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Mar Drugs
2020 May 29;186:. doi: 10.3390/md18060286.
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Characterization of the Hydrolysis Kinetics of Fucosylated Glycosaminoglycan in Mild Acid and Structures of the Resulting Oligosaccharides.
Liu X
,
Zhang Z
,
Mao H
,
Wang P
,
Zuo Z
,
Gao L
,
Shi X
,
Yin R
,
Gao N
,
Zhao J
.
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: Mild acid hydrolysis is a common method for the structure analysis of fucosylated glycosaminoglycan (FG). In this work, the effects of acid hydrolysis on the structure of FG from S. variegatus (SvFG) and the reaction characteristic were systemically studied. The degree of defucosylation (DF) and molecular weights (Mw) of partial fucosylated glycosaminoglycans (pFs) were monitored by 1H NMR and size-exclusion chromatography, respectively. The kinetic plots of DF, degree of desulfation (DS) from fucose branches, and degree of hydrolysis (DH) of the backbone are exponentially increased with time, indicating that acid hydrolysis of SvFG followed a first-order kinetics. The kinetic rate constants kDF, kDS, and kDH were determined to be 0.0223 h-1, 0.0041 h-1, and 0.0005 h-1, respectively. The structure of the released sulfated fucose branches (FucS) from SvFG and HfFG (FG from H. fuscopunctata) was characterized by 1D/2D NMR spectroscopy, suggesting the presence of six types of fucose: α/β Fuc2S4S, Fuc3S4S, Fuc3S, Fuc4S, Fuc2S, and Fuc. The Fuc3S4S was more susceptible to acid than Fuc2S4S, and that the sulfate ester in position of O-2 and O-3 than in O-4 of fucose. The structure characteristic of pF18 indicated the cleavage of backbone glycosidic bonds. The APTT prolonged activity reduced with the decrease of the DF and Mw of the pFs, and became insignificant when its DF was 87% with Mw of 3.5 kDa.
31600649, 81773737, 81703374 National Natural Science Foundation of China, 2016ACA138 Key Projects of Technological Innovation of Hubei Province, CTZ18015 Fundamental Research Funds for the Central University, South-Central University for Nationalities
Figure 1. 1H NMR spectra of SvFG (A) and HfFG (B) and their structures (C). Labels I, III, V represent type I (Fuc2S4S), III (Fuc3S4S), V (Fuc4S) of FucS, respectively.
Figure 2. Part of the 1H NMR spectra of pFs at different periods of partial acid hydrolysis at 60 °C (A); the integral variation of proton signals at 5.6, 3.13, 1.2 ppm as hydrolysis time course (B).
Figure 3. Time course (0–36 h) of average molecular weight (Mw) of pF1–12 (A) and temperature course (50, 60, 70 and 80 °C) of Mw of pF13–16 (B).
Figure 4. 1H (A) and 13C (B) NMR spectra of pF18.
Figure 5. 1H /13C NMR (A, B), 1H-1H COSY spectra of Sc8 (C) and the structures of Sc8 (D).
Figure 6. The variation of sulfated patterns of fucose released from SvFG with the reaction time (A) and temperature (C), the fitting curve of degree of desulfation in Scs versus reaction time (B) and temperature (D). Percentage proportions of Fuc2S4S (■), Fuc2S (●), Fuc4S (◂), Fuc3S4S (▴), Fuc3S (○), and Fuc (▸) were determined from the 1H NMR spectra of the acid-released and corresponding acid-resistant fragment.
Figure 7. 1H NMR spectra of Sc19 (A), Sc20 (B), Sc9 (C), and Sc18 (D). Sc9 and Sc19 were the side chains obtained at 60 °C for 12 h from SvFG and HfFG, respectively; Sc18 and Sc20 were the side chains obtained at 100 °C for 2 h from SvFG and HfFG, respectively.
Figure 8. Plot of degree of defucosylation (DF) (A), degree of desulfation (DS) in fucose branches (B), and degree of hydrolysis (DH) of the backbone glycosidic bonds (C) as a function of hydrolysis time.
Figure 9. APTT (activated partial thromboplastin time) prolongation activities of heparin, LMWH (low-molecular-weight heparin), native SvFG, and its partial fucosylated derivatives (pF10–12, pF17–18) (A); Effects of molecular weight, degree of fucosylation, and –SO3− content in pFs on their APTT prolonged activities (B).
