| In this thesis, the waste generated during the preparation of heparin from porcine small intestinal mucosa was used as material. Fristly, the waste was extracted with 60℃ hot water, trypsin (EC3.4.21.4),2709 alkaline protease (CAS:9014-01-1), pepsin (CAS:9001-75-6) and subtilisin (EC3.4.21.14) respectively. Five kinds of crude mucins Ims-W, Ims-Y, Ims-YJ, Ims-A and Ims-N were obtained in the yields of 0.30%,1.21%,4.03%,0.34% and 1.22%, respectively. Afterwards, physicochemical properties of crude mucins were analyzed and monosaccharide compositions were determined using reversed-phase high performance liquid chromatography (RP-HPLC). It was shown that the molecular weights of Ims-W, Ims-Y, Ims-A and Ims-N were 137.1 kDa,102.8 kDa,66.5 kDa,684 kDa and 839 kDa, respectively. The corresponding protein contents were determined to be 27.24% to 56.78%, while contents of sulfate group were all less than 5%. The results of monosaccharide composition analysis demonstrated that all the crude mucins were mostly composed of galactosamine (GalN), glucosamine (GlcN), galactose (Gal), fucose (Fuc), N-acetylneuraminicacid (NeuAc) and N-Glycolylneuraminic acid (NeuGc). Because of different extraction process, the sialic acid contents of crude mucins existed great differences, and contents of NeuAc and NeuGc were generally 4.61%-12.71% and 2.91%-17.58%.Ims-Y and Ims-YJ both extracted with protease hydrolysis method were selected for futher purification and structural characterization using mass spectrometry (MS) method. According to their charge distribution, the crude mucins Ims-Y and Ims-YJ were fractionated into six components by strong anion exchange chromatography, namely Ims-Y-0.6, Ims-Y-0.8, Ims-Y-1.0, Ims-YJ-0.6, Ims-YJ-0.8 and Ims-YJ-1.0. The yield of each fraction was determined to be 73.21%,3.22%, 5.35%,83.33%,1.67% and 2.89%, respectively. The analysis of physicochemical properties indicated that the protein contents of Ims-Y-0.6, Ims-Y-0.8,Ims-Y-1.0, Ims-YJ-0.6, Ims-YJ-0.8 and Ims-YJ-1.0 were 20.34%,30.91%、10.90%,31.06%, 10.78%and 13.96%, sulfate group contents were 3.43%,8.71%,13.92%,3.16%,9.13% and 17.21%, and molecular weight were763 kDa,24 kDa,31 kDa,658 kDa,21 kDa and 22 kDa, respectively. It was shown in monosaccharide composition analysis showed that six fractions were mostly composed of GalN, GIcN, Gal and Fuc, while Ims-Y-1.0 and Ims-YJ-1.0 also contained 13%-16%glucuronic acid (GlcA). In addition, both Ims-Y-0.6 and Ims-YJ-0.6 possessed NeuAc and NeuGc, and their contents were distributed between 11.82% and 36.14%. However, the other four fractions didn’t have NeuAc and NeuGc.In order to analyze the precise structures of Ims-Y-0.6 and Ims-YJ-0.6, reductive (3-elimination was used to release the glycans attached to their core proteins. The reductive oligosaccharide mixtures were isolated and purified with a Bio-gel P2 gel filtration chromatography. According to the results of Bio-gel P2 purification, electrospray ionization-collision induced dissociation-tandem mass spectrometry (ESI-CID-MS/MS) technology was used to analyze the structure of Ims-Y-0.6. Following the ion patterns of O-glycans reported in previous literatures, the core structure, sulfate position, sialic acid position, fucose position and linkage type of the glycans were identified by ESI-CID-MS/MS to finally 26 kinds of oligosaccharides The results indicated that Ims-Y-0.6 was a highly-sialylated blood groups H epitope mucin with its GlcNAc residues sulfated at C-6 position.Finally, in this thesis, the phagocytic capacity of Raw264.7 macrophages was investigated by the neutral red phagocytosis assay, which was affected by the versatile mucins derived from the waste during the preparation of heparin from pocine small intestinal mucosa. The results indicated that these mucins could activate macrophages, promote their phagocytic capacity at certain concentration and enhance immune activity. The results obtained in this thesis could provid a useful reference for further development and utilization of the high-glycosylated mucins. |
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