Isolation, Purification And Structure Of Glycosaminoglycan From Thelenota Ananas

The components of the body wall of Thelenota ananas harvested in Shandong Provincewere analyzed. A spectrophotometric method detecting the GAG in matrix wasestablished. GAG was isolated and purified from the body wall of Thelenota ananas,and its structure was analyzed.The moisture in the body wall of Thelenota ananas was 8.52%. Other componentswere gross protein 83.78%, crude fat 3.24%, glycogen 7.83% and ash 4.56% respectivelybased on dry flesh weight. The composition of amino acids in the protein was perfectexcept Ile and Cys lower. The percentage of unsaturated fat was 27.85% in total fats,with a high content of EPA. Moreover some minerals and microelements such as K, Ca,Mg, Zn and Mn were rich.The dimethylmethylene blue assay for GAG in sea cucumber tissue was established.The optimum pH was 3.00, A520 was read during 4-6min. A good calibration curve (A=0.0104c + 0.0118, r = 0.9996) was prepared in the range of 5~30 mg.L-1 of GAG. Theprocedure was directly applied for the rapid and reproducible quantitativedetermination of GAG in papain digests of sea cucumber tissues with high sensitivityand accuracy. The content of GAG in the body wall was Holothuria nobilis 1.35%,Thelenota ananas 2.32%, Stichopus japonicus 4.22% and Stichopus chloronotus 7.10%respectively assayed with dimethylmethylene blue method.Crude GAG was extracted and isolated from the body wall of Thelenota ananas bymeans of enzymic and alkaline hydrolysis, and ethanol (45%, v/v) and KAc (1.2 mol.L-1)precipitation. Crude GAG was purified through DEAE-Cellulose column and SepharoseCL-4B column. The fraction S4 was homogeneous showed by UV, agarose gelelectrophoresis and HPGPC, and its Mw was 218289.S4 was composed of 21.61% glucuronic acid, 25.42% GalNAc, 18.55% fucose and 39.80%sulfate。IR spectra suggested that S4 had α-and β-glycosidic bonds. The opticalrotation suggested that the fucose mainly had L anomeric configuration,and glucuronicacid and GalNAc mainly had D anomeric configuration。The structure of S4 were studied by periodate oxidation, paritial hydrolysis,desulfation and NMR. The main chain of S4 was →4)-β-D-GlcpUA-(1→3)-( β-D-GalpNAc)-(1→, part of the GlcUAn was sulfated at position O-6. Disaccharide unitsof sulfated fucopyranosyl units was linked to approximatedly one-half of theglucuronic acid moieties through the O-3 position of the acid.