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. 2021 Sep 17;297(4):101207. doi: 10.1016/j.jbc.2021.101207

Figure 1.

Figure 1

Purification, electrophoretic mobility, and 1D1H NMR spectra of the polyanionic glycosidic fractions obtained from Pentacta pygmaea.A, crude polysaccharides (20 mg) obtained from the body wall of the sea cucumber P. pygmaea through nonspecific proteolytic (papain) digestion were fractionated in a DEAE cellulose column (2.5 × 20 cm) equilibrated with 100 mM sodium acetate buffer (pH 6.0). Multiple fractions (1 ml each) were obtained using a linear gradient of NaCl, from 0 to 3 M (---). Fractions were individually analyzed by Metachromasy using 1,9-dimethylmethylene blue (Abs 525 nm, ○) for the presence of sulfated glycan, by Dubois reaction (Abs 490 nm, •) for hexose, by Ehrlich assay (Abs 420 nm, Δ) for sialic acid, and by Carbazole reaction (Abs 550 nm, ▲) for uronic acid. The fractions corresponding to the respective peaks, labeled as nonsulfated glycan and the P. pygmaea-derived fucosylated chondroitin sulfate (PpFucCS), were pooled, dialyzed, and lyophilized for further characterization. B, molecular weight distribution of the purified PpFucCS was analyzed by polyacrylamide gel electrophoresis along with a series of molecular markers: low-molecular-weight heparin (LMWH) (∼8 kDa), unfractionated heparin (UFH) (∼15 kDa), chondroitin sulfate-A (CS-A) (∼40 kDa), chondroitin sulfate-C (∼60 kDa), and the sea cucumber Isostichopus badionotus-derived fucosylated chondroitin sulfate (IbFucCS) and sulfated fucan (IbSF). Samples (10 μg/each) were loaded on a 12% polyacrylamide gel and stained by 0.1% (w/v) toluidine blue (in 1% acetic acid) after electrophoretic migration. C, one-dimensional (1D) 1H NMR spectrum (δH expansion 6.0–0.0 ppm) of the purified PpFucCS were recorded in D2O, at 50 °C, on a 600 MHz Bruker NMR instrument. The NMR signals corresponding to characteristic peaks were properly labeled, as seen for the four composing α-fucose (Fuc) units (2,4S, 2,4S, 4S, and 0S) and the three composing β-N-acetylgalactosamine (GalNAc, N) units (4S, 6S, and 0S), all based on the diagnostic 1H signals present in the anomeric region, glucuronic acid (U) and GalNAc and Fuc methyl protons. S denotes sulfation in which the preceding number indicates the site or lack of occurrence.