC lyases, as described previously [6]. Intact and enzymedegraded glycans were analyzed by agarose gel electrophoresis, as described earlier. Deaminative cleavage with nitrous acid - deaminative cleavage was performed by nitrous acid at pH 1.5, as described [27]. Briefly, total glycans extracted from ascidians tissues ( 20 g) were incubated with 5 L of fresh generated HNO2 at room temperature for 90 min. The reaction mixtures were then neutralized with 1.0 M Na2CO3. Intact and nitrous acid-degraded glycans were analyzed by agarose gel electrophoresis.Analysis of the disaccharides formed by digestion of the 1-(5-(Aminomethyl)-2-nitrophenyl)ethanol dermatan sulfate with chondroitin ABC lyaseThe inhibitory activity of ascidian DSs was measured in 96-well plate. Briefly, 30 L of 68 nM HCII in 0.02 M Tris-HCl, pH 7.4/0.15 M NaCl/PEG 1 mg/mL were incubated with 30 L of ascidian DSs at different concentrations. After incubating for 2 minutes,.